Patent application title: METHOD FOR PRODUCING MONATIN USING AN L-AMINO ACID AMINOTRANSFERASE
Inventors:
Yasuaki Takakura (Kawasaki-Shi, JP)
Hiroomi Ogino (Kawasaki-Shi, JP)
Masakazu Sugiyama (Kawasaki-Shi, JP)
Masakazu Sugiyama (Kawasaki-Shi, JP)
Kenichi Mori (Kawasaki-Shi, JP)
Kenichi Mori (Kawasaki-Shi, JP)
Eri Tabuchi (Kawasaki-Shi, JP)
Koki Ishikawa (Kawasaki-Shi, JP)
Uno Tagami (Kawasaki-Shi, JP)
Hidemi Fujii (Kawasaki-Shi, JP)
Assignees:
Ajinomoto Co., Inc.
IPC8 Class: AC12P1304FI
USPC Class:
435106
Class name: Chemistry: molecular biology and microbiology micro-organism, tissue cell culture or enzyme using process to synthesize a desired chemical compound or composition preparing alpha or beta amino acid or substituted amino acid or salts thereof
Publication date: 2014-08-21
Patent application number: 20140234916
Abstract:
The present invention provides a methodology for improving a yield of
2R,4R-Monatin. Specifically, the present invention provides a method for
producing 2S,4R-Monatin or a salt thereof, comprising contacting 4R-IHOG
with an L-amino acid aminotransferase in the presence of an L-amino acid
to form the 2S,4R-Monatin; a method for producing 2R,4R-Monatin or a salt
thereof, comprising isomerizing the 2S,4R-Monatin to form the
2R,4R-Monatin; and the like. These production methods may further
comprise condensing indole-3-pyruvate and pyruvate to form the 4R-IHOG,
and deaminating a tryptophan to form the indole-3-pyruvate.Claims:
1.-30. (canceled)
31. A method for producing 2R,4R-Monatin or a salt thereof, comprising the following (I) and (II): (I) contacting 4R form of 4-(indole-3-yl-methyl)-4-hydroxy-2-oxoglutaric acid (4R-IHOG) with an L-amino acid aminotransferase in the presence of an L-amino acid to form the 2S,4R-Monatin, wherein the L-amino acid aminotransferase consists of an amino acid sequence having 95% or more identity to the amino acid sequence of SEQ ID NO:2; and (II) isomerizing the 2S,4R-Monatin to form the 2R,4R-Monatin.
32. The production method of claim 31, wherein a keto acid formed from the L-amino acid due to the action of the L-amino acid aminotransferase is contacted with a decarboxylase to degrade said keto acid.
33. The production method of claim 31, wherein the L-amino acid is L-aspartate.
34. The production method of claim 33, wherein oxaloacetate formed from the L-aspartate due to the action of the L-amino acid aminotransferase is contacted with an oxaloacetate decarboxylase to degrade said oxaloacetate to irreversibly form pyruvate.
35. The production method of claim 31, wherein the L-amino acid aminotransferase is derived from a microorganism belonging to the genus Arthrobacter, Bacillus, Candida, Corynebacterium, Lodderomyces, Micrococcus, Microbacterium, Nocardia, Pseudomonas, Rhizobium, Stenotrophomonas, Dietzia, Ochrobactrum, Brevundimonas, Burkholderia, Carnimonas, Yarrowia, Clostridium, Deinococcus, Eubacterium, Lactobacillus, Methanothermobacter, Phormidium, Pyrococcus, Rhodococcus, Saccharomyces, Saccharophagus, Sinorhizobium, Thermoanaerobacter, Thermotoga or Thermus.
36. The production method of claim 35, wherein the L-amino acid aminotransferase is derived from Arthrobacter sp., Bacillus altitudinis, Bacillus cellulosilyticus, Bacillus pumilus, Bacillus sp., Candida norvegensis, Candida inconspicua, Corynebacterium ammoniagenes, Corynebacterium glutamicum, Lodderomyces elongisporus, Micrococcus luteus, Microbacterium sp., Nocardia globerula, Pseudomonas chlororaphis, Pseudomonas citronocllolis, Pseudomonas fragi, Pseudomonas putida, Pseudomonas synxantha, Pseudomonas taetrolens, Pseudomonas sp., Rhizobium radiobacter, Rhizobium sp., Stenotrophomonas sp., Dietzia maxis, Ochrobactrum pseudogrignonense, Brevundimonas diminuta, Burkholderia sp., Carnimonas sp., Yarrowia lypolytica, Clostridium cellulolyticum, Deinococcus geothermalis, Eubacterium rectale, Lactobacillus acidophilus, Methanothermobacter thermautotrophicus, Phormidium lapideum, Pyrococcus horikoshii, Rhodococcus erythropolis, Saccharomyces cerevisiae, Saccharophagus degradans, Sinorhizobium meliloti, Thermoanaerobacter tengcongensis, Thermotoga maritima, or Thermus thermophilus.
37. The production method of claim 31, wherein the L-amino acid aminotransferase comprises one or more mutations of amino acid residues selected from the group consisting of the amino acid residues at position 39, position 109, position 128, position 150, position 258, position 287, position 288, position 289, position 303, position 358 and position 431 in the amino acid sequence of SEQ ID NO:2.
38. The production method of claim 37, wherein the one or more mutations of amino acid residues are selected from the group consisting of: i) substitution of the lysine at position 39 with an arginine; ii) substitution of the serine at position 258 with a glycine; iii) substitution of the glutamine at position 287 with a glutamic acid; iv) substitution of the threonine at position 288 with a glycine; v) substitution of the isoleucine at position 289 with an alanine; vi) substitution of the aspartic acid at position 109 with a glycine; vii) substitution of the histidine at position 150 with a tyrosine; viii) substitution of the phenylalanine at position 303 with a leucine; ix) substitution of the aspartic acid at position 358 with a tyrosine; x) substitution of the serine at position 431 with a threonine; and xi) substitution of the glutamic acid at position 128 with a glycine.
39. The production method of claim 31, wherein the 4R-IHOG is contacted with the L-amino acid aminotransferase using a transformant that expresses the L-amino acid aminotransferase.
40. The production method of claim 31, further comprising condensing indole-3-pyruvate and pyruvate to form the 4R-IHOG.
41. The production method of claim 40, wherein the indole-3-pyruvate and the pyruvate are condensed by contacting the indole-3-pyruvate and the pyruvate with an aldolase.
42. The production method of claim 40, wherein at least part of the pyruvate used in the formation of the 4R-IHOG is from pyruvate formed from the oxaloacetate due to the action of an oxaloacetate decarboxylase.
43. The production method of claim 40, further comprising deaminating a tryptophan to form the indole-3-pyruvate.
44. The production method of claim 43, wherein the tryptophan is deaminated by contacting the tryptophan with a deamination enzyme.
45. The production method of claim 41, wherein the production of 2S,4R-Monatin or the salt thereof is carried out in one reactor.
46. The production method of claim 40, wherein the production of the 2S,4R-Monatin or the salt thereof is carried out in one reactor.
47. The production method of claim 41, wherein the 2S,4R-Monatin is isomerized in the presence of an aromatic aldehyde.
48. The production method of claim 41, wherein the salt is a sodium salt or a potassium salt.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority from U.S. provisional Patent Application No. 61/477,402, filed on Apr. 20, 2011, the entire contents of which are incorporated herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to a method for producing Monatin using an L-amino acid aminotransferase, and the like.
BACKGROUND ART
[0003] Monatin [4-(indole-3-yl-methyl)-4-hydroxy-glutamic acid] is a compound that is one of amino acids contained in roots of Schlerochitom ilicifolius that is a shrub in South Africa and is particularly expected as a low calorie sweetener because of having sweetness one thousand and several hundreds times sweeter than sucrose (see Patent Document 1). The Monatin has asymmetric carbon atoms at positions 2 and 4, and a naturally occurring stereoisomer of Monatin is a 2S,4S-isomer. Naturally non-occurring three stereoisomers have been synthesized by organic chemistry processes. All of these stereoisomers are excellent in sweetness, and expected to be used as the sweeteners.
[0004] Several methods have been reported as the methods for producing the Monatin (e.g., see Patent Document 2). However, all of the reported methods require a step of multiple stages, and thus, it is required to improve a synthetic yield of the Monatin.
[0005] Specifically, for the method for producing the Monatin, the following method for producing 2R,4R-Monatin by synthesizing indole-3-pyruvate (hereinafter referred to as "IPA" as needed) from L-tryptophan (L-Trp), synthesizing 4R form of 4-(indole-3-yl-methyl)-4-hydroxy-2-oxoglutaric acid (hereinafter referred to as "4R-IHOG" as needed) from the resulting IPA and pyruvate, and subsequently subjecting the obtained 4R-IHOG to an oximation reaction, a reduction reaction and an epimerization-crystallization method has been known (conventional method (1)) (see Patent Document 2).
[0006] However, an aldolase step (second step) is an equilibrium reaction, and thus, a satisfactory yield is not always obtained in this reaction.
##STR00001##
[0007] In order to improve the yield of the 2R,4R-Monatin, the method for producing the 2R,4R-Monatin by a one-pot enzymatic reaction has been invented (conventional method (2)) (see Patent Documents 3 to 6).
[0008] Patent Document 1: JP Sho-64-25757-A
[0009] Patent Document 2: International Publication WO2003/059865
[0010] Patent Document 3: International Publication WO2007/133184
[0011] Patent Document 4: International Publication WO2005/042756
[0012] Patent Document 5: US Patent Application Publication No. 2006/0252135 Specification
[0013] Patent Document 6: US Patent Application Publication No. 2008/020434 Specification
SUMMARY OF INVENTION
Problem to be Solved by the Invention
[0014] The object of the present invention is to provide a method for producing Monatin with a good yield.
Means for Solving Problem
[0015] As a result of an extensive study, the present inventors have found that the above problem can be solved by using an L-amino acid aminotransferase, and completed the present invention. No L-amino acid aminotransferase that acts upon 4R-IHOG has been known so far.
[0016] Accordingly, the present invention is as follows.
[1] A method for producing 2S,4R-Monatin or a salt thereof, comprising contacting 4R-IHOG with an L-amino acid aminotransferase in the presence of an L-amino acid to form the 2S,4R-Monatin. [2] The production method of [1], further comprising contacting a keto acid with a decarboxylase to degrade the keto acid, wherein the keto acid is formed from the L-amino acid due to action of the L-amino acid aminotransferase. [3] The production method of [1], wherein the L-amino acid is L-aspartate. [4] The production method of [3], further comprising contacting oxaloacetate with an oxaloacetate decarboxylase to irreversibly form pyruvate, wherein the oxaloacetate is formed from the L-aspartate by action of the L-amino acid aminotransferase. [5] The production method of [1], wherein the L-amino acid aminotransferase is derived from a microorganism belonging to genus Arthrobacter, genus Bacillus, genus Candida, genus Corynebacterium, genus Lodderomyces, genus Micrococcus, genus Microbacterium, genus Nocardia, genus Pseudomonas, genus Rhizobium, genus Stenotrophomonas, genus Dietzia, genus Ochrobactrum, genus Brevundimonas, genus Burkholderia, genus Carnimonas, genus Yarrowia, genus Clostridium, genus Deinococcus, genus Eubacterium, genus Lactobacillus, genus Methanothermobacter, genus Phormidium, genus Pyrococcus, genus Rhodococcus, genus Saccharomyces, genus Saccharophagus, genus Sinorhizobium, genus Thermoanaerobacter, genus Thermotoga or genus Thermus. [6] The production method of [5], wherein the L-amino acid aminotransferase is derived from a microorganism belonging to Arthrobacter sp., Bacillus altitudinis, Bacillus cellulosilyticus, Bacillus pumilus, Bacillus sp., Candida norvegensis, Candida inconspicua, Corynebacterium ammoniagenes, Corynebacterium glutamicum, Lodderomyces elongisporus, Micrococcus luteus, Microbacterium sp., Nocardia globerula, Pseudomonas chlororaphis, Pseudomonas citronocllolis, Pseudomonas fragi, Pseudomonas putida, Pseudomonas synxantha, Pseudomonas taetrolens, Pseudomonas sp., Rhizobium radiobacter, Rhizobium sp., Stenotrophomonas sp., Dietzia marls, Ochrobactrum pseudogrignonense, Brevundimonas diminuta, Burkholderia sp., Carnimonas sp., Yarrowia lypolytica, Clostridium cellulolyticum, Deinococcus geothermalis, Eubacterium rectale, Lactobacillus acidophilus, Methanothermobacter thermautotrophicus, Phormidium lapideum, Pyrococcus horikoshii, Rhodococcus erythropolis, Saccharomyces cerevisiae, Saccharophagus degradans, Sinorhizobium meliloti, Thermoanaerobacter tengcongensis, Thermotoga maritima, or Thermus thermophilus. [7] The production method of [1], wherein the L-amino acid aminotransferase consists of an amino acid sequence showing 90% or more identity to the amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:48, SEQ ID NO:53, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, or SEQ ID NO:111. [8] The production method of [7], wherein the L-amino acid aminotransferase comprises one or more mutations of amino acid residues selected from the group consisting of the amino acid residues at position 39, position 109, position 128, position 150, position 258, position 287, position 288, position 289, position 303, position 358 and position 431 in the amino acid sequence represented by SEQ ID NO:2. [9] The production method of [8], wherein the one or more mutations of amino acid residues are selected from the group consisting of: i) substitution of the lysine at position 39 with an arginine; ii) substitution of the serine at position 258 with a glycine; iii) substitution of the glutamine at position 287 with a glutamic acid; iv) substitution of the threonine at position 288 with a glycine; v) substitution of the isoleucine at position 289 with an alanine; vi) substitution of the aspartic acid at position 109 with a glycine; vii) substitution of the histidine at position 150 with a tyrosine; viii) substitution of the phenylalanine at position 303 with a leucine; ix) substitution of the aspartic acid at position 358 with a tyrosine; x) substitution of the serine at position 431 with a threonine; and xi) substitution of the glutamic acid at position 128 with a glycine. [10] The production method of [1], wherein the 4R-IHOG is contacted with the L-amino acid aminotransferase using a transformant that expresses the L-amino acid aminotransferase. [11] The production method of [1], further comprising condensing indole-3-pyruvate and pyruvate to form the 4R-IHOG. [12] The production method of [11], the indole-3-pyruvate and the pyruvate are condensed by contacting the indole-3-pyruvate and the pyruvate with an aldolase. [13] The production method of [11], wherein at least part of the pyruvate used in the formation of the 4R-IHOG is from pyruvate formed from the oxaloacetate due to action of the oxaloacetate decarboxylase. [14] The production method of [11], further comprising deaminating a tryptophan to form the indole-3-pyruvate. [15] The production method of [14], wherein the tryptophan is deaminated by contacting the tryptophan with a deamination enzyme. [16] The production method of [11] or [14], wherein the production of the 2S,4R-Monatin or the salt thereof is carried out in one reactor. [17] A method for producing 2R,4R-Monatin or a salt thereof, comprising the following (I) and (II): (I) performing the method of [1] to form the 2S,4R-Monatin; and (II) isomerizing the 2S,4R-Monatin to form the 2R,4R-Monatin. [18] The production method of [17], wherein the 2S,4R-Monatin is isomerized in the presence of an aromatic aldehyde. [19] The production method of [17], wherein the salt is a sodium salt or a potassium salt. [20] An L-amino acid aminotransferase that is a protein selected form the group consisting of the following (A)-(D): (A) a protein consisting of the amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:48, SEQ ID NO:53, or SEQ ID NO:61; (B) a protein comprising the amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:48, SEQ ID NO:53, or SEQ ID NO:61; (C) a protein consisting of an amino acid sequence showing 90% or more identity to the amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:48, SEQ ID NO:53, or SEQ ID NO:61, and having an L-amino acid aminotransferase activity; and (D) a protein consisting of an amino acid sequence comprising mutation of one or several amino acid residues, which is selected from the group consisting of deletion, substitution, addition and insertion of the amino acid residues in the amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:48, SEQ ID NO:53, or SEQ ID NO:61, and having an L-amino acid aminotransferase activity. [21] The L-amino acid aminotransferase of [20], wherein the L-amino acid aminotransferase comprises one or more mutations of amino acid residues selected from the group consisting of the amino acid residues at position 39, position 109, position 128, position 150, position 258, position 287, position 288 and position 289, position 303, position 358 and position 431 in the amino acid sequence represented by SEQ ID NO:2. [22] The L-amino acid aminotransferase of [21], wherein the one or more mutations of amino acid residues are selected from the group consisting of: i) substitution of the lysine at position 39 with an arginine; ii) substitution of the serine at position 258 with a glycine; iii) substitution of the glutamine at position 287 with a glutamic acid; iv) substitution of the threonine at position 288 with a glycine; v) substitution of the isoleucine at position 289 with an alanine; vi) substitution of the aspartic acid at position 109 with a glycine; vii) substitution of the histidine at position 150 with a tyrosine; viii) substitution of the phenylalanine at position 303 with a leucine; ix) substitution of the aspartic acid at position 358 with a tyrosine; x) substitution of the serine at position 431 with a threonine; and xi) substitution of the glutamic acid at position 128 with a glycine. [23] A polynucleotide selected from the group consisting of the following (a)-(e): (a) a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO:1, SEQ ID NO:47, SEQ ID NO:52, or SEQ ID NO:60; (b) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO:1, SEQ ID NO:47, SEQ ID NO:52, or SEQ ID NO:60; (c) a polynucleotide consisting of a nucleotide sequence showing 90% or more identity to the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:47, SEQ ID NO:52, or SEQ ID NO:60, and encoding a protein having an L-amino acid aminotransferase activity; (d) a polynucleotide that hybridizes under a stringent condition with a polynucleotide consisting of the nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO:1, SEQ ID NO:47, SEQ ID NO:52, or SEQ ID NO:60, and encodes a protein having an L-amino acid aminotransferase activity; and (e) a polynucleotide encoding the L-amino acid aminotransferase of [20]. [24] An expression vector comprising the polynucleotide of [23]. [25] A transformant introduced with the expression vector of [24]. [26] A method for producing an L-aminotransfearase, comprising culturing the transformant of [25] in a medium to obtain the L-amino acid aminotransferase. [27] A method of producing 2S,4R-Monatin or a salt thereof, comprising contacting 4R-IHOG with the L-amino acid aminotransferase of [20] in the presence of an L-amino acid to form the 2S,4R-Monatin. [28] A method for producing 2R,4R-Monatin or a salt thereof, comprising the following (I') and (II'): (I') performing the method of [27] to form the 2S,4R-Monatin; and (II') isomerizing the 2S,4R-Monatin to form the 2R,4R-Monatin. [29] The production method of [28], wherein the 2S,4R-Monatin is isomerized in the presence of an aromatic aldehyde. [30] The production method of [28], wherein the salt is a sodium salt or a potassium salt.
Effect of the Invention
[0017] The method of the present invention can contribute to improvement of the yield of the Monatin by producing the 2S,4R-Monatin with a good yield from 4R-IHOG using the L-amino acid aminotransferase. The method of the present invention has an advantage that it is not necessary to use an expensive D-amino acid (D-Asp and the like) as a substrate when the 2S,4R-Monatin is formed from IHOG or that it is not necessary to add an enzyme such as racemase to form the D-amino acid from an L-amino acid. In the method of the present invention, when performing not only the reaction to form the 2S,4R-Monatin from 4R-IHOG (third step) but also the reaction to form IPA from L-Trp (first step) and the reaction to form 4R-IHOG from IPA (second step), whole reaction equilibrium can be defined in the third step and the reaction equilibrium in the second step can be largely shifted to a direction to form 4R-IHOG. In this case, the method of the present invention makes it possible to produce the 2S,4R-Monatin with a very good yield by avoiding a by-product of L-Trp (progress of a reverse reaction of the first step).
BRIEF DESCRIPTION OF DRAWINGS
[0018] FIG. 1 is a view showing one example of the production method of the present invention. Trp: tryptophan; IPA: indole-3-pyruvate; IHOG: 4-(indole-3-yl-methyl)-4-hydroxy-2-oxoglutaric acid; Monatin: 4-(indole-3-yl-methyl)-4-hydroxy-glutamic acid.
[0019] FIG. 2 is a view showing one example of the production method of the present invention. Abbreviations are the same as in FIG. 1; and
[0020] FIG. 3 is a view showing a preferable example of the production method of the present invention. L-Trp: L-tryptophan; L-Asp: L-aspartic acid; OAA: oxaloacetate; PA: pyruvate; and the other abbreviations are the same as in FIG. 1.
[0021] FIG. 4 is a graph showing a reaction of forming 2S,4R-Monatin from L-Trp in 400 ml scale using the L-amino acid aminotransferase mutant (ID166). SR-Monatin: 2S,4R-Monatin; SS-Monatin: 2S,4S-Monatin; IHOG: 4R-IHOG; Trp: L-Trp.
[0022] FIG. 5 is a graph showing a reaction of forming 2S,4R-Monatin from L-Trp in 80 ml scale using the L-amino acid aminotransferase mutant (ID189). The abbreviations are similar to those of FIG. 4.
[0023] FIG. 6 is a graph showing a reaction of forming 2S,4R-Monatin from L-Trp in 80 ml scale using the L-amino acid aminotransferase mutant (ID296). The abbreviations are similar to those of FIG. 4.
BEST MODES FOR CARRYING OUT THE INVENTION
(1) Method for Producing 2S,4R-Monatin or a Salt Thereof
[0024] The present invention provides a method (1) for producing 2S,4R-Monatin or a salt thereof. The production method of the present invention can be classified into (1-1) a method for producing the 2S,4R-Monatin from 4R-IHOG, (1-2) a method for producing the 2S,4R-Monatin from IPA and pyruvate, and (1-3) a method for producing the 2S,4R-Monatin from tryptophan. The methods (1-1), (1-2) and (1-3) are common in contacting 4R-IHOG with an L-amino acid aminotransferase in the presence of the L-amino acid to form the 2S,4R-Monatin.
(1-1) Method for Producing 2S,4R-Monatin from 4R-IHOG
[0025] This method comprises contacting 4R-IHOG with the L-amino acid aminotransferase in the presence of the L-amino acid to form the 2S,4R-Monatin (reaction 1). By contacting 4R-IHOG with the L-amino acid aminotransferase in the presence of the L-amino acid, an amino group in the L-amino acid can be transferred to 4R-IHOG to form the 2S,4R-Monatin.
##STR00002##
[0026] The kinds of the L-amino acid is not particularly limited as long as the amino group in the L-amino acid can be transferred to 4R-IHOG that is an objective substrate by the L-amino acid aminotransferase. Various L-amino acids such as L-α-amino acids are known as such an L-amino acid. Specifically, such an L-amino acid includes L-aspartic acid, L-alanine, L-lysine, L-arginine, L-histidine, L-glutamic acid, L-asparagine, L-glutamine, L-serine, L-threonine, L-tyrosine, L-cysteine, L-valine, L-leucine, L-isoleucine, L-proline, L-phenylalanine, L-methionine and L-tryptophan. A salt form of the L-amino acid may be added to a reaction solution. The concentration of the L-amino acid in a reaction solution is, for example, 1 mM to 3 M, preferably 20 mM to 1 M, more preferably 100 mM to 500 mM.
[0027] In one embodiment, the L-amino acid aminotransferase may be a protein derived from a microorganism such as a bacterium, actinomycete or yeast. The classification of the microorganisms can be carried out by a classification method well-known in the art, e.g., a classification method used in the database of NCBI (National Center for Biotechnology Information) (http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=91347). Examples of the microorganisms from which the L-amino acid aminotransferase is derived include microorganisms belonging to genus Arthrobacter, genus Bacillus, genus Candida, genus Corynebacterium, genus Lodderomyces, genus Micrococcus, genus Microbacterium, genus Nocardia, genus Pseudomonas, genus Rhizobium, genus Stenotrophomonas, genus Dietzia, genus Ochrobactrum, genus Brevundimonas, genus Burkholderia, genus Carnimonas, genus Yarrowia, genus Clostridium, genus Deinococcus, genus Eubacterium, genus Lactobacillus, genus Methanococcus, genus Methanothermobacter, genus Phormidium, genus Pyrococcus, genus Rhodococcus, genus Saccharomyces, genus Saccharophagus, genus Sinorhizobium, genus Thermoanaerobacter, genus Thermotoga, and genus Thermus.
[0028] Specifically, examples of the microorganisms belonging to genus Arthrobacter include Arthrobacter sp.
[0029] Examples of the microorganisms belonging to genus Bacillus include Bacillus altitudinis, Bacillus cellulosilyticus, Bacillus pumilus, and Bacillus sp. Examples of the microorganisms belonging to genus Candida include Candida norvegensis and Candida inconspicua. Examples of the microorganisms belonging to genus Corynebacterium include Corynebacterium ammonia genes, and Corynebacterium glutamicum. Examples of the microorganisms belonging to genus Lodderomyces include Lodderomyces elongisporus. Examples of the microorganisms belonging to genus Micrococcus include Micrococcus luteus. Examples of the microorganisms belonging to genus Microbacterium include Microbacterium sp. Examples of the microorganisms belonging to genus Nocardia include Nocardia globerula.
[0030] Examples of the microorganisms belonging to genus Pseudomonas include Pseudomonas chlororaphis (e.g., Pseudomonas chlororaphis subsp. chlororaphis), Pseudomonas citronocllolis, Pseudomonas fragi, Pseudomonas putida, Pseudomonas synxantha, Pseudomonas taetrolens, and Pseudomonas sp.
[0031] Examples of the microorganisms belonging to genus Rhizobium include Rhizobium radiobacter and Rhizobium sp. Examples of the microorganisms belonging to genus Stenotrophomonas include Stenotrophomonas sp. Examples of the microorganisms belonging to genus Dietzia include Dietzia marls. Examples of the microorganisms belonging to genus Ochrobactrum include Ochrobactrum pseudogrignonense. Examples of the microorganisms belonging to genus Brevundimonas include Brevundimonas diminuta. Examples of the microorganisms belonging to genus Burkholderia include Burkholderia sp. Examples of the microorganisms belonging to genus Carnimonas include Carnimonas sp. Examples of the microorganisms belonging to genus Yarrowia include Yarrowia lypolytica.
[0032] Examples of the microorganisms belonging to genus Clostridium include Clostridium cellulolyticum. Examples of the microorganisms belonging to genus Deinococcus include Deinococcus geothermalis. Examples of the microorganisms belonging to genus Eubacterium include Eubacterium rectale. Examples of the microorganisms belonging to genus Lactobacillus include Lactobacillus acidophilus. Examples of the microorganisms belonging to genus Methanococcus include Methanococcus jannaschii. Examples of the microorganisms belonging to genus Methanothermobacter include Methanothermobacter thermautotrophicus. Examples of the microorganisms belonging to genus Phormidium include Phormidium lapideum. Examples of the microorganisms belonging to genus Pyrococcus include Pyrococcus horikoshii. Examples of the microorganisms belonging to genus Rhodococcus include Rhodococcus erythropolis. Examples of the microorganisms belonging to genus Saccharomyces include Saccharomyces cerevisiae. Examples of the microorganisms belonging to genus Saccharophagus include Saccharophagus degradans. Examples of the microorganisms belonging to genus Sinorhizobium include Sinorhizobium meliloti. Examples of the microorganisms belonging to genus Thermoanaerobacter include Thermoanaerobacter tengcongensis. Examples of the microorganisms belonging to genus Thermotoga include Thermotoga maritima. Examples of the microorganisms belonging to genus Thermus include Thermus thermophilus.
[0033] In another embodiment, the L-amino acid aminotransferase may be a naturally occurring protein or an artificial mutant protein. Such an L-amino acid aminotransferase includes those consisting of an amino acid sequence having high homology (e.g., similarity, identity) to an amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:48, SEQ ID NO:53, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, or SEQ ID NO:111, and having an L-amino acid aminotransferase activity. The term "L-amino acid aminotransferase activity" refers to an activity of transferring the amino group in the L-amino acid to 4R-IHOG that is the objective substrate for forming the 2S,4R Monatin that is an objective compound having the amino group. Specifically, the L-amino acid aminotransferase includes a protein consisting of the amino acid sequence showing 80% or more, preferably 90% or more, more preferably 95% or more and particularly preferably 98% or more or 99% or more homology (e.g., similarity, identity) to the amino acid sequence represented by SEQ ID NO:2, and having the L-amino acid aminotransferase activity.
[0034] The homology of the amino acid sequences and nucleotide sequences can be determined using algorithm BLAST by Karlin and Altschul (Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)) or FASTA by Pearson (Methods Enzymol., 183, 63 (1990)). Programs referred to as BLASTP and BLASTN (see http://www.ncbi.nlm.nih.gov) have been developed based on this algorithm BLAST. Thus, the homology of the amino acid sequences and the nucleotide sequences may be calculated using these programs with default setting. A numerical value obtained when matching count is calculated as a percentage by using GENETYX Ver. 7.0.9 that is software from GENETYX Corporation and using full length polypeptide chains encoded in ORF with setting of Unit Size to Compare=2 may be used as the homology of the amino acid sequences. The lowest value among the values derived from these calculations may be employed as the homology of the amino acid sequences and the nucleotide sequences.
[0035] In further another embodiment, the L-amino acid aminotransferase may be a protein consisting of an amino acid sequence comprising mutation (e.g., deletion, substitution, addition and insertion) of one or several amino acid residues in the amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:48, SEQ ID NO:53, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, or SEQ ID NO:111, and having the L-amino acid aminotransferase activity. The mutation of one or several amino acid residues may be introduced into one region or multiple different regions in the amino acid sequence. The term "one or several amino acid residues" indicate a range in which a three dimensional structure and the activity of the protein are not largely impaired. The term "one or several amino acid residues" in the case of the protein denote, for example, 1 to 100, preferably 1 to 80, more preferably 1 to 50, 1 to 30, 1 to 20, 1 to 10 or 1 to 5 amino acid residues. Such mutation may be attributed to naturally occurring mutation (mutant or variant) based on individual difference, species difference and the like of the microorganism carrying a gene encoding the L-amino acid aminotransferase.
[0036] A position of the amino acid residue to be mutated in the amino acid sequence is apparent to those skilled in the art. Specifically, a person skilled in the art can recognize the correlation between the structure and the function by 1) comparing the amino acid sequences of the multiple proteins having the same kind of activity (e.g., the amino acid sequence represented by SEQ ID NO:2, and amino acid sequences of other L-amino acid aminotransferase), 2) clarifying relatively conserved regions and relatively non-conserved regions, and then 3) predicting a region capable of playing an important role for its function and a region incapable of playing the important role for its function from the relatively conserved regions and the relatively non-conserved regions, respectively. Therefore, a person skilled in the art can specify the position of the amino acid residue to be mutated in the amino acid sequence of the L-amino acid aminotransferase.
[0037] When an amino acid residue is mutated by the substitution, the substitution of the amino acid may be conservative substitution. As used herein, the term "conservative substitution" means that a certain amino acid residue is substituted with an amino acid residue having an analogous side chain. Families of the amino acid residues having the analogous side chain are well-known in the art. Examples of such families include an amino acid having a basic side chain (e.g., lysine, arginine or histidine), an amino acid having an acidic side chain (e.g., aspartic acid or glutamic acid), an amino acid having a non-charged polar side chain (e.g., asparagine, glutamine, serine, threonine, tyrosine or cysteine), an amino acid having a non-polar side chain (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine or tryptophan), an amino acid having a β-position branched side chain (e.g., threonine, valine or isoleucine), an amino acid having an aromatic side chain (e.g., tyrosine, phenylalanine, tryptophan or histidine), an amino acid having a hydroxyl group (e.g., alcoholic or phenolic)-containing side chain (e.g., serine, threonine or tyrosine), and an amino acid having a sulfur-containing side chain (e.g., cysteine or methionine). Preferably, the conservative substitution of the amino acids may be the substitution between aspartic acid and glutamic acid, the substitution among arginine, lysine and histidine, the substitution between tryptophan and phenylalanine, the substitution between phenylalanine and valine, the substitution among leucine, isoleucine and alanine, and the substitution between glycine and alanine.
[0038] In further another embodiment, the L-amino acid aminotransferase may be a protein encoded by DNA that hybridizes under a stringent condition with a nucleotide sequence complementary to a nucleotide sequence represented by SEQ ID NO:2, SEQ ID NO:47, SEQ ID NO:52, SEQ ID NO:60, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, or SEQ ID NO:110, and having the L-amino acid aminotransferase activity. The "stringent condition" refers to the condition where a so-called specific hybrid is formed whereas no non-specific hybrid is formed. Although it is difficult to clearly quantify this condition, one example of this condition is the condition where a pair of polynucleotides with high homology (e.g., identity), for example, a pair of polynucleotides having the homology of 80% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 90% or more are hybridized whereas a pair of polynucleotides with lower homology than that are not hybridized. Specifically, such a condition includes hybridization in 6×SSC (sodium chloride/sodium citrate) at about 45° C. followed by one or two or more washings in 0.2×SSC and 0.1% SDS at 50 to 65° C.
[0039] In a preferred embodiment, the L-amino acid aminotransferase may be L-amino acid aminotransferase mutant in which one or more (e.g., one or two) of any amino acid residues selected from the group consisting of the amino acid residues at position 39, position 109, position 128, position 150, position 258, position 287, position 288, position 289, position 303, position 358, and position 431 in the amino acid sequence represented by SEQ ID NO:2 are mutated (e.g., substituted). Preferred examples of the L-amino acid aminotransferase mutant comprise one or more (e.g., one or two) substitutions selected from the group consisting of:
i) substitution of the lysine at position 39 with an arginine; ii) substitution of the serine at position 258 with a glycine; iii) substitution of the glutamine at position 287 with a glutamic acid; iv) substitution of the threonine at position 288 with a glycine; v) substitution of the isoleucine at position 289 with an alanine; vi) substitution of the aspartic acid at position 109 with a glycine; vii) substitution of the histidine at position 150 with a tyrosine; viii) substitution of the phenylalanine at position 303 with a leucine; ix) substitution of the aspartic acid at position 358 with a tyrosine; x) substitution of the serine at position 431 with a threonine; and xi) substitution of the glutamic acid at position 128 with a glycine.
[0040] For the combination of the substitution of one or more (e.g., one or two) of any amino acid residues selected from the group consisting of the amino acid residues at position 39, position 109, position 128, position 150, position 258, position 287, position 288, position 289, position 303, position 358 and position 431 in the amino acid sequence represented by SEQ ID NO:2, the combined mutations as shown below can be introduced although the combination of the amino acid substitutions which can be utilized in the present invention is not limited to the following:
a) T288G
b) S258G/I289A
c) K39R/T288G
d) Q287E/T288G
e) K39R/D109R/T288G/S431T
f) K39R/D109R/T288G/F303L
g) D109R/Q287E/T288G/F303L
h) D109R/S258G/I289A/F303L
i) D109R/Q287E/T288G/S431T
j) D109R/S258G/I289A/S431T
k) K39R/D109R/E128G/T288G/F303L
l) K39R/D109G/E128G/I288G/F303L
m) D109R/E128G/Q287E/T288G/F303L
n) D109R/E128G/S258G/I289A/S431T
o) D109G/E128G/Q287E/T288G/F303L
p) D109G/E128G/S258G/I289A/F303L
q) K39R/D109G/H150Y/T288G/F303L/D358Y/S431T
r) K39R/D109G/E128G/H150Y/T288G/F303L/D358Y
s) D109G/H150Y/Q287E/T288G/F303L/D35BY/S431T
t) D109G/H150Y/S258G/I289A/F303L/D358Y/S431T
u) D109G/E128G/H150Y/Q287E/T288G/F303L/D358Y or
v) D109G/E128G/H150Y/S258G/I289A/F303L/D358Y
[0041] In one embodiment, the contact of 4R-IHOG with the L-amino acid aminotransferase can be accomplished by allowing 4R-IHOG and the L-amino acid aminotransferase extracted from an L-amino acid aminotransferase-producing microorganism (extracted enzyme) to coexist in a reaction solution. Examples of the L-amino acid aminotransferase-producing microorganism include the microorganisms that naturally produce the L-amino acid aminotransferase (e.g., the aforementioned microorganisms), and transformants that express the L-amino acid aminotransferase. Specifically, examples of the extracted enzyme include a purified enzyme, a crude enzyme, an immorbilized enzyme, a cuture broth, and a treated product of the culture broth (e.g., an L-amino acid aminotransferase-containing fraction prepared from the above enzyme-producing microorganism, and a disrupted product of and a lysate of the above enzyme-producing microorganism). Examples of the treatment for obtaining the treated product of the culture broth from the culture broth include a heat treatment (42° C. to 80° C., pH 3 to 12, 1 minute to 24 hours), a solvent treatment (e.g, xylene, toluene, ethanol, isopropylalcohol), a surfactant (e.g., Tween 20, Triton X-100), and a treatment with a bacteriolytic enzyme (e.g., lysozyme treatment). Alternatively, the culture broth is subjected to a reaction after retaining it with adjusting temperature, pH and the like to enhance an enzymatic activity detected in the broth. In this case, the temperature may be set at 4° C. to 60° C., preferably 20° C. to 37° C. In addition, the pH may be set at 3 to 12, preferably 7 to 9. The time may be set for about 5 minutes to 20 days, preferably about 1 hour to 7 days. During retaining the broth, aeration and agitation may be or may not be carried out.
[0042] In another embodiment, the contact of 4R-IHOG with the L-amino acid aminotransferase can be accomplished by allowing 4R-IHOG and the L-amino acid aminotransferase-producing microorganism to coexist in the reaction solution (e.g., culture medium).
[0043] The reaction solution used in the production method (1) of the present invention is not particularly limited as long as the objective reaction progresses, and for example, water and buffer are used. Examples of the reaction solution include Tris buffer, phosphate buffer (e.g., KH2PO4), carbonate buffer, borate buffer and acetate buffer. The concentration of the buffer may be, for example, 0.1 mM to 10 M, preferably 1 mM to 1 M. When the L-amino acid aminotransferase-producing microorganism is used in the production method of the present invention, the culture medium may be used as the reaction solution. Such a culture medium can be prepared using a medium described later. The reaction solution used in the production method of the present invention may further comprise pyridoxal phosphate (PLP) as a coenzyme. A salt form of PLP may be added to the reaction solution. The concentration of PLP in the reaction solution may be, for example, 1 μM to 100 mM, preferably 10 μM to 1 mM. When the reaction solution comprises PLP, an effect to form 2R,4R-Monatin from the 2S,4R-Monatin can be expected by an isomerization reaction which can be catalyzed by PLP (e.g., see Example 11).
[0044] A pH value of the reaction solution used in the production method (1) of the present invention is not particularly limited as long as the objective reaction progresses, and is, for example, pH 5 to 10, is preferably pH 6 to 9 and is more preferably pH 7 to 8.
[0045] A reaction temperature in the production method (1) of the present invention is not particularly limited as long as the objective reaction progresses, and is, for example, 10 to 50° C., is preferably 20 to 40° C. and is more preferably 25 to 35° C.
[0046] A reaction time period in the production method (1) of the present invention is not particularly limited as long as the time period is sufficient to form the 2S,4R-Monatin, and is, for example, 2 to 100 hours, is preferably 4 to 50 hours and is more preferably 8 to 25 hours.
[0047] When a transformant that expresses the L-amino acid aminotransferase is used as the L-amino acid aminotransferase-producing microorganism, this transformant can be made by, for example, making an expression vector of the L-amino acid aminotransferase, and then introducing this expression vector into a host. For example, the transformant that expresses the L-amino acid aminotransferase can be obtained by making the expression vector incorporating DNA having the nucleotide sequence represented by SEQ ID NO:1, and introducing it into an appropriate host. For example, various prokaryotic cells including bacteria belonging to genus Escherichia such as Escherichia coli, genus Corynebacterium (e.g., Corynebacterium glutamicum) and genes Bacillus (e.g., Bacillus subtilis), and various eukaryotic cells including genus Saccharomyces (e.g., Saccharomyces cerevisiae), genus Pichia (e.g., Pichia stipitis) and genus Aspergillus (e.g., Aspergillus oryzae) can be used as the host for expressing the L-amino acid aminotransferase. For the host, a strain having deletion of a certain gene may be used. Examples of such a gene which may be deleted include AspC, an L-amino acid aminotransferase derived from a host, an aldolase derived from a host, a deamination enzyme derived from a host. Examples of the transformants include a transformant carrying a vector in its cytoplasm, and a transformant introduced with a gene of interest into its genome.
[0048] An L-amino acid aminotransferase-producing microorganism can be cultured using certain culture apparatus (e.g., a test tube, a flask, or a jar fermenter) in a medium having the composition mentioned below. The culture condition can be set appropriately. Specifically, the culture temperature may be 25° C. to 37° C., pH may be 6.5 to 7.5, the culture time may be 1 hour to 100 hours. The cultivation may be carried out with controlling the concentration of dissolved oxygen. In this case, the concentration of dissolved oxygen (DO value) in the culture solution may be utilized as an indicator of the controlling.
[0049] The condition on aeration and agitation can be controlled such that relative concentration of dissolved oxygen (DO value) in the case of the concentration of oxygen in air being considered 21% is not less than 1% to 10%, preferably 3% to 8%. The cultivation may be batch cultivation or fed-batch cultivation. In the case of the fed-batch cultivation, a sugar source solution and a solution containing phosphate can be continuously or discontinuously added in a sequential manner to continue the cultivation.
[0050] The hosts to be transformed are as described above. Describing Escherichia coli in detail, the host can be selected from Escherichia coli K12 strain subspecies, Escherichia coli JM109, DH5α, HB101, BL21 (DE3) strains and the like. Methods for performing the transformation and methods for selecting the transformant are described in Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor press (2001/01/15) and the like. A method for making transformed Escherichia coli and producing a certain enzyme by the use thereof will be specifically described below as one example.
[0051] As a promoter for expressing DNA encoding the L-amino acid aminotransferase, the promoter typically used for producing a heterogeneous protein in E. coli can be used, and includes potent promoters such as PhoA, PhoC, T7 promoter, lac promoter, trp promoter, trc promoter, tac promoter, PR and PL promoters of lambda phage, and T5 promoter. PhoA, PhoC and lac are preferred. As the vector, pUC (e.g., pUC19, pUC18), pSTV, pBR (e.g., pBR322), pHSG (e.g., pHSG299, pHSG298, pHSG399, pHSG398), RSF (e.g., RSF1010), pACYC (e.g., pACYC177, pACYC184), pMW (e.g., pMW119, pMW118, pMW219, pMW218), pQE (e.g., pQE30) and derivatives thereof, and the like may be used. The vectors of phage DNA may also be utilized as the other vectors. Further, the expression vector containing the promoter and capable of expressing the inserted DNA sequence may be used. Preferably, the vector may be pUC, pSTV or pMW.
[0052] A terminator that is a transcription termination sequence may be ligated to downstream of an L-amino acid aminotransferase gene. Examples of such a terminator include T7 terminator, fd phage terminator, T4 terminator, a terminator of a tetracycline resistant gene, and a terminator of an E. coli trpA gene.
[0053] So-called multiple copy types are preferable as the vector for introducing the L-amino acid aminotransferase gene into E. coli, and include plasmids having a replication origin derived from ColE1, such as pUC type plasmids, pBR322 type plasmids or derivatives thereof. Here, the "derivatives" means those in which modification is given to the plasmids by substitution, deletion, insertion, addition and/or inversion of nucleotides. The "modification" as referred to here also includes the modification by mutagenic treatments by mutagenic agents and UV irradiation, or natural mutation, or the like.
[0054] For selecting the transformant, it is preferable that the vector has a marker such as an ampicillin resistant gene. As such a plasmid, the expression vectors carrying the strong promoter are commercially available (e.g., pUC types (supplied from TAKARA BIO Inc.), pPROK types (supplied from Clontech), pKK233-2 (supplied from Clontech)).
[0055] The L-amino acid aminotransferase is expressed by transforming E. coli with the obtained expression vector and culturing this E. coli.
[0056] A medium such as M9-casamino acid medium and LB medium typically used for culturing E. coli may be used as the medium. The medium may contain a certain carbon source, a nitrogen source, and a coenzyme (e.g., pyridoxine hydrochloride). Specifically, peptone, yeast extract, NaCl, glucose, MgSO4, ammonium sulfate, potassium dihydrogen phosphate, ferric sulfate, manganese sulfate, thiamine, hydrolysate of soy with hydrochloric acid, Disfoam GD113-K (NOF Corporation) and the like may be used. Culture conditions and production induction conditions are appropriately selected depending on types of the marker and the promoter in the used vector, the host bacterium and the like.
[0057] The following methods and the like are available for recovering the L-amino acid aminotransferase. The L-amino acid aminotransferase can be obtained as a disrupted product or a lysate by collecting the L-amino acid aminotransferase-producing microorganism followed by disrupting (e.g., sonication, homogenization) or lysing (e.g., lysozyme treatment) the microbial cells. Also, the purified enzyme, the crude enzyme, the L-amino acid aminotransferase-containing fraction, or the like can be obtained by subjecting such a disrupted product or lysate to techniques such as extraction, precipitation, filtration and column chromatography.
[0058] In a preferred embodiment, the production method of the present invention further comprises contacting a keto acid (R--COCOOH) formed from the L-amino acid (e.g., L-α-amino acid) by action of the L-amino acid aminotransferase with a decarboxylase to degrade the keto acid (see the reaction 1'). By promoting the degradation of the keto acid formed from the L-amino acid by an amino group transfer reaction, it is possible to shift the equilibrium of the reaction to form the 2S,4R-Monatin from 4R-IHOG so that the 2S,4R-Monatin is formed in a larger amount.
##STR00003##
[0059] The decarboxylase used in the present invention is the enzyme that catalyzes a decarboxylation reaction of the keto acid. The decarboxylation reaction by the decarboxylase can be irreversible. Various enzymes are known as the decarboxylase used for the irreversible decarboxylation reaction of the keto acid, and examples thereof include an oxaloacetate decarboxylase derived from Pseudomonas stutzeri (Arch Biochem Biophys., 365, 17-24, 1999) and a pyruvate decarboxylase derived from Zymomonas mobilis (Applied Microbiology and Biotechnology, 17, 152-157, 1983).
[0060] In a particularly preferred embodiment, the production method of the present invention comprises contacting oxaloacetate (CAA) formed from L-aspartic acid (L-Asp) by action of the L-amino acid aminotransferase with the oxaloacetate decarboxylase to form the pyruvate (PA) (see the reaction 1''). By promoting the irreversible formation of the pyruvate from the oxaloacetate, it is possible to shift the equilibrium of the reaction to form the 2S,4R-Monatin from 4R-IHOG so that the 2S,4R-Monatin is formed in a larger amount. A salt form of L-aspartic acid may be added to the reaction solution. The concentration of L-aspartate in the reaction solution is 1 mM to 3 M, preferably 20 mM to 1 M, more preferably 100 mM to 500 mM.
##STR00004##
[0061] The oxaloacetate decarboxylase used in the present invention is the enzyme that catalyzes the decarboxylation reaction of the oxaloacetate to form the pyruvate. The decarboxylation reaction by the oxaloacetate decarboxylase can be irreversible. Various enzymes are known as the oxaloacetate decarboxylase used for the irreversible decarboxylation reaction of the oxaloacetate. Examples of such an oxaloacetate decarboxylase include the oxaloacetate decarboxylase derived from Pseudomonas stutzeri (Arch Biochem Biophys., 365, 17-24, 1999), the oxaloacetate decarboxylase derived from Klebsiella aerogenes (FEBS Lett., 141, 59-62, 1982), and the oxaloacetate decarboxylase derived from Sulfolobus solfataricus (Biochim Biophys Acta., 957, 301-311, 1988).
[0062] When the decarboxylase is used in the production of the 2S,4R-Monatin from 4R-IHOG, the contact of the keto acid formed from the L-amino acid with the decarboxylase can be accomplished by allowing the keto acid and the decarboxylase extracted from a decarboxylase-producing microorganism (extracted enzyme) or the decarboxylase-producing microorganism to coexist in the reaction solution (e.g., culture medium). Examples of the decarboxylase-producing microorganism include microorganisms that naturally produce the decarboxylase and transformants that express the decarboxylase. Examples of the extracted enzyme include a purified enzyme, a crude enzyme, an immobilized enzyme, a culture broth, and a treated product of the culture broth (e.g., a decarboxylase-containing fraction prepared from the above decarboxylase-producing microorganism, and a disrupted product of and a lysate of the above decarboxylase-producing microorganism). Examples of the treatment for obtaining the treated product of the culture broth from the culture broth include a heat treatment (42° C. to 80° C., pH 3 to 12, 1 minute to 24 hours), a solvent treatment (e.g, xylene, toluene, ethanol, isopropylalcohol), a surfactant (e.g., Tween 20, Triton X-100), and a treatment with a bacteriolytic enzyme (e.g., lysozyme treatment). Alternatively, the culture broth is subjected to a reaction after retaining it with adjusting temperature, pH and the like to enhance an enzymatic activity detected in the broth. In this case, the temperature may be set at 4° C. to 60° C., preferably 20° C. to 37° C. The pH may be set at 3 to 12, preferably 7 to 9. The time may be set for about 5 minutes to 20 days, preferably about 1 hour to 7 days. During retaining the broth, aeration and agitation may be or may not be carried out.
[0063] When both the L-amino acid aminotransferase and the decarboxylase are used in the production of the 2S,4R-Monatin from 4R-IHOG, the L-amino acid aminotransferase and the decarboxylase may be provided in the reaction solution in the following manner:
[0064] L-amino acid aminotransferase (extracted enzyme) and decarboxylase (extracted enzyme);
[0065] L-amino acid aminotransferase-producing microorganism and decarboxylase (extracted enzyme);
[0066] L-amino acid aminotransferase (extracted enzyme) and decarboxylase-producing microorganism;
[0067] L-amino acid aminotransferase-producing microorganism and decarboxylase-producing microorganism; and
[0068] L-amino acid aminotransferase- and decarboxylase-producing microorganism.
[0069] Preferably, the L-amino acid aminotransferase- and decarboxylase-producing microorganism may be a transformant. Such a transformant can be made by i) introducing an expression vector of the L-amino acid aminotransferase into the decarboxylase-producing microorganism, ii) introducing an expression vector of the decarboxylase into the L-amino acid aminotransferase-producing microorganism, (iii) introducing a first expression vector of the L-amino acid aminotransferase and a second expression vector of the decarboxylase into a host microorganism, and (iv) introducing an expression vector of the L-amino acid aminotransferase and the decarboxylase into the host microorganism. Examples of the expression vector of the L-amino acid aminotransferase and the decarboxylase include i') an expression vector containing a first expression unit composed of a first polynucleotide encoding the L-amino acid aminotransferase and a first promoter operatively linked to the first polynucleotide, and a second expression unit composed of a second polynucleotide encoding the decarboxylase and a second promoter operatively linked to the second polynucleotide; and ii') an expression vector containing a first polynucleotide encoding the L-amino acid aminotransferase, a second polynucleotide encoding the decarboxylase and a promoter operatively linked to the first polynucleotide and the second polynucleotide (vector capable of expressing polycistronic mRNA). The first polynucleotide encoding the L-amino acid aminotransferase may be located upstream or downstream the second polynucleotide encoding the decarboxylase.
(1-2) Method for Producing 2S,4R-Monatin from IPA and Pyruvate
[0070] The production method of the present invention may further comprise condensing IPA and the pyruvate to form 4R-IHOG in order to prepare 4R-IHOG. The condensation of IPA and the pyruvate can be carried out by the organic chemistry process, or an enzymatic method using an aldolase. The method for forming 4R-IHOG by condensing IPA and the pyruvate by the organic chemistry process is disclosed in, for example, International Publication WO2003/059865 and US Patent Application Publication No. 2008/0207920. The method for forming 4R-IHOG by condensing IPA and the pyruvate by the enzymatic method using the aldolase is disclosed in, for example, International Publication WO2003/056026, JP 2006-204285-A, US Patent Application Publication No. 2005/0244939 and International Publication WO2007/103989. Therefore, in the present invention, these methods can be used in order to prepare 4R-IHOG from IPA and the pyruvate.
[0071] IPA used for the preparation of 4R-IHOG is an unstable compound. Therefore, the condensation of IPA and the pyruvate may be carried out in the presence of a stabilizing factor for IPA. Examples of the stabilizing factor for IPA include superoxide dismutase (e.g., see International Publication WO2009/028338) and mercaptoethanol (e.g., see International Publication WO2009/028338). For example, the transformant expressing the superoxide dismutase is disclosed in International Publication WO2009/028338. Thus, such a transformant may be used in the method of the present invention.
[0072] The reaction to form 4R-IHOG from IPA and the pyruvate and the reaction to form the 2S,4R-Monatin from 4R-IHOG may be progressed separately or in parallel. These reactions may be carried out in one reactor. When these reactions are carried out in one reactor, these reactions can be carried out by adding the substrates and the enzymes sequentially or simultaneously. Specifically, when the reaction to form 4R-IHOG from IPA and the pyruvate by the enzymatic method using the aldolase and the reaction to form the 2S,4R-Monatin from 4R-IHOG by the L-amino acid aminotransferase are carried out, (1) IPA, the pyruvate and the aldolase, and (2) the L-amino acid and the L-amino acid aminotransferase may be added in one reactor sequentially or simultaneously. A salt form of pyruvate (e.g., sodium salt) may be added to the reaction solution. Pyruvate may be added to the reaction solution in any manner (e.g., batch method, or feed method). The concentration of pyruvate in the reaction solution may be, for example, 0.1 mM to 10 M, preferably 1 mM to 1 M.
[0073] In a preferred embodiment, the production method of the present invention is combined with the above reaction 1'' as follows. In this case, the pyruvate irreversibly formed from the oxaloacetate is utilized for the preparation of 4R-IHOG. In other words, at least a part of the pyruvate used for the formation of 4R-IHOG can be from the pyruvate formed from the oxaloacetate by action of the oxaloacetate decarboxylase. In this case, it should be noted that an initial amount of the pyruvate in the reaction system is not necessarily important if an amount of the L-amino acid present in the reaction system is sufficient because the pyruvate is formed from the oxaloacetate in conjunction with the formation of the 2S,4R-Monatin. Therefore, the larger amount of the L-amino acid may be added to the reaction system compared with the pyruvate.
##STR00005##
[0074] When the aldolase is used in the production of 4R-IHOG from IPA and the pyruvate, the contact of IPA and the pyruvate with the aldolase can be accomplished by allowing IPA, the pyruvate and the aldolase extracted from an aldolase-producing microorganism (extracted enzyme) or the aldolase-producing microorganism to coexist in the reaction solution (e.g., culture medium). Examples of the aldolase-producing microorganism include microorganisms that naturally produce the aldolase and transformants that express the aldolase. Examples of the extracted enzyme include a purified enzyme, a crude enzyme, an immobilized enzyme, a culture broth, and a treated product of the culture broth (e.g., an aldolase-containing fraction prepared from the above aldolase-producing microorganism, a disrupted product of and a lysate of the above aldolase-producing microorganism). Examples of the treatment for obtaining the treated product of the culture broth from the culture broth include a heat treatment (42° C. to 80° C., pH 3 to 12, 1 minute to 24 hours), a solvent treatment (e.g, xylene, toluene), a surfactant treatment. The culture broth may be used under a condition of 4° C. to 60° C., pH 3 to 12, and 5 minutes to 20 days (with or without aeration and agitation). The aldolase-producing microorganism may further express other enzyme(s) (e.g., superoxide dismutase, L-amino acid aminotransferase, decarboxylase). Alternatively, a microorganism that produces the other enzyme in addition to the aldolase-producing microorganism may be allowed to coexist in the reaction solution. Those described in the production method (1-1) of the present invention can be used as the reaction solution.
[0075] Preferably, the aldolase-, L-amino acid aminotransferase- and decarboxylase-producing microorganism may be a transformant. The expression of the aldolase, the L-amino acid aminotransferase and the decarboxylase may be carried out using the same transformant, or it may be carried out with a combination of two transformants, or the three enzymes may be expressed in separate transformants. the aldolase, L-amino acid aminotransferase and decarboxylase genes are expressed in the same transformant, these genes may be integrated into its chromosome, or the aldolase, L-amino acid aminotransferase and decarboxylase genes are inserted to one vector. Alternatively, an expression vector of the L-amino acid aminotransferase may be introduced to a microorganism which produces the decarboxylase and aldolase, or a first expression vector of the L-amino acid aminotransferase and a second expression vector of the decarboxylase and the aldolase may be introduced to a host microorganism. Examples of the expression vector of the aldolase, the L-amino acid aminotransferase and the decarboxylase include i') an expression vector containing a first expression unit composed of a first polynucleotide encoding the L-amino acid aminotransferase and a first promoter operatively linked to the first polynucleotide, a second expression unit composed of a second polynucleotide encoding the decarboxylase and a second promoter operatively linked to the second polynucleotide, and a third expression unit composed of a third polynucleotide encoding the decarboxylase and a third promoter operatively linked to the third polynucleotide; and ii') an expression vector containing a first expression unit composed of a first polynucleotide encoding the L-amino acid aminotransferase, a second polynucleotide encoding the decarboxylase and a promoter operatively linked to the first polynucleotide and the second polynucleotide, and a second expression unit composed of a third polynucleotide encoding the aldolase and a promoter operatively linked to the third polynucleotide (a vector capable of expressing a polycistronic mRNA). The positions of genes encoding the L-amino acid aminotransferase, the decarboxylase and the aldolase on a plasmid are not particularly limited.
[0076] Various conditions such as the temperature, the pH value and the time period in the reaction can be appropriately established as long as the objective reaction can progress. For example, the conditions of the enzymatic method using the aldolase may be the same as those described in the production method (1-1) of the present invention.
(1-3) Method for Producing 2S,4R-Monatin or a Salt Thereof from Tryptophan or a Salt Thereof
[0077] The production method of the present invention may further comprise deaminating a tryptophan (Trp) in order to prepare IPA. Trp includes L-Trp, D-Trp and a mixture of L-Trp and D-Trp. The deamination of Trp can be performed by the organic chemistry technique and the enzymatic method using a deamination enzyme.
[0078] Various methods are known as the method for deaminating Trp to form IPA by the organic chemistry technique. Examples of such a method include the method in which the tryptophan is used as a starting material and reacted with pyridine aldehyde in the presence of a base for dehydration of a proton acceptor (e.g., see JP Sho-62-501912 and International Publication WO1987/000169), and the method of subjecting to acid hydrolysis after a condensation reaction using indole and ethyl-3-bromopyruvate ester oxime as raw materials (e.g., European Patent Application Publication No. 421946).
[0079] As used herein, the term "deamination enzyme" refers to the enzyme capable of forming IPA from Trp. The formation of IPA from Trp is essentially conversion of the amino group (--NH2) in Trp to an oxy group (═O). Therefore, the enzymes that catalyze this reaction are sometimes termed as other names such as an amino acid deaminase, an aminotransferase and an amino acid oxidase. Therefore, the term "deamination enzyme" means any enzyme that can form IPA from Trp, and the enzymes having the other name (e.g., amino acid deaminase, aminotransferase, amino acid oxidase) which catalyze the reaction to form IPA from Trp are also included in the "deamination enzyme."
[0080] Examples of the method for forming IPA from Trp using the amino acid deaminase or an amino acid deaminase-producing microorganism include the method disclosed in International Publication WO2009/028338. A general formula of the reaction catalyzed by the amino acid deaminase includes the following formula: Amino acid+H2O→2-oxo acid+NH3.
[0081] Examples of the method for forming IPA from Trp using the aminotransferase or an aminotransferase-producing microorganism include the methods disclosed in East Germany Patent DD 297190, JP Sho-59-95894-A, International Publication WO2003/091396 and US Patent Application Publication No. 2005/028226.
[0082] Examples of the method for forming IPA from Trp using the L-amino acid oxidase or an L-amino acid oxidase-producing microorganism include the methods disclosed in U.S. Pat. No. 5,002,963, John A. Duerre et al. (Journal of Bacteriology 1975, vol. 121, No. 2, p 656-663), JP Sho-57-146573, International Publication WO2003/056026 and International Publication WO2009/028338. The general formula of the reaction catalyzed by the amino acid oxidase includes the following formula: Amino acid+O2+H2O→2-Oxo acid+H2O2+NH3. For the purpose of suppressing the degradation of the compound by hydrogen peroxide as the by-product produced at that time, a hydrogen peroxide-degrading enzyme such as a catalase may be added in the reaction solution.
[0083] The reaction to form IPA from Trp, the reaction to form 4R-IHOG from IPA and the pyruvate and the reaction to form 2S,4R-Monatin from 4R-IHOG may be progressed separately or in parallel. These reactions may be carried out in one reactor. When these reactions are carried out in one reactor, these reactions can be carried out by adding the substrates and the enzymes sequentially or simultaneously. Specifically, when the reaction to deaminate Trp by the enzymatic method using the deamination enzyme to form IPA, the reaction to form 4R-IHOG from IPA and the pyruvate by the enzymatic method using the aldolase, and the reaction to form 2S,4R-Monatin from 4R-IHOG by the L-amino acid aminotransferase are carried out, (1) Trp and the deamination enzyme, (2) the pyruvate and the aldolase, and (3) the L-amino acid and the L-amino acid aminotransferase may be added in one reactor sequentially or simultaneously.
[0084] When the deamination enzyme is used in the production of IPA from Trp, the contact of Trp with the deamination enzyme can be accomplished by allowing Trp and the deamination enzyme extracted from a deamination enzyme-producing microorganism (extracted enzyme) or the deamination enzyme-producing microorganism to coexist in the reaction solution. Examples of the deamination enzyme-producing microorganism include microorganisms that naturally produce the deamination enzyme and transformants that express the deamination enzyme. For example, the pTB2 strain described in Example 2 of WO 2009/028338 (the modified strain of E. coli introduced with the amino acid deaminase gene derived from the strain of Providencia rettgeri) may be used. An operative promoter (e.g., phoA, phoC, trp, lac, or tac promoter) may be linked to the deaminase gene in the plasmid. When E. coli is used as a host, a plasmid capable of expressing a deaminase may be introduced to a host having a deletion of a certain gene such as aspC gene. Examples of the extracted enzyme include a purified enzyme, a crude enzyme, an immobilized enzyme, a cuture broth, and a treated product of the culture broth (e.g., a deamination enzyme-containing fraction prepared from the above deamination enzyme-producing microorganism, a disrupted product of and a lysate of the above deamination enzyme-producing microorganism). Examples of the treatment for obtaining the treated product of the culture broth from the culture broth include a heat treatment (42° C. to 80° C., pH 3 to 12, 1 minute to 24 hours), a solvent treatment (e.g, xylene, toluene, ethanol, isopropylalcohol), a surfactant (e.g., Tween 20, Triton X-100), and a treatment with a bacteriolytic enzyme (e.g., lysozyme treatment). Alternatively, the culture broth is subjected to a reaction after retaining it with adjusting temperature, pH and the like to enhance an enzymatic activity detected in the broth. In this case, the temperature may be set at 4° C. to 60° C., preferably 20° C. to 37° C. In addition, the pH may be set at 3 to 12, preferably 7 to 9. The time may be set for about 5 minutes to 20 days, preferably about 1 hour to 7 days. During retaining the broth, aeration and agitation may be or may not be carried out. The deamination enzyme-producing microorganism may further express the other enzyme(s) (e.g., aldolase, superoxide dismutase, L-amino acid aminotransferase, decarboxylase). Alternatively, the other enzyme-producing microorganism in addition to the deamination enzyme-producing microorganism may be allowed to coexist in the reaction solution. Those described in the production method (1-1) of the present invention can be used as the reaction solution. Trp is preferably L-trp. A salt form of Trp may be added to the reaction solution. The concentration of Trp in the reaction solution is, for example, 1 mM to 3 M, preferably 20 mM to 1 M, more preferably 20 mM to 300 mM.
[0085] Various conditions such as the temperature, the pH value and the time period in the reaction can be appropriately established as long as the objective reaction can progress. For example, the conditions of the enzymatic method using the deamination enzyme may be the same as those described in the production method (1-1) of the present invention.
[0086] In a preferred embodiment, when the production method (1-3) of the present invention is carried out in one reactor, the deaminase, the aldolase, the L-amino acid aminotransferase, and the oxaloacetate decarboxylase, and/or one or more transformants expressing them are used. The superoxide dimustase, and/or a transformant expressing it may be further used. These enzymes may be mutants. For an expression system of the enzymes, the aforementioned transformants can be used. Specifically, a transformant carrying the expression vector of a gene of interest in its cytoplasm, a transformant introduced with a gene of interest on its genome, and a transformant which carries the expression vector of a gene of interest in its cytoplasm, and which is introduced with a gene of interest on its genome. For an expression vector used in the preparation of the transformant, the aforementioned expression vector can be used.
[0087] In a preferred embodiment, when the production method (1-3) of the present invention is carried out in one reactor, a reaction solution containing a certain concentrations of L-Trp, L-Asp, PA, a buffer (e.g., phosphate buffer, Tris buffer) and PLP can be used. The concentration of L-Trp is, for example, 1 mM to 3 M, preferably 10 mM to 1 M, more preferably 50 mM to 300 mM. The concentration of L-Asp is, for example, 1 mM to 3 M, preferably 100 mM to 1 M, more preferably 200 mM to 400 mM. L-Asp may be a salt form (e.g., sodium salt, potassium salt) or a free form. When L-Asp is used in a free form, pH may be appropriately adjusted after supplying it in the reaction solution. In this case, an alkaline solution (e.g., NaOH aqueous solution, KOH aqueous solution) may be used for the adjustment of pH. The concentration of PA is, for example, 1 mM to 3 M, preferably 10 mM to 100 mM. PA may be a salt form (e.g., sodium salt, potassium salt) or a free form. When PA is used in a free form, pH may be adjusted after supplying it in the reaction solution. The concentration of PLP is, for example, 1 μM to 100 mM, preferably 10 μM to 1 mM. The reaction solution may further contain magnesium, phosphate, and antifoaming agent. When magnesium is used as a salt, the salt form of magnesium is not particularly limited, and examples of the salt form include magnesium chloride and magnesium sulfate. The concentration of magnesium is, for example, 0.1 mM to 100 mM, preferably 0.5 mM to 5 mM. In addition, the phosphate is used as a salt, the salt form of the phosphate is not particularly limited, and examples of the salt form include a potassium salt (e.g., monopotassium salt, dipotassium salt, tripotassium salt) and a sodium salt (e.g., monosodium salt, disodium salt, trisodium salt). The concentration of the phosphate is, for example, 1 mM to 100 mM, preferably 10 mM to 50 mM. The antifoaming agent is not particularly limited, and examples of the antifoaming agent include GD113K. The concentration of the antifoaming agent is not particularly limited, and is 0.0001% to 1% (v/v), preferably 0.001% to 0.1% (v/v). The reaction condition such as pH, temperature, aeration, agitation and time can be appropriately set. The pH of the reaction solution is, for example, 5 to 10, preferably 6 to 9, more preferably 7 to 8. The control of pH during the reaction may be achieved by adding an acid or alkaline appropriately. The acid or alkaline used in this case is not particularly limited, and examples of the acid or alkaline include hydrochloric acid, phosphoric acid, sulfuric acid, ammonium gas, ammonium aqueous solution, NaOH aqueous solution, and KOH aqueous solution. The concentration of the acid or alkaline used in the adjustment of pH is not particularly limited. It is, for example, 0.1 N to 20 N, preferably 3 N to 12 N, when a solution of the acid or alkaline is used. The reaction temperature is, for example, 10° C. to 50° C., preferably 20° C. to 40° C., more preferably 25° C. to 35° C. When a container capable of controlling aeration and agitation (e.g., jar fermenter) is used for the reaction, the concentration of dissolved oxygen in the reaction solution can be set by controlling the conditions on aeration and agitation. A person skilled in the art can set the conditions on aeration and agitation according to the used container. For example, when a jar fermenter with a volume of 1 litter is used, the condition on aeration is, for example, 1/200 to 1 vvm, preferably 1/100 to 1/10 vvm. The condition on agitation is, for example, 100 rpm to 1000 rpm, preferably 400 rpm to 700 rpm. Examples of the enzyme to be added to the reaction include a purified enzyme, a microorganism expressing an enzyme, a treated product of a microorganism expressing an enzyme, a cuture broth containing a microorganism expressing an enzyme, and a treated product of a culture broth containing a microorganism expressing an enzyme. Examples of the treatment for obtaining the treated product of the culture broth from the culture broth include a heat treatment (42° C. to 80° C., pH 3 to 12, 1 minute to 24 hours), a solvent treatment (e.g, xylene, toluene, ethanol, isopropylalcohol), a surfactant (e.g., Tween 20, Triton X-100), and a treatment with a bacteriolytic enzyme (e.g., lysozyme treatment). Alternatively, the culture broth is subjected to a reaction after retaining it with adjusting temperature, pH and the like to enhance an enzymatic activity detected in the broth. In this case, the temperature of the culture broth may be 4° C. to 60° C., preferably 20° C. to 37° C. The pH of the culture broth may be 3 to 12, preferably 7 to 9. The retaining time may be about 5 minutes to 20 days, preferably about 1 hour to 7 days. During retaining the broth, aeration and agitation may be or may not be carried out.
[0088] Each enzyme to be added to the reaction solutions can be appropriately determined by measuring an activity of each enzyme previously. The deaminease activity, the aldolase activity, the L-amino acid aminotransferase activity, and the oxaloacetate decarboxylase activity can be measured by the following methods.
[0089] Deaminase activity: 10 mM L-Phe, 100 mM NH4Cl, 100 mM Tris-HCl (pH 8.0), 0.25 mM NADH and phenylalanine dehydrogenase (manufactured by UNITIKA, derived from Thermoactinomyces intermedius) at 25° C. The activity is calculated from the reduction of the absorbance measured at 340 nm.
[0090] L-amino acid aminotransferase activity (L-Asp/α-KG activity): 100 mM L-Asp-Na-laq, 10 mM α-KG-2Na, 50 μM PLP, 100 mM Tris-HCl (pH 8.0), 0.25 mM NADH and 2 U/mL of MDH at 25° C. The activity is calculated from the reduction of the absorbance at 340 nm. Malic dehydrogenase from porcine heart (Sigma) was used as MDH.
[0091] Aldolase activity: 2 mM 4-phenyl-4-hydroxy-2-oxo glutarate (PHOG), 100 mM Tris-HCl (pH 7.0), 1 mM MgCl2, 0.25 mM NADH, 10 U/ml lactate dehydrogenase (manufactured by ORIENTAL YEAST Co., Ltd., derived from Leuconostoc mesenteroides) at 25° C. The activity is calculated from the reduction of the absorbance at 340 nm.
[0092] Oxaloacetate decarboxylase activity: 1 mM oxaloacetate, 100 mM Tris-HCl (pH 8.0), 0.25 mM NADH, 10 U/ml lactate dehydrogenase (manufactured by ORIENTAL YEAST Co., Ltd., derived from Leuconostoc mesenteroides) at 25° C. The activity is calculated from the reduction of the absorbance at 340 nm.
[0093] Based on the enzymatic activities determined as mentioned above, the amounts of enzymes to be added to the reaction solution may be as follows. The amount of the deaminase to be added to the reaction solution is, for example, 0.1 to 20 U/ml, preferably 0.5 to 2 U/ml. The amount of the aldolase to be added to the reaction solution is, for example, 1 to 1000 U/ml, preferably 10 to 100 U/ml. The amount of the L-amino acid aminotransferase to be added to the reaction solution is, for example, 1 to 1000 U/ml, preferably 10 to 100 U/ml. The amount of the oxaloacetate decarboxylase to be added to the reaction solution is, for example, 0.01 U/ml or more, preferably 0.1 U/ml or more. Each substrate may be added to a reaction system by a batch method or a feed method. The enzyme, the microorganism expressing the enzyme, the treated product of the microorganism expressing the enzyme, the cuture broth containing the microorganism expressing the enzyme, and the treated product of the culture broth containing the microorganism expressing the enzyme may also be added to the reaction system by a batch method or a feed method. The reaction time is, for example, 2 to 100 hours, preferably 4 to 50 hours, more preferably 8 to 25 hours. The reaction solution may be sterilized under an appropriate condition (e.g., temperature, pH, time).
[0094] When the production method (1-2) of the present invention is carried out in one reactor, such a production method can be carried out similar to the production method (1-3) of the present invention.
[0095] The purified 2S,4R-Monatin can be obtained by taking advantage of known purification methods such as column treatment, crystallization treatment and extraction treatment for a 2S,4R-Monatin-containing reaction solution obtained by any of the production methods (1-1), (1-2) and (1-3) of the present invention. The purified 2S,4R-Monatin can be provided to a method (2) for producing 2R,4R-Monatin or a salt thereof. The 2S,4R-Monatin-containing reaction solution obtained by any of the production methods (1-1), (1-2) and (1-3) of the present invention can also be directly provided to the method (2) for producing the 2R,4R-Monatin or the salt thereof.
(2) Method for Producing 2R,4R-Monatin or a Salt Thereof
[0096] The present invention provides a method (2) for producing 2R,4R-Monatin or the salt thereof. The production method of the present invention comprises performing the production method (1) of the present invention to form the 2S,4R-Monatin or a salt thereof, and isomerizing the 2S,4R-Monatin or the salt thereof to form 2R,4R-Monatin or a salt thereof.
[0097] The isomerization of the 2S,4R-monatin to the 2R,4R-Monatin can be performed by any method that enables the isomerization (e.g., see International Publication WO2005/082850 and International Publication WO03/059865). However, in terms of enhancing a yield of the 2R,4R-Monatin, the isomerization of the 2S,4R-Monatin is preferably performed by epimerization-crystallization (e.g., see International Publication WO2005/082580). The epimerization-crystallization is a method in which the isomerization reaction and the crystallization are performed simultaneously. In this case, the isomerization reaction at position 2 to convert the 2S,4R-Monatin into the 2R,4R-Monatin and the crystallization of the converted 2R,4R-Monatin are performed simultaneously by the epimerization-crystallization.
[0098] In the epimerization-crystallization, the isomerization reaction may be performed in the presence of an aldehyde. The aldehyde includes an aliphatic aldehyde and an aromatic aldehyde, and the aromatic aldehyde is preferred. A purified 2S,4R-Monatin or a 2S,4R-Monatin-containing reaction solution may be used as the 2S,4R-Monatin used for the isomerization reaction.
[0099] For the aliphatic aldehyde, for example, a saturated or unsaturated aldehyde having 1 to 7 carbon atoms, such as formaldehyde, acetaldehyde, propionaldehyde, n-butyl aldehyde, 1-butyl aldehyde, n-valeraldehyde, capronaldehyde, n-heptylaldehyde, acrolein or methacrolein can be used.
[0100] For the aromatic aldehyde, the aromatic aldehyde such as benzaldehyde, salicylaldehyde, m-hydroxybenzaldehyde, p-hydroxybenzaldehyde, o-nitrobenzaldehyde, p-nitrobenzaldehyde, 5-nitrosalicylaldehyde, 3,5-dichlorosalicylaldehyde, anisaldehyde, o-vanillin, vanillin, furfural, pyridoxal or 5-phosphate pyridoxal can be used. Particularly, pyridoxal, 5-nitrosalicylaldehyde, or 3,5-dichlorosalicylaldehyde is preferred as the aromatic aldehyde.
[0101] The aldehyde can be used in the range of 0.01 to 1 mol equivalent and more preferably 0.05 to 0.5 mol equivalent to the Monatin present in the system.
[0102] The epimerization-crystallization is performed in the presence of the aldehyde, and a mixed solvent of water and an organic solvent is used as a solvent. The organic solvent miscible with the water is used as the organic solvent, and particularly, alcohol such as methanol, ethanol, propanol or isopropanol is preferred. Two or more different kinds of organic solvents may be used in mixture. A volume ratio of the organic solvent to the water is set in the range of preferably 1:0.01 to 1:1 and more preferably 1:0.1 to 1:0.5 (organic solvent:water).
[0103] The temperature in the epimerization-crystallization is set in the range of preferably 0 to 100° C. and more preferably 40 to 80° C. The time period for performing the epimerization-crystallization is set in the range of preferably 10 hours to one week and more preferably 15 hours to 96 hours.
[0104] The pH value is set in the range of 4 to 13, preferably 4.5 to 10 and more preferably 5 to 9. The pH value can be adjusted using an acid or an alkali. The acid to be used is not particularly limited, and an organic acid such as acetic acid, or an inorganic acid such as hydrochloric acid or sulfuric acid can be used. The alkali is not also particularly limited, and an alkali metal hydroxide such as sodium hydroxide or potassium hydroxide, or an organic base such as ammonia or amine can be used.
[0105] Each compound obtained by the above method can be isolated and purified by optionally combining known separation and purification procedures such as concentration, reduced pressure concentration, solvent extraction, crystallization, recrystallization, solvent transfer, a treatment with activated charcoal, and treatments with chromatography and the like using ion exchange resin or synthetic adsorption resin. The salts of the compound used in the method of the present invention and the compound (objective compound) produced by the method of the present invention can be produced, for example, by adding the inorganic acid or the organic acid to the objective compound according to the method publicly known per se. The objective compound and the salt thereof may be hydrate, and both hydrate and non-hydrate are included in the scope of the present invention. The compounds (e.g., Trp, IPA, 4R-IHOG, 2S,4R-Monatin) used for the production methods of the present invention may be the forms of various salts such as sodium salts, potassium salts and ammonium salts. The compounds (e.g., IPA, 4R-IHOG, 2S,4R-Monatin, 2R,4R-Monatin) obtained by the production method of the present invention may also be the forms of various salts.
[0106] The present invention will be described in detail by the following Examples, but the present invention is not limited by these Examples.
EXAMPLES
Analytical Condition of HPLC
[0107] In Examples 1 to 7, if HPLC analysis was performed, the HPLC analysis was performed under the condition shown in the Example.
[0108] In Examples 8 to 15, the HPLC analysis was performed under the condition shown below.
[0109] Detector: Ultraviolet absorption spectrometer (measured wavelength: 210 nm)
[0110] Column temperature: 40° C.
[0111] Column: CAPCELLPAK C18 Type MGII, inner diameter: 3 mm, length: 25 cm, and particle diameter: 5 μm, Shiseido Co., Ltd.
[0112] Mobile phase: Solution A (aqueous solution of 20 mM potassium dihydrogen phosphate:acetonitrile=95:5) and solution B (aqueous solution of 20 mM potassium dihydrogen phosphate:acetonitrile=60:40)
[0113] Gradient program: See the following Table 1
TABLE-US-00001 TABLE 1 Gradient program Time (min) Mobile phase A (%) Mobile phase B (%) 0.0 100 0 15.0 100 0 40.0 0 100 45.0 0 100 45.1 100 0
[0114] Flow: 0.45 mL/minute
[0115] Injection amount: 20 IAL
[0116] Analysis time period: 60 minutes
Example 1
Formation of 2S,4R-Monatin from 4R-IHOG Using Extraction Solution from Bacillus altitudinis AJ1616 Microbial Cells
[0117] Bacillus altitudinis AJ1616 was streaked on CM2G agar medium (10 g/L of yeast extract, 10 g/L of polypeptone, 5 g/L of glucose, 5 g/L of sodium chloride, 15 g/L of agar, pH 7.0), and cultured at 30° C. for 2 days.
[0118] One loopful of the resulting microbial cells was inoculated to 3 mL of an enzyme production medium (10 g/L of yeast extract, 10 g/L of polypeptone, 1 g/L of glucose, 3 g/L of dipotassium hydrogen phosphate, 1 g/L of potassium dihydrogen phosphate, 0.1 g/L of magnesium sulfate heptahydrate, 5 g/L of ammonium sulfate) in a test tube, which was then cultured with shaking at 30° C. for 16 hours. The microbial cells were collected from 2 mL of the cultured medium by centrifugation, washed with and suspended in 20 mM Tris-HCl (pH 7.6) to prepare 1 mL of a microbial cell suspension.
[0119] 1 g of glass beads (0.1 mm) was added to 1 mL of this microbial cell suspension, and the microbial cells were disrupted using a multi beads shocker (Yasui Kikai Co., Ltd.). The resulting disrupted cell solution was centrifuged to use a supernatant as a microbial cell extract.
[0120] A 2S,4R-Monatin synthesis reaction solution (0.1 mL) (9.5 mM 4R-IHOG, 0.5 mM 4S-IHOG, 100 mM L-Asp, 50 μM PLP, 100 mM Tris-HCl, pH 8.0) was prepared so that 0.05 mL of the Bacillus altitudinis AJ1616 microbial cell extract was contained. The reaction solution was reacted at 30° C. for 20 hours. After termination of the reaction, the formed 2S,4R-Monatin was quantified, and its concentration was 0.21 mM.
[0121] The 2S,4R-Monatin was quantified using UPLC (Waters). The analytical condition is as follows.
[0122] Mobile phase: 20 mM KH2PO4/asetonitrile=100/5
[0123] Flow rate: 0.15 mL/minute
[0124] Column temperature: 40° C.
[0125] Detection: UV 210 nm
[0126] Column: ACQUITY UPLC BEH C18, 2.1×50 mm, 1.7 μm (Waters).
Example 2
Purification of Aminotransferase Derived from Bacillus altitudinis AJ1616
[0127] An aminotransferase for forming the 2S,4R-Monatin was purified from a soluble fraction of Bacillus altitudinis AJ1616 as follows. The reaction for synthesizing 2S,4R-Monatin and the quantification of 2S,4R-Monatin were performed in the same manner as in Example 1.
(1) Preparation of Soluble Fraction
[0128] Bacillus altitudinis AJ1616 was streaked on CM2G agar medium (10 g/L of yeast extract, 10 g/L of polypeptone, 5 g/L of glucose, 5 g/L of sodium chloride, 15 g/L of agar, pH 7.0), and cultured at 30° C. for 2 days.
[0129] One loopful of the resulting microbial cells was inoculated to 160 mL of TB (Terrific Broth) medium in a 500 mL Sakaguchi flask, which was then cultured with shaking at 30° C. for 16 hours. The microbial cells were collected from about 2000 mL of the cultured medium by centrifugation, washed with and suspended in 20 mM Tris-HCl (pH 7.6), 100 mM NaCl, and then disrupted by sonication at 4° C. for 30 minutes. Microbial cell debris was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as a soluble fraction.
(2) Anion Exchange Chromatography
[0130] The above soluble fraction was applied onto an anion exchange chromatography column HiLoad 26/10 Q Sepharose HP (supplied from GE Health Care Bioscience, CV=53 mL) equilibrated with 20 mM Tris-HCl (pH 7.6), 100 mM NaCl, and adsorbed to the carrier. Proteins that had not been adsorbed to the carrier (unadsorbed proteins) were washed out with 20 mM Tris-HCl (pH 7.6), 100 mM NaCl, and subsequently the adsorbed proteins were eluted by linearly changing the concentration of NaCl from 100 mM to 500 mM at a flow rate of 8 mL/minute. A 2S,4R-Monatin forming activity was measured in each fraction, and detected in the fractions corresponding to about 200 mM NaCl.
(3) Hydrophobic Chromatography
[0131] The fractions in which the 2S,4R-Monatin forming activity had been detected were combined, and ammonium sulfate and Tris-HCl (pH 7.6) were added thereto at final concentrations of 1.4 M and 20 mM, respectively. This solution was applied to a hydrophobic chromatography column HiLoad 16/10 Phenyl Sepharose HP (supplied from GE Health Care Bioscience, CV=20 mL) equilibrated with 1.4 M ammonium sulfate, 20 mM Tris-HCl (pH 7.6), and adsorbed to the carrier. Unadsorbed proteins that had not been adsorbed to the carrier were washed out with 1.4 M ammonium sulfate, 20 mM Tris-HCl (pH 7.6), and subsequently, a 2S,4R-Monatin forming enzyme was eluted by linearly changing the concentration of ammonium sulfate from 1.4 M to 0 M at a flow rate of 3 mL/minute. The 2S,4R-Monatin forming activity was measured in each fraction, and detected in the fractions corresponding to about 1.0 M ammonium sulfate.
(4) Gel Filtration Chromatography
[0132] The fractions in which the 2S,4R-Monatin forming activity had been detected were combined and concentrated using Amicon Ultra-15 30K (Millipore). The resulting concentrated solution was diluted with 20 mM Tris-HCl (pH 7.6), 150 mM NaCl. This solution was applied to a gel filtration column HiLoad 16/60 Superdex 200 pg (supplied from GE Health Care Bioscience, CV=120 mL) equilibrated with 20 mM Tris-HCl (pH 7.6), 150 mM NaCl, and eluted at a flow rate of 1 mL/minute. This manipulation confirmed the 2S,4R-Monatin forming activity in a location estimated as a molecular weight of about 120 kDa.
(5) Anion Exchange Chromatography
[0133] The fractions in which the 2S,4R-Monatin forming activity had been detected were combined and applied to an anion exchange chromatography column Mono Q 5/5 (supplied from Pharmacia (GE Health Care Bioscience), CV=1 mL) equilibrated with 20 mM Tris-HCl, 100 mM NaCl (pH 7.6), and adsorbed to the carrier. Proteins that had not been adsorbed to the carrier (unadsorbed proteins) were washed out with 20 mM Tris-HCl (pH 7.6), 100 mM NaCl, and subsequently the adsorbed proteins were eluted by linearly changing the concentration of NaCl from 100 mM to 500 mM at a flow rate of 0.5 mL/minute. The 2S,4R-Monatin forming activity was measured in each fraction, and detected in the fractions corresponding to about 200 mM NaCl.
(6) SDS-PAGE
[0134] The obtained fractions were subjected to SDS-PAGE, and a band around 45 kDa was observed in the active fraction. This band was subjected to analysis of an N-terminal amino acid sequence as a candidate for the aminotransferase for forming the 2S,4R-Monatin. The band was also subjected to the analysis of an internal amino acid sequence.
Example 3
Determination of N-Terminal and Internal Amino Acid Sequences of Aminotransferase Derived from Bacillus altitudinis AJ1616
[0135] The purified enzyme solution obtained in Example 2 was subjected to the analysis of the N-terminal amino acid sequence, and the sequence SGFTALSEAELNDLY (SEQ ID NO:4) was obtained as the N-terminal amino acid sequence. The sample in SDS-PAGE gel was treated with trypsin (pH 8.0, 35° C., 20 hours), and subsequently subjected to reverse phase HPLC to separate peptide fragments. The amino acid sequences in the fractionated fractions were analyzed, and the sequence QLDLSMGMLDVV (SEQ ID NO:5) was obtained as the internal amino acid sequence. Both the N-terminal amino acid sequence and the internal amino acid sequence exhibited high homology to the aminotransferase derived from Bacillus pumilus SAFR-032 (YP001487343).
Example 4
Cloning of Aminotransferase Gene Derived from Bacillus altitudinis AJ1616
[0136] Bacillus altitudinis AJ1616 was cultured in the same manner as in Example 1. The microbial cells were collected from the cultured medium by centrifugation, and genomic DNA was extracted.
[0137] A DNA fragment including an aminotransferase gene was amplified by PCR using the obtained genomic DNA as a template. For primers, the primer Bp-u300-f (5'-ctcaggaagcaggcgcaaaaagattaattt-3' (SEQ ID NO:6) and the primer Bp-d200-r (5'-ggatgctgtctttgtcatcccaaagtggat-3' (SEQ ID NO:7) were used, which were designed from DNA sequences of upstream 300 bp and downstream 200 bp in the aminotransferase gene with reference to the genomic DNA sequence of Bacillus pumilus SAFR-032 (CP000813). PCR was performed using KOD-plus-ver. 2 (Toyobo) under the following condition.
TABLE-US-00002 1 cycle 94° C., 2 min 25 cycles 98° C., 10 sec 55° C., 10 sec 68° C., 60 sec 1 cycle 68° C., 60 sec 4° C.
[0138] A nucleotide sequence of about 1800 bp of the amplified DNA fragment was determined, and the nucleotide sequence was shown to include 1308 bp of ORF that had the high homology to the aminotransferase gene derived from Bacillus pumilus SAFR-032 (NC 009848). The homology was 89% in the DNA sequences and 93% in the amino acid sequences.
[0139] The N-terminal amino acid sequence and the internal amino acid sequence obtained in Example 3 were found in this sequence. Thus, it was thought that the aminotransferase gene having the 2S,4R-Monatin forming activity could have been acquired.
Example 5
Expression of Aminotransferase Derived from Bacillus altitudinis AJ1616 in E. coli
[0140] (1) Construction of Plasmid Expressing Aminotransferase Derived from Bacillus altitudinis AJ1616
[0141] A DNA fragment including the aminotransferase gene derived from Bacillus altitudinis AJ1616 was amplified by PCR using the genomic DNA of Bacillus altitudinis AJ1616 as the template. The primer 1616AT-Nde-f (5'-ggaattccatATGAGCGGTTTTACAGCGTT-3': SEQ ID NO:8) and the primer 1616-xho-r (5'-gtcaaggagtttttctcgagTACCGTTGGTGCTGATTGAC-3': SEQ ID NO:9) were used as the primers. A NdeI sequence in the aminotransferase gene was converted using the primer 1616-delNde-f (5'-GGATTGAAGGAACAcATGAAAAAGCATGC-3': SEQ ID NO:10) and the primer 1616-delNde-r (5'-GCATGCTTTTTCATgTGTTCCTTCAATCC-3': SEQ ID NO:11). PCR was performed using KOD-plus-ver. 2 (Toyobo) under the following condition.
TABLE-US-00003 1 cycle 94° C., 2 min 25 cycles 98° C., 10 sec 55° C., 10 sec 68° C., 60 sec 1 cycle 68° C., 60 sec 4° C.
[0142] The resulting DNA fragment of about 1300 bp was treated with restriction enzymes NdeI and XhoI, and then ligated to pET-22b (Novagen) likewise treated with NdeI and XhoI. E. coli JM109 was transformed with this solution containing the ligated product, the objective plasmid was extracted from ampicillin resistant colonies, and this plasmid was designated as pET-22-1616AT-His. This plasmid expresses the aminotransferase derived from Bacillus altitudinis AJ1616 which has the His-tag to C-terminus end (1616AT-His).
(2) Purification of 1616AT-His from E. coli Expression Strain
[0143] The constructed expression plasmid pET-22-1616AT-His was introduced into E. coli BL21 (DE3). One loopful of the resulting transformant was inoculated to 160 mL of Overnight Express Instant TB Medium (Novagen) containing 100 mg/L of ampicillin in a 500 mL Sakaguchi flask, and cultured with shaking at 37° C. for 16 hours. After the termination of the cultivation, microbial cells were collected from about 1000 mL of the resulting cultured medium by centrifugation, washed with and suspended in 20 mM Tris-HCl (pH 7.6), 100 mM NaCl and 20 mM imidazole, and disrupted by sonication at 4° C. for 30 minutes. Microbial cell debris was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as a soluble fraction.
[0144] The obtained soluble fraction was applied to a His-tag protein purification column HisPrep FF 16/10 (supplied from Pharmacia (GE Health Care Bioscience), CV=20 mL) equilibrated with 20 mM Tris-HCl (pH 7.6), 100 mM NaCl and 20 mM imidazole, and adsorbed to the carrier. Proteins that had not been adsorbed to the carrier (unadsorbed proteins) were washed out with 20 mM Tris-HCl (pH 7.6), 100 mM NaCl and 20 mM imidazole, and subsequently the adsorbed proteins were eluted by linearly changing the concentration of imidazole from 20 mM to 250 mM at a flow rate of 3 mL/minute.
[0145] The obtained fractions were combined and concentrated using Amicon Ultra-15 30K (Millipore). The concentrated solution was diluted with 20 mM Tris-HCl (pH 7.6), 100 mM NaCl, and applied to the anion exchange chromatography column HiLoad 16/10 Q Sepharose HP (supplied from GE health Care Bioscience, CV=20 mL) equilibrated with 20 mM Tris-HCl (pH 7.6), 100 mM NaCl, and adsorbed to the carrier. The proteins that had not been adsorbed to the carrier (unadsorbed proteins) were washed out with 20 mM Tris-HCl (pH 7.6), 100 mM NaCl, and subsequently the adsorbed proteins were eluted by linearly changing the concentration of NaCl from 100 mM to 500 mM at a flow rate of 3 mL/minute.
[0146] The 2S,4R-Monatin forming activity was measured in each eluted fraction, and the fractions in which the 2S,4R-Monatin forming activity had been confirmed were combined and concentrated using Amicon Ultra-15 30K (Millipore). The concentrated solution was diluted with 20 mM Tris-HCl (pH 7.6) to use as a 1616AT-His solution.
Example 6
Synthesis Reaction of 2S,4R-Monatin, Using 1616AT-His
[0147] The 2S,4R-Monatin was quantified by HPLC analysis. The analytical condition was as follows.
[0148] Mobile phase: 20 mM KH2PO4/acetonitrile-100/5
[0149] Flow rate: 1.0 mL/minute
[0150] Column temperature: 40° C.
[0151] Detection: UV 280 nm
[0152] Column: CAPCELL PAK MGII, 4.6×150 mm, 3 μm, (Shiseido Co., Ltd.)
(1) Synthesis of 2S,4R-Monatin from 4R-IHOG
[0153] The 1616AT-His solution prepared so as to contain 0.5 mg of 1616AT-His (Example 5) was added to 0.1 mL of the reaction solution (9.5 mM 4R-IHOG, 0.5 mM 4S-IHOG, 80 mM L-Asp, 50 μM PLP, 100 mM Tris-HCl, pH 8.0), and then reacted at 25° C. for 12 hours. After the termination of the reaction, the formed 2S,4R-Monatin was quantified, and its concentration was 8.6 mM.
(2) Synthesis of 2S,4R-Monatin from Indole Pyruvate (IPA) and Pyruvate (PA)
[0154] A reaction mixture was prepared so as to contain 0.5 mg of 1616AT-His (the 1616AT-His solution in Example 5 was used), 0.01 mg of SpAld (a solution having an aldolase activity, the preparation method of the solution is explained in detail below, see also JP 2006-204285-A) and 1 U of oxaloacetate decarboxylase (Sigma, 04878) in 0.1 mL of a reaction solution (50 mM IPA, 100 mM PA, 100 mM L-Asp, 1 mM MgCl2, 50 μM PLP, 100 mM Tris-HCl, 100 mM potassium phosphate buffer, pH 8.0), and reacted at 25° C. for 2 hours. After the termination of the reaction, the formed 2S,4R-Monatin was quantified, and its concentration was 5.0 mM.
(3) Synthesis of 2S,4R-Monatin from L-Trp
[0155] A reaction mixture was prepared so as to contain 5 mg of 1616AT-His (the 1616AT-His solution in Example 5 was used), 0.2 mg of SpAld, 0.4 mL of the cultured medium (TB medium) of pTB2 strain (a bacterial strain capable of expressing a deamination enzyme, the preparation method of the bacterial strain is explained in detail below, see also WO2009/028338) in the Sakaguchi flask, 200 U of superoxide dismutase (Sigma, S8160) and 10 U of oxaloacetate decarboxylase (Sigma, 04878) in 1.0 mL of a reaction solution (50 mM L-Trp, 100 mM PA, 400 mM L-Asp, 1 mM MgCl2, 50 μM PLP, 100 mM Tris-HCl, 100 mM potassium phosphate buffer, pH 6.5), and reacted at 25° C. for 12 hours. The reaction was performed using a test tube with shaking at 140 rpm. After the termination of the reaction, the formed 2S,4R-Monatin was quantified, and its concentration was 22 mM (44% of yield).
[0156] SpAld was prepared by the following method.
[0157] A DNA fragment including a SpAld gene was amplified by PCR using plasmid DNA, ptrpSpALD described in Example 5 in JP 2006-204285-A as the template. The primer SpAld-f-NdeI (5'-GGAATTCCATATGACCCAGACGCGCCTCAA-3': SEQ ID NO:12) and the primer SpAld-r-HindIII (5'-GCCCAAGCTTTCAGTACCCCGCCAGTTCGC-3': SEQ ID NO:13) were used. E. coli rare codons (6L-ctc, 13L-ctc, 18P-ccc, 38P-ccc, 50P-ccc, 77P-ccc, 81P-ccc and 84R-cga) in an aldolase gene were converted to 6L-ctg, 13L-ctg, 18P-ccg, 38P-ccg, 50P-ccg, 77P-ccg, 81P-ccg and 84R-cgc, respectively. When 6L was converted, the primer 6L-f (5'-ACCCAGACGCGCCTGAACGGCATCATCCG-3': SEQ ID NO:14) and the primer 6L-r (5'-CGGATGATGCCGTTCAGGCGCGTCTGGGT-3': SEQ ID NO:15) were used. When 13L was converted, the primer 13L-f (5'-ATCATCCGCGCTCTGGAAGCCGGCAAGCC-3': SEQ ID NO:16) and the primer 13L-r (5'-GGCTTGCCGGCTTCCAGAGCGCGGATGAT-3': SEQ ID NO:17) were used. When 18P was converted, the primer 18P-f (5'-GAAGCCGGCAAGCCGGCTTTCACCTGCTT-3': SEQ ID NO:18) and the primer 18P-r (5'-AAGCAGGTGAAAGCCGGCTTGCCGGCTTC-3': SEQ ID NO:19) were used. When 38P was converted, the primer 38P-f (5'-CTGACCGATGCCCCGTATGACGGCGTGGT-3': SEQ ID NO:20) and the primer 38P-r (5'-ACCACGCCGTCATACGGGGCATCGGTCAG-3': SEQ ID NO:21) were used. When 50P was converted, the primer 50P-f (5'-ATGGAGCACAACCCGTACGATGTCGCGGC-3': SEQ ID NO:22) and the primer 50P-r (5'-GCCGCGACATCGTACGGGTTGTGCTCCAT-3': SEQ ID NO:23) were used. When 77P, 81P and 84P were converted, the primer 77P-81P-84R-f (5'-CGGTCGCGCCGTCGGTCACCCCGATCGCGCGCATCCCGGCCA-3': SEQ ID NO:24) and the primer 77P-81P-84R-r (5'-TGGCCGGGATGCGCGCGATCGGGGTGACCGACGGCGCGACCG-3': SEQ ID NO:25) were used. PCR was performed using KOD-plus (Toyobo) under the following condition.
TABLE-US-00004 1 cycle 94° C., 2 min 25 cycles 94° C., 15 sec 55° C., 15 sec 68° C., 60 sec 1 cycle 68° C., 60 sec 4° C.
[0158] The resulting DNA fragment of about 900 bp was treated with the restriction enzymes NdeI and HindIII, and ligated to pSFN Sm_Aet (Examples 1, 6 and 12 in International Publication WO2006/075486) likewise treated with NdeI and HindIII. E. coli JM109 was transformed with this solution containing the ligated product. The objective plasmid was extracted from ampicillin resistant strains, and this plasmid was designated as pSFN-SpAld.
[0159] One loopful of E. coli JM 109/pSFN-SpAld that was the bacterial strain carrying the constructed plasmid pSFN-SpAld was inoculated to 50 mL of LB liquid medium containing 100 mg/L of ampicillin in a 500 mL Sakaguchi flask, and cultured with shaking at 36° C. for 8 hours. After the termination of the culture, 0.0006 mL of the obtained cultured medium was added to 300 mL of a seed liquid medium (10 g of glucose, 5 g of ammonium sulfate, 1.4 g of potassium dihydrogen phosphate, 0.45 g of hydrolyzed soybeans as a nitrogen amount, 1 g of magnesium sulfate heptahydrate, 0.02 g of iron (II) sulfate heptahydrate, 0.02 g of manganese (II) sulfate pentahydrate, 1 mg of thiamin hydrochloride, 0.1 mL of Disfoam GD-113K (NOF Corporation), pH 6.3, made to one liter with water) containing 100 mg/L of ampicillin in a 1000 mL volume of jar fermenter, and seed cultivation was started. The seed cultivation was performed at 33° C. with ventilation at 1/1 vvm with stirring at 700 rpm and controlling pH at 6.3 with ammonia until glucose was consumed. Then, 15 mL of the cultured medium obtained as above was added to 285 mL of a main liquid medium (15 g of glucose, 5 g of ammonium sulfate, 3.5 g of phosphoric acid, 0.45 g of hydrolyzed soybeans as the nitrogen amount, 1 g of magnesium sulfate heptahydrate, 0.05 g of iron (II) sulfate heptahydrate, 0.05 g of manganese (II) sulfate pentahydrate, 1 mg of thiamin hydrochloride, 0.1 mL of Disfoam GD-113K (NOF Corporation), pH 6.3, made to 0.95 L with water) containing 100 mg/L of ampicillin in a 1000 mL volume of jar fermenter, and main cultivation was started. The main cultivation was performed at 36° C. with ventilation at 1/1 vvm, pH was controlled to 6.3 with ammonia, and stirring was controlled at 700 rpm or more so that the concentration of dissolved oxygen was 5% or more. After glucose contained in the main medium was consumed, the cultivation was continued with dropping a glucose solution at 500 g/L for total 50 hours.
[0160] Microbial cells were collected by centrifugation from 100 mL of the obtained cultured medium, washed with and suspended in 20 mM Tris-HCl (pH 7.6), and disrupted by sonication at 4° C. for 30 minutes. Microbial cell debris was removed from the disrupted solution by centrifugation, and the obtained supernatant was used as a soluble fraction.
[0161] The above soluble fraction was applied to the anion exchange chromatography column HiLoad 26/10 Q Sepharose HP (supplied from GE health Care Bioscience, CV=53 mL) equilibrated with 20 mM Tris-HCl (pH 7.6), and adsorbed to the carrier. The proteins that had not been adsorbed to the carrier (unadsorbed proteins) were washed out with 20 mM Tris-HCl (pH 7.6), and subsequently, the adsorbed proteins were eluted by linearly changing the concentration of NaCl from 0 mM to 500 mM at a flow rate of 8 mL/minute. Fractions having an aldolase activity were combined, and ammonium sulfate and Tris-HCl (pH 7.6) were added thereto at final concentrations of 1 M and 20 mM, respectively.
[0162] The resulting solution was applied to the hydrophobic chromatography column HiLoad 16/10 Phenyl Sepharose HP (supplied from GE health Care Bioscience, CV=20 mL) equilibrated with 1 M ammonium sulfate, 20 mM Tris-HCl (pH 7.6), and adsorbed to the carrier. The proteins that had not been adsorbed to the carrier were washed out with 1 M ammonium sulfate, 20 mM Tris-HCl (pH 7.6), and subsequently, the adsorbed proteins were eluted by linearly changing the concentration of ammonium sulfate from 1 M to 0 M at a flow rate of 3 mL/minute. The fractions having the aldolase activity were combined and concentrated using Amicon Ultra-15 10K (Millipore). The obtained concentrated solution was diluted with 20 mM Tris-HCl (pH 7.6), and used as a SpAld solution. The aldolase activity was measured as an aldol degradation activity using PHOG as the substrate under the following condition.
[0163] Reaction condition: 50 mM Phosphate buffer (pH 7.0), 2 mM PHOG, 0.25 mM NADH, 1 mM MgCl2, 16 U/mL lactate dehydrogenase, an absorbance at 340 nm was measured at 25° C.
[0164] pTB2 strain was prepared by the following method.
[0165] One loopful of pTB2 strain described in Example 2 in International Publication WO2009/028338 was inoculated to 50 mL of the TB liquid medium containing 100 mg/L of ampicillin in a 500 mL Sakaguchi flask, and cultured with shaking at 37° C. for 16 hours. The obtained cultured medium was used as the cultured medium of pTB2 strain in the Sakaguchi flask (TB medium).
Example 7
Synthesis of 2S,4R-Monatin by Microorganisms Having 2S,4R-Monatin Forming Activity
(1) Synthesis of 2S,4R-Monatin by Bacteria
[0166] Rhizobium radiobacter LAT1, Rhizobium radiobacter AJ11568, Dietzia maris AJ2788, Stenotrophomonas sp. AJ3447, Stenotrophomonas sp. AJ13127, Pseudomonas chlororaphis subsp. chlororaphis NBRC3904, Micrococcus luteus NBRC3067, Stenotrophomonas sp. AJ11634, Pseudomonas putida NBRC12668, Ochrobactrum pseudogrignonense AJ3735, Stenotrophomonas sp. AJ1591, Stenotrophomonas sp. AJ3839, Brevundimonas diminuta AJ3958, Pseudomonas citronocllolis ATCC13674, Arthrobacter sp. AJ1436, Rhizobium sp. AJ12469, Rhizobium radiobacter AJ2777, Burkholderia sp. AJ3084, Microbacterium sp. AJ2787, Pseudomonas taetrolens ATCC4683, Rhizobium radiobacter ATCC4452, Rhizobium radiobacter AJ2557, Carnimonas sp. AJ3230, Rhizobium radiobacter NBRC12667, Pseudomonas fragi NBRC3458, Rhizobium radiobacter NBRC12664, Corynebacterium ammoniagenes NBRC12072, Pseudomonas sp. AJ1594, Rhizobium radiobacter ATCC6466, Pseudomonas synxantha NBRC3912, Rhizobium radiobacter ATCC4720, or Pseudomonas sp. AJ2438 was applied onto a nutrient broth (NB) agar medium or the CM2G agar medium (10 g/L of yeast extract, 10 g/L of polypeptone, 5 g/L of glucose, 5 g/L of NaCl, 15 g/L of agar, pH 7.0), and cultured at 30° C. for 2 days.
[0167] One loopful of the obtained microbial cells was inoculated to 3 mL of an enzyme production medium (10 g/L of yeast extract, 10 g/L of polypeptone, 1 g/L of glucose, 3 g/L of dipotassium hydrogen phosphate, 1 g/L of potassium dihydrogen phosphate, 0.1 g/L of magnesium sulfate heptahydrate, 5 g/L of ammonium sulfate) in a test tube, which was then cultured with shaking at 30° C. for 16 hours. The microbial cells were collected from 2 mL of the cultured medium by centrifugation, washed with and suspended in 20 mM Tris-HCl (pH 7.6) to prepare 1 mL of a microbial cell suspension.
[0168] Then, 1 g of glass beads (0.1 mm) was added to 1 mL of this microbial cell suspension, and the microbial cells were disrupted using the multi beads shocker (Yasui Kikai Co., Ltd.). The resulting disrupted cell solution was centrifuged to use a supernatant as a microbial cell extract.
[0169] The reaction of synthesizing 2S,4R-Monatin and the quantification of 2S,4R-Monatin were performed in the same manner as in Example 1, and amounts of the 2S,4R-Monatin which was formed were as follows (Table 2)
TABLE-US-00005 TABLE 2 Amount of 2S,4R-Monatin which was produced Amount of 2S,4R-Monatin Microorganism which was formed Rhizobium radiobacter LAT1 3.8 mM Rhizobium radiobacter AJ11568 3.5 mM Dietzia maris AJ2788 3.2 mM Stenotrophomonas sp. AJ3447 2.7 mM Stenotrophomonas sp. AJ13127 2.7 mM Pseudomonas chlororaphis subsp 2.6 mM chlororaphis NBRC3904 Micrococcus luteus NBRC3067 2.3 mM Stenotrophomonas sp. AJ11634 2.2 mM Pseudomonas putida NBRC12668 2.2 mM Ochrobactrum pseudogrignonense AJ3735 2.2 mM Stenotrophomonas sp. AJ1591 2.1 mM Stenotrophomonas sp. AJ3839 2.1 mM Brevundimonas diminuta AJ3958 2.0 mM Pseudomonas citronocllolis ATCC13674 1.9 mM Arthrobacter sp. AJ1436 1.7 mM Rhizobium sp. AJ12469 1.6 mM Rhizobium radiobacter AJ2777 1.5 mM Burkholderia sp. AJ3084 1.5 mM Microbacterium sp. AJ2787 1.5 mM Pseudomonas taetrolens ATCC4683 1.4 mM Rhizobium radiobacter ATCC4452 1.4 mM Rhizobium radiobacter AJ2557 1.4 mM Carnimonas sp. AJ3230 1.4 mM Rhizobium radiobacter NBRC12667 1.3 mM Pseudomonas fragi NBRC3458 1.3 mM Rhizobium radiobacter NBRC12664 1.3 mM Corynebacterium ammoniagenes 1.2 mM NBRC12072 Pseudomonas sp. AJ1594 1.2 mM Rhizobium radiobacter ATCC6466 1.2 mM Pseudomonas synxantha NBRC3912 1.1 mM Rhizobium radiobacter ATCC4720 1.1 mM Pseudomonas sp. AJ2438 1.0 mM
(2) Synthesis of 2S,4R-Monatin by Actinomycete
[0170] Nocardia globerula ATCC21022 was applied onto a YMPG agar medium (3 g/L of yeast extract, 3 g/L of malt extract, 5 g/L of polypeptone, 10 g/L of glucose, 15 g/L of agar, pH 7.0), and cultured at 30° C. for 2 days.
[0171] One loopful of the obtained microbial cells was inoculated to 3 mL of a YMPG medium (3 g/L of yeast extract, 3 g/L of malt extract, 5 g/L of polypeptone, 10 g/L of glucose, pH 7.0) in a test tube, and cultured with shaking at 30° C. for 16 hours. The microbial cells were collected from 2 mL of the cultured medium by centrifugation, washed with and suspended in 20 mM Tris-HCl (pH 7.6) to prepare 1 mL of a microbial cell suspension.
[0172] Then, 1 g of glass beads (0.1 mm) was added to 1 mL of this microbial cell suspension, and the microbial cells were disrupted using the multi beads shocker (Yasui Kikai Co., Ltd.). The resulting disrupted cell solution was centrifuged to use a supernatant as a microbial cell extract.
[0173] The reaction of synthesizing 2S,4R-Monatin and the quantification of 2S,4R-Monatin were performed in the same manner as in Example 1, and amount of the 2S,4R-Monatin which was formed was as follows (Table 3)
TABLE-US-00006 TABLE 3 Amount of 2S,4R-Monatin which was formed Amount of 2S4R-Monatin Microoganism which was formed Nocardia globerula ATCC21022 0.57 mM
(3) Synthesis of 2S,4R-Monatin by Yeast
[0174] Lodderomyces elongisporus CBS2605, Candida norvegensis NBRC0970, Candida inconspicua NBRC0621 or Yarrowia lypolytica NBRC0746 was applied onto a YPD agar medium (10 g/L of yeast extract, 20 g/L of polypeptone, 20 g/L of glucose, 15 g/L of agar), and cultured at 30° C. for 2 days.
[0175] One loopful of the obtained microbial cells was inoculated to 3 mL of a YPD medium (10 g/L of yeast extract, 20 g/L of polypeptone, 20 g/L of glucose) in a test tube, and cultured with shaking at 30° C. for 16 hours. The microbial cells were collected from 2 mL of the cultured medium by centrifugation, washed with and suspended in 20 mM Tris-HCl (pH 7.6) to prepare 1 mL of a microbial cell suspension.
[0176] Then, 1 g of glass beads (0.5 mm) was added to 1 mL of this microbial cell suspension, and the microbial cells were disrupted using the multi beads shocker (Yasui Kikai Co., Ltd.). The resulting disrupted cell solution was centrifuged to use a supernatant as a microbial cell extract.
[0177] The reaction of synthesizing 2S,4R-Monatin and the quantification of 2S,4R-Monatin were performed in the same manner as in Example 1, and amount of the 2S,4R-Monatin which was formed were as follows (Table 4)
TABLE-US-00007 TABLE 4 Amount of 2S,4R-Monatin which was formed Amount of 2S4R-Monatin Microorganism which was formed Lodderomyces elongisporus CBS2605 0.57 mM Candida norvegensis NBRC0970 0.55 mM Candida inconspicua NBRC0621 0.52 mM Yarrowia lypolytica NBRC0746 0.52 mM
Example 8
Production of 2S,4R-Monatin Potassium Salt Dihydrate
[0178] After 149.00 g of ethanol was added to a reduction reaction concentrated solution (containing 36.62 g (125.28 mmol) of Monatin, (2S,4R):(2R,4R)=32:68), 0.25 g of 2R,4R-Monatin potassium salt monohydrate was added as seed crystals, and the mixture was stirred at 56° C. for 4 hours to perform preferential crystallization of the 2R,4R-Monatin potassium salt monohydrate. The crystallized crystals were separated by filtration (wet crystals 31.27 g) to obtain 225.80 g of a mother solution (containing 22.41 g (76.68 mmol) of Monatin, (2S,4R):(2R,4R)=53:47). This mother solution was cooled to 10° C. and stirred for 5 hours to crystallize 2S,4R-Monatin potassium salt dihydrate. The crystals were separated by filtration (wet crystals 32.74 g), and dried under reduced pressure to yield 9.88 g (15.68 mmol) of the objective 2S,4R-Monatin potassium salt dihydrate (HPLC purity: 55.5%). Then, 9.35 g of the crude crystals were dissolved in 25.37 g of water, and 58.99 g of ethanol was added to this dissolved solution, which was stirred at 25° C. for 5 hours to refine the 2S,4R-Monatin potassium salt dehydrate by crystallization. The crystals were separated by filtration (wet crystals 4.49 g), and dried under reduced pressure to yield 3.75 g (9.62 mmol) of the objective 2S,4R-Monatin potassium salt dihydrate (HPLC purity: 96.0%).
[0179] A water content and a potassium content of the obtained crystals (2S,4R-Monatin potassium salt dihydrate) were analyzed by a water measurement method and a cation analysis method using ion chromatography. Details of the performed water measurement method and cation analysis method are shown below.
(Water Measurement Method)
[0180] Measurement apparatus: Hiranuma Automatic Water Measurement Apparatus AQV-2000 (supplied from Hiranuma Sangyo Corporation)
[0181] Measurement condition: Titration solution=Hydranal Composite 5K (supplied from Riedel de Haen)
(Cation Analysis Method)
[0182] Apparatus: Tosoh IC2001
[0183] Column: TSKgel SuperIC-Cation (4.6×150 mm)
[0184] Guard column: TSKgel SuperIC-Cation (1 cm)
[0185] Suppress gel: TSKgel TSKsuppressIC-C
[0186] Column temperature: 40° C.
[0187] Eluant flow: 0.7 mL/minute
[0188] Sample injection amount: 30 μL
[0189] Detection: Electric conductivity
[0190] Eluant composition: 2.2 mM methanesulfonic acid+1.0 mM 18-crown-6-ether+0.5 mM histidine mixed aqueous solution
[0191] 1H NMR (400 MHz, D2O) δ:2.11 (dd, J=19.0, 27.0 Hz, 1H), 2.39 (dd, J=5.0, 27.0 Hz, 1H), 3.14 (s, 2H), 3.90 (dd, J=5.0, 19.0 Hz, 1H), 7.06 (m, 1H), 7.13 (m, 1H), 7.15 (s, 1H), 7.40 (d, 8.5 Hz, 1H), 7.6 (d, 8.5 Hz, 1H)
[0192] ESI-MS Calculated value: C14H16N2O5=292.11
[0193] ESI-MS Analyzed value: C14H16N2O5=290.9 [M-H].sup.
Example 9
Isomerization Reaction Using 5-Nitrosalicylaldehyde
[0194] 0.15 g (0.38 mmol) of the 2S,4R-Monatin potassium salt dihydrate was added to 10.0 g of an aqueous solution of 70% ethanol, and completely dissolved at 60° C. 7.6 mg (0.045 mmol) of 5-nitrosalicylaldehyde and 7.5 μL (0.13 mmol) of acetic acid were added to that dissolved solution, and stirred at 60° C. for 48 hours. The reaction solution was analyzed and quantified by HPLC, and a molar ratio of 2S,4R-Monatin and 2R,4R-Monatin in the reaction solution was 1:2.1.
Example 10
Isomerization Reaction Using Pyridoxal Hydrochloride Salt
[0195] 0.15 g (0.38 mmol) of the 2S,4R-Monatin potassium salt dihydrate was added to 10.0 g of the aqueous solution of 70% ethanol, and completely dissolved at 60° C. 9.1 mg (0.045 mmol) of pyridoxal hydrochloride and 7.5 μL (0.13 mmol) of acetic acid were added to that dissolved solution, and stirred at 60° C. for 48 hours. The reaction solution was analyzed and quantified by HPLC, and the molar ratio of 2S,4R-Monatin and 2R,4R-Monatin in the reaction solution was 1:1.3.
Example 11
Isomerization Reaction Using Pyridoxal 5-Phosphate Monohydrate
[0196] 0.15 g (0.38 mmol) of the 2S,4R-Monatin potassium salt dihydrate was added to 10.0 g of the aqueous solution of 70% ethanol, and completely dissolved at 60° C. 12.8 mg (0.048 mmol) of pyridoxal 5-phosphate monohydrate and 7.5 μL (0.13 mmol) of acetic acid were added to that dissolved solution, and stirred at 60° C. for 48 hours. The reaction solution was analyzed and quantified by HPLC, and the molar ratio of 2S,4R-Monatin and 2R,4R-Monatin in the reaction solution was 1:1.1.
Example 12
Isomerization Reaction Using Salicylaldehyde
[0197] 0.15 g (0.38 mmol) of the 2S,4R-Monatin potassium salt dihydrate was added to 10.0 g of the aqueous solution of 70% ethanol, and completely dissolved at 60° C. 5.3 mg (4.6 μL, 0.043 mmol) of salicylaldehyde and 7.5 μL (0.13 mmol) of acetic acid were added to that dissolved solution, and stirred at 60° C. for 48 hours. The reaction solution was analyzed and quantified by HPLC, and the molar ratio of 2S,4R-Monatin and 2R,4R-Monatin in the reaction solution was 1:0.6.
Example 13
Isomerization Reaction Using 3,5-Dichlorosalicylaldehyde
[0198] 0.15 g (0.38 mmol) of the 2S,4R-Monatin potassium salt dihydrate was added to 10.0 g of the aqueous solution of 70% ethanol, and completely dissolved at 60° C. 8.1 mg (0.042 mmol) of 3,5-dichlorosalicylaldehyde and 7.5 μL (0.13 mmol) of acetic acid were added to that dissolved solution, and stirred at 60° C. for 48 hours. The reaction solution was analyzed and quantified by HPLC, and the molar ratio of 2S,4R-Monatin and 2R,4R-Monatin in the reaction solution was 1:1.5.
Example 14
Production of 2R,4R-Monatin Potassium Salt Monohydrate by Isomerization-Crystallization Using 2S,4R-Monatin Potassium Salt Dihydrate as Starting Material
[0199] The 2S,4R-Monatin potassium salt dihydrate is added to an aqueous solution of 20% ethanol and completely dissolved at 60° C. 5 molar percent 5-Nitrosalicylaldehyde relative to the 2S,4R-Monatin, and 30 molar percent acetic acid relative to the 2S,4R-Monatin are added to this dissolved solution, and stirred for 48 hours. Ethanol at a final concentration of 70% is added to this reaction solution (2S,4R-Monatin:2R,4R-Monatin=1:2.1), subsequently one percent 2R,4R-Monatin potassium salt monohydrate relative to the 2R,4R-Monatin in the reaction solution is added as the seed crystals thereto, and the mixture is stirred at 60° C. for 48 hours to perform the isomerization-crystallization. The crystallized crystals are separated by filtration, and dried under reduced pressure to yield the objective 2R,4R-Monatin potassium salt monohydrate.
Example 15
Isomerization Reaction Using Glyoxylic Acid
[0200] 0.15 g (0.38 mmol) of the 2S,4R-Monatin potassium salt dihydrate was added to 10.0 g of the aqueous solution of 70% ethanol, and completely dissolved at 60° C. 5.1 mg (0.069 mmol) of glyoxylic acid and 7.5 μL (0.13 mmol) of acetic acid were added to that dissolved solution, and stirred at 60° C. for 48 hours. The reaction solution was analyzed and quantified by HPLC, and the molar ratio of 2S,4R-Monatin and 2R,4R-Monatin in the reaction solution was 1:0.07
Example 16
Production of L-Amino Acid Aminotransferase (LAT) Mutants Derived from AJ1616 Strain and Measurement of Specific Activity for Various Keto Acids
(1) Production of Mutated LAT-Expressing Plasmid by Site-Directed Mutagenesis
[0201] Plasmids expressing a mutated LAT derived from AJ1616 strain were produced by site-directed mutagenesis in accordance with protocols of QuickChange Site-Directed Mutagenesis Kit supplied from Stratagene. One set of primers designed so that a mutation (substitution) was introduced into a target nucleotide residue and became complementary in respective chains of double stranded DNA was synthesized. The produced mutants and the nucleotide sequences of the primers used for the production of the mutants are shown in Tables 5 and 6, respectively. The mutant plasmids were produced using pET22-AJ1616LAT-His(C) as the template under the following PCR condition:
TABLE-US-00008 1 cycle 95° C., 1 min 18 cycles 95° C., 30 sec 55° C., 1 min 68° C., 8 min after completion of the cycles 4° C.
[0202] The template pET22-AJ1616LAT-His(C) was cleaved by treating with the restriction enzyme Dpn I (37° C., one hour) cleaving by recognition of methylated DNA, and subsequently E. coli JM109 was transformed with the resulting reaction solution. The plasmid was collected from the transformant, and it was confirmed by sequencing the nucleotides that the mutation (substitution) of the target nucleotide residue had been introduced. ID136 that was a double mutant of S258C/I289A was constructed by making an S258G mutant plasmid followed by repeating the same manipulation using the primers for introducing an I289A mutation. ID189 that was a double mutant of K39R/T288G was constructed by making an ID166 (T288G) mutant plasmid followed by repeating the same manipulation using the primers for introducing a K39R mutation. ID296 that was a double mutant of Q287E/T288G was constructed by making a T288G mutant plasmid followed by repeating the same manipulation using the primers for introducing a Q287E/T288G mutation.
TABLE-US-00009 TABLE 5 Mutants which were prepared ID Mutants ID136 S258G/I289A ID166 T288G ID189 K39R/T288G ID296 Q287E/T288G
TABLE-US-00010 TABLE 6 Nucleotide sequences of primers used for introducing mutation Nucleotide sequences Mutants Primer names (SEQ ID NOs) K39R K39R_FW gacatgtctagagggcgtccttcaccaaaacag (SEQ ID NO: 26) K39R_RV ctgttttggtgaaggacgccctctagacatgtc (SEQ ID NO: 27) S258G S258G_FW gttcgcctctactggtaaaattacgttccc (SEQ ID NO: 28) S258G_RV gggaacgtaattttaccagtagaggcgaac (SEQ ID NO: 29) T288G T288G_FW cagctatcagttcaaggcattgggccagataaaatc (SEQ ID NO: 30) T288G_RV gattttatctggcccaatgccttgaactgatagctg (SEQ ID NO: 31) I289A I289A_FW ctatcagttcaaaccgctgggccagataaaatc (SEQ ID NO: 32) I289A_RV gattttatctggcccagcggtttgaactgatag (SEQ ID NO: 33) Q287E/ Q287E_T288G_FW Cagctatcagttgaaggcattgggccag T288G (SEQ ID NO: 34) Q287E_T288G_RV ctggcccaatgccttcaactgatagctg (SEQ ID NO: 35)
(2) Expression and Purification of Mutated LAT
[0203] E. coli JM109 (DE3) was transformed with the obtained mutant AJ1616 LAT-expressing plasmid to produce a mutant AJ1616 LAT-expressing strain. Microbial cells of the mutant AJ1616 LAT-expressing strain pET22-AJ1616LATmut-His(C)/E. coli JM109 (DE3) that was grown on an LB-amp (100 mg/L) plate were inoculated to 100 mL of Overnight Express Instant TB Medium (Novagen) containing 100 mg/L of ampicillin, and cultured with shaking at 37° C. for 16 hours using a Sakaguchi flask. After completion of the cultivation, the microbial cells were collected from the resulting medium by centrifugation, washed with and suspended in 20 mM Tris-HCl (pH 7.6), 300 mM NaCl and 10 mM imidazole, followed by being sonicated. Microbial cell debris was removed from the disrupted suspension by centrifugation, and the resulting supernatant was used as a soluble fraction. The resulting soluble fraction was applied onto a His-tagged protein purification column, His TALON superflow 5 ml Cartridge (Clontech) equilibrated with 20 mM Tris-HCl (pH 7.6), 300 mM NaCl and 10 mM imidazole, and absorbed to the carrier. Proteins that had not been absorbed to the carrier (unabsorbed proteins) were washed out with 20 mM Tris-HCl (pH 7.6), 300 mM NaCl and 10 mM imidazole, and subsequently, the absorbed proteins were eluted using 20 mM Tris-HCl (pH 7.6), 300 mM NaCl and 150 mM imidazole at a flow rate of 5 mL/minute. Resulting fractions were combined, and the combined fraction was concentrated using Amicon Ultra-15 30K (Millipore). The concentrated fraction was diluted with 20 mM Tris-HCl (pH 7.6) to use as a mutant AJ1616 LAT solution. If necessary, the purification was performed by increasing the amount of the medium and the number of the His TALON columns to be connected.
(3) Measurement of Protein Concentration
[0204] A protein concentration was measured using a protein assay CBB solution (diluted to 5 folds for the use) supplied from Nacalai Tesque. The protein concentration was calculated by preparing a standard curve using solutions containing 0.05, 0.1, 0.25 and 0.5 mL/mL BSA as the standards.
(4) Measurement of Activity for L-Asp/α-KG, L-Asp/PA and L-Asp/±MHOG by colorimetric assay
[0205] The activity of AJ1616 LAT for various substrates was measured. 100 mM L-Asp was used as an amino donor substrate in a transamination reaction, and a specific activity for 10 mM various keto acids was measured by a colorimetric assay.
[0206] Activity for L-Asp/α-KG (α-ketoglutaric acid): measured in 100 mM L-Asp-Na, 10 mM α-KG-2Na, 50 μM PLP, 100 mM Tris-HCl (pH 8.0), 0.25 mM NADH, and 2 U/mL MDH at 25° C. The activity was calculated from the reduction of absorbance at 340 nm. Malic dehydrogenase from porcine heart (Sigma) was used as MDH. The activity for L-Asp/α-KG is shown in the column "α-KG" of the aminotransferase activity in Table 9.
[0207] Activity for L-Asp/PA: measured in 100 mM L-Asp-Na, 10 mM PA-2Na, 50 μM PLP, 100 mM Tris-HCl (pH 8.0), 0.25 mM NADH, and 2 U/mL MDH (same as above) at 25° C. The activity was calculated from the reduction of the absorbance at 340 nm. The activity for L-Asp/PA is shown in the column "PA" of the aminotransferase activity in Table 9.
[0208] Activity for L-Asp/(±)-MHOG (4-hydroxy-4-methyl-2-ketoglutarate): measured in 100 mM L-Asp-Na, 10 mM (±)-MHOG, 50 μM PLP, 100 mM Tris-HCl (pH 8.0), 0.25 mM NADH, 2 U/mL MDH and 10 U/mL LDH at 25° C. The activity was calculated from the reduction of the absorbance at 340 nm. D-Lactate dehydrogenase from Leuconostoc mesenteroides (Oriental Yeast) was used as LDH. LDH was added in order to remove PA in a trace amount contaminated in (±)-MHOG. The activity for L-Asp/(±)-MHOG is shown in the column "(±)-MHOG" of the aminotransferase activity in Table 9.
(5) Measurement of Activity for L-Asp/4R-IHOG and L-Asp/IPA
[0209] The activity of forming 2S,4R-Monatin from 4R-IHOG, which was an objective activity, and the activity of forming a byproduct L-Trp from IPA were measured. 100 mM L-Asp was used as the amino donor substrate in the transamination reaction, the transamination reaction to 10 mM keto acid was performed. The amount of formed amino acid was quantified by UPLC or HPLC, and the specific activity was calculated.
[0210] Activity for L-Asp/4R-IHOG (10 mM): measured in 100 mM L-Asp-Na, 10 mM 4R-IHOG (containing 4S-IHOG in a trace amount), 50 μM PLP, and 100 mM Tris-HCl (pH 8.0) at 25° C. Formed 2S,4R-Monatin and 2S,4S-Monatin were quantified by UPLC analysis. The reaction was stopped using a 200 mM citrate Na solution (pH 4.5). The activity for L-Asp/4R-IHOG is shown in the column "4R-IHOG" of the aminotransferase activity in Table 9.
[0211] Activity for L-Asp/IPA: measured in 100 mM L-Asp-Na, 10 mM IPA, 50 μM PLP, and 100 mM Tris-HCl (pH 8.0) (pH was adjusted to 8.0 with 1 N NaOH after preparing the reaction solution) at 25° C. Formed Trp was quantified by the UPLC analysis. The reaction was stopped using the 200 mM citrate Na solution (pH 4.5). The activity for L-Asp/IPA is shown in the column "IPA" of the aminotransferase activity in Table 9.
[0212] Formed Monatin and Trp were quantified using ACQUITY UPLC system supplied from Waters. A measurement condition is shown below. The reaction in 0.2 mL was performed for 15 minutes, and then stopped. The reaction solution after stopping the reaction was centrifuged, and then about 0.2 mL of the supernatant was subjected to the UPLC analysis. Results obtained by measurement using serial dilutions in which the concentrations of the samples and a blank fell into the range of 0.01 to 0.05 mM were employed as activity values.
TABLE-US-00011 TABLE 7 UPLC Column: ACQUITY UPLC HSS T3 2.1 × 50 mm Column Temp.: 40° C. Sample Temp.: 4° C. Detection: UV 210 nm Injection vol.: 5 μl Mobile Phase A: 20 mM KH2PO4 (Filt.) Mobile Phase B: ACN Flow rate: 0.5 ml/min Method: 20 mM KH2PO4_05_HSS Time (min) A (%) B (%) 0 96 4 1.9 96 4 2.0 60 40 2.2 60 40 2.3 96 4 3.0 96 4
[0213] 2S,4R-Monatin, 2S,4S-Monatin and Trp can be quantified separately at 1.1 minutes, 1.5 minutes and 1.3 minutes, respectively.
[0214] The quantification using HPLC under the following analysis condition was also performed in conjunction with the above.
[0215] HPLC condition (quantification condition for Monatin, Trp, IPA, IAA (indole acetate), IAD (indole aldehyde))
[0216] Column: CAPCELL PAK C18 TYPE MGII 3 μm, 4.6 mm×150 mm (Shiseido)
[0217] Column temperature: 40° C.
[0218] Detection wavelength: 280 nm
[0219] Flow rate: 1.0 mL/minute
[0220] Mobile phase: A: 20 mM KH2PO4/CH3CN=100/5, B: CH3CN
TABLE-US-00012 TABLE 8 Time (min) A (%) B (%) 0 100 0 6 100 0 11 90 10 25 90 10 26 100 0 30 100 0
(6) Results of Measuring Specific Activity of AJ1616 Strain LAT Mutants Against Various Keto Acids
[0221] The results of the specific activity against 10 mM keto acid are shown in Table 9, which were measured with the produced mutant and L-Asp as the amino donor. The objective activity of forming 2S,4R-Monatin using 4R-IHOG as the substrate was enhanced in any of the produced mutants. Concerning relative values of side reaction relative to the objective activity, the activity of producing the byproduct L-Trp, the activity of producing the byproduct MHG (4-hydroxy-4-methyl glutamate), and the activity of producing the byproduct L-Ala, relative to the objective activity (activity of forming 2S,4R-Monatin) were reduced in any of the mutants.
TABLE-US-00013 TABLE 9 Specific activities of mutants relative to various keto acids. Relative values of side reaction relative to Aminotransferase activity of forming activity (U/mg) 2S,4R-Monatin (SR) ID Mutants α-KG PA ±MHOG 4R-IHOG IPA Trp/SR MHG/SR Ala/SR WT WT 235 0.45 1.3 0.92 0.11 0.12 1.45 0.49 136 S258G/I289A 14 0.06 0.09 6.8 0.54 0.08 0.01 0.01 166 T288G 184 0.23 2.1 6.7 0.28 0.04 0.31 0.03 189 K39R/T288G 90 0.23 1.6 9.7 0.31 0.03 0.17 0.02 296 Q287E/T288G 50 0.18 1.5 11.1 0.24 0.02 0.14 0.02
Example 17
Construction of E. coli JM109 ΔaspC Strain and Production of Broth Containing Expressed Deaminase
[0222] E. coli JM109 ΔaspC was constructed by following methods. E. coli JM109/pKD46 was cultured at 30° C. overnight on the LB-amp (100 mg/L) plate. Obtained microbial cells were inoculated to 50 mL of LB (containing 100 mg/L of Amp and 10 mM L-arabinose). This was cultured with shaking at 30° C. using the Sakaguchi flask. When OD610 became about 0.6, a cultivation temperature was changed to 37° C. and the cultivation was continued with shaking for additional one hour. The microbial cells were collected from the resulting medium by centrifugation, washed with 10% glycerol, and collected again by centrifugation. These were suspended in 10% glycerol to use as competent cells.
[0223] Amplification by PCR was performed with pMW118-attL-cat-attR as the template using the primer aspC-L1 (5'-TTTGAGAACATTACCGCCGCTCCTGCCGACCCGATTCTGGGCtgaagcctgcttttttat-3': SEQ ID NO:36) and the primer aspC-R1 (5'-CAGCACTGCCACAATCGCTTCGCACAGCGGAGCCATGTTATCcgctcaagttagtataaa-3: SEQ ID NO:37). The resulting PCR product was extracted from agarose to use as a DNA fragment for aspC gene disruption. PCR was performed using KOD-plus-ver. 2 (Toyobo).
[0224] The competent cells were transformed with the purified DNA fragment, and an objective transformant was selected on an LB-Cm (20 mg/L) plate at 37° C. It was confirmed by colony PCR that attL-cat-attR was inserted into the aspC gene region of the transformant. The primers used are the primer aspC-up (5'-AACCTCTTGGCAACGGTAAAAAAGCTGAAC-3': SEQ ID NO: 38), the primer attL-1 (5'-TAGTGACCTGTTCGTTGC-3': SEQ ID NO:39), the primer aspC-down (5'-GCCTGCGCAAAGTCGTATGTTTGGTCTGGA-5': SEQ ID NO:40), and the primer attR-1 (5'-TTACGTTTCTCGTTCAGC-3': SEQ ID NO:41). Z-taq (TAKARA) was used for PCR.
[0225] The obtained transformant was inoculated to 3 mL of LB (Cm 20 mg/L), and cultured with shaking at 37° C. for 6 hours. Microbial cells were collected from the resulting medium by centrifugation, washed with 10% glycerol, and the microbial cells were collected again by centrifugation. These were suspended in 10% glycerol to use as competent cells.
[0226] The competent cells were transformed with pMW-intxis-ts in order to remove the Cm resistant gene sequence inserted in the genomic DNA. An objective transformant was selected on the LB-amp (100 mg/L) plate at 30° C. The obtained transformant was cultured on the LB plate at 42° C. overnight, and the microbial cells were streaked on the LB-amp (100 mg/L) plate and on the LB-Cm (20 mg/L) plate, respectively and cultured at 37° C. The transformant was confirmed not to grow on both the plate containing Amp and the plate containing Cm. Further the removal of the Cm resistant gene was confirmed by colony PCR using the primer aspC-up (5'-AACCTCTTGGCAACGGTAAAAAAGCTGAAC-3': SEQ ID NO:38) and the primer aspC-down (5'-GCCTGCGCAAAGTCGTATGTTTGGTCTGGA-5': SEQ ID NO:40). Z-tag (TAKARA) was used for PCR.
[0227] The obtained strain was designated as an aspC-deficient strain, E. coli JM109ΔaspC. A deaminase-expressing strain pTB2/E. coli JM109ΔaspC was constructed by transforming E. coli JM109ΔaspC with a deaminase-expressing plasmid, pTB2. This bacterial strain was cultured on the LB-amp (100 mg/L) at 37° C. overnight. The obtained microbial cells were inoculated to 100 mL of TB-amp (100 mg/L) and cultured with shaking at 37° C. for 16 hours using the Sakaguchi flask. The resulting medium was used as Ps_aad broth.
Example 18
Construction of Oxaloacetate Decarboxylase-Expressing Strain
[0228] Synthesis of an OAA decarboxylase gene derived from Pseudomonas putida KT2440 strain was asked GenScript, and a plasmid DNA in which a DNA fragment including the OAA decarboxylase gene had been inserted in pUC57 was obtained. A frequency of codon usage was optimized for expression in E. coli (see SEQ ID NOS:42 and 43). This plasmid was cleaved with NdeI and XhoI, inserted into pET22b cleaved with NdeI and XhoI, and the resulting plasmid was designated as pET22-PpODC-His(C). E. coli BL21 (DE3) was transformed with the resulting plasmid to obtain a PpODC-His(C)-expressing strain, pET22-PpODC-His(C)/E. coli BL21 (DE3). Microbial cells of the PpODC-His(C)-expressing strain, pET22-PpODC-His(C)/E. coli BL21 (DE3) grown on the LB-amp (100 mg/L) plate were inoculated to 100 mL of Overnight Express Instant TB Medium (Novagen), and cultured with shaking at 30° C. for 16 hours using the Sakaguchi flask. After the termination of cultivation, microbial cells were collected from the resulting medium, and washed with and suspended in 20 mM Tris-HCl (pH 7.6), 300 mM NaCl and 10 mM imidazole, followed by being sonicated. Microbial cell debris was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as a soluble fraction. The resulting soluble fraction was applied onto a His-tagged protein purification column, His TALON superflow 5 ml Cartridge (Clontech) equilibrated with 20 mM Tris-HCl (pH 7.6), 300 mM NaCl and 10 mM imidazole, and absorbed to the carrier. Proteins that had not been absorbed to the carrier (unabsorbed proteins) were washed out with 20 mM Tris-HCl (pH 7.6), 300 mM NaCl and 10 mM imidazole, and subsequently, the absorbed proteins were eluted using 20 mM Tris-HCl (pH 7.6), 300 mM NaCl and 150 mM imidazole at a flow rate of 5 mL/minute. Resulting fractions were combined, and the obtained solution was concentrated using Amicon Ultra-15 10K (Millipore). The obtained solution was diluted with 20 mM Tris-HCl (pH 7.6) to use as a PpODC solution.
[0229] An ODC activity was measured under the condition shown below.
[0230] The measurement of the ODC activity was performed under the following condition.
[0231] 10 mM OAA, 100 mM Tris-HCl (pH 8.0), 0.25 mM NADH and 10 U/mL of LDH at 25° C. The activity was calculated from the reduction of the absorbance at 340 nm. D-Lactate dehydrogenase from Leuconostoc mesenteroides (Oriental Yeast) was used as LDH. The reaction and the analysis on a scale of 1 mL were performed, and activity values in serial dilutions in which a measured value [(sample Δ340 nm/min)-(blank Δ340 nm/min)] fell onto the range of 0.05 to 0.15 were employed. The enzyme was diluted with 20 mM Tris-HCl (pH 7.6) and 0.01% BSA.
Example 19
One-Pot Synthesis Reaction of 2S,4R-Monatin from 100 mM L-Trp (WT, ID136, ID166)
[0232] A reaction was performed for 22 hours using the purified mutant AJ1616 LAT under the following condition. The reaction was performed in a volume of 1 mL using a test tube. Sampling was performed after 14, 18 and 22 hours. The sample was diluted with TE buffer, which was then ultrafiltrated using an Amicon Ultra-0.5 mL centrifugation type filter 10 kDa, and a filtrate was analyzed. HPLC was used for the analysis.
[0233] Reaction condition: 100 mM L-Trp, 50 mM PA-Na, 300 mM L-Asp-Na, 1 mM MgCl2, 50 μM PLP, 100 mM Tris-HCl, 20 mM KPB, pH 7.0, 40% Ps_aad broth, 0.2 mg/mL of purified SpAld enzyme, 10 U/mL of commercially available OAA DCase enzyme, 2 U/mL of purified mutant AJ1616 LAT enzyme (vs 10 mM 4R-IHOG), and 200 U/mL of commercially available SOD enzyme at 25° C. at 140 rpm.
[0234] Methods for preparing the enzyme subjected to the reaction are described below.
[0235] Ps_aad broth: Prepared according to the method described in Example 17.
[0236] Purified SpAld enzyme: A jar cultivation of the SpAld-expressing strain was performed according to the method described in Example 6, and the thermal treatment at 60° C. was further performed for one hour. Microbial cells were collected from 100 mL of the resulting medium after the thermal treatment by centrifugation, and washed with and suspended in 20 mM Tris-HCl (pH 7.6), followed by being sonicated. Microbial cell debris was removed from the disrupted solution by centrifugation. The resulting supernatant was used as a soluble fraction. Ammonium sulfate and Tris-HCl (pH 7.6) were added so that this soluble fraction contained 1 M ammonium sulfate and 20 mM Tris-HCl (pH 7.6). This solution was applied onto a hydrophobic chromatography column HiLoad 26/10 Phenyl Sepharose HP (supplied from GE Healthcare Bioscience, CV=53 mL) equilibrated with 1 M ammonium sulfate and 20 mM Tris-HCl (pH 7.6), and absorbed to the carrier. Unabsorbed proteins which had not been absorbed to the carrier were washed out with 1 M ammonium sulfate and 20 mM Tris-HCl (pH 7.6). Subsequently, the absorbed proteins were eluted by linearly changing the concentration of ammonium sulfate from 1 M to 0 M at a flow rate of 8 mL/minute. Fractions in which the activity had been detected were combined, and the obtained solution was concentrated using Amicon Ultra-15 10k (Millipore). The resulting concentrated solution was diluted with 20 mM Tris-HCl (pH 7.6) to use as an SpAld solution. A PHOG degrading activity measurement method was used for measuring the aldolase activity (measured in 2 mM PHOG, 50 mM KPB, 1 mM MgCl2, 0.25 mM NADH, and 16 U/mL of LDH at 25° C. (pH 7.0). The activity was calculated from the reduction of the absorbance at 340 nm). D-Lactate dehydrogenase from Leuconostoc mesenteroides (Oriental Yeast) was used as LDH.
[0237] Mutant AJ1616 LAT: Microbial cells of the mutant AJ1616 LAT-expressing strain, pET22-AJ1616LATmut-His(C)/E. coli JM109 (DE3) grown on the LB-amp (100 mg/L) plate were inoculated to 100 mL of Overnight Express Instant TB Medium (Novagen) containing 100 mg/L of ampicillin, and cultured with shaking at 37° C. for 16 hours using the Sakaguchi flask. After the termination of cultivation, the microbial cells were collected from the resulting medium, and washed with and suspended in 20 mM Tris-HCl (pH 7.6), 300 mM NaCl and 10 mL imidazole, followed by being sonicated. Microbial cell debris was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as a soluble fraction. The resulting soluble fraction was applied onto a His-tagged protein purification column, His TALON superflow 5 ml Cartridge (Clontech) equilibrated with 20 mM Tris-HCl (pH 7.6), 300 mM NaCl and 10 mM imidazole, and absorbed to the carrier. Proteins that had not been absorbed to the carrier (unabsorbed proteins) were washed out with 20 mM Tris-HCl (pH 7.6), 300 mM NaCl and 10 mM imidazole, and subsequently, the absorbed proteins were eluted using 20 mM Tris-HCl (pH 7.6), 300 mM NaCl and 150 mM imidazole at a flow rate of 5 mL/minute. Resulting fractions were combined, and the obtained solution was concentrated using Amicon Ultra-15 30K (Millipore). The concentrated solution was diluted with 20 mM Tris-HCl (pH 7.6) to use as a mutant AJ1616 LAT solution. If necessary, the purification was performed by increasing the amount of the culture medium and the number of the His TALON columns to be connected.
[0238] OAA DCase: Oxaloacetate decarboxylase from Pseudomonas sp. (Sigma) was used. The value described by the manufacturer was used as the amount of the enzyme (U).
[0239] SOD: Superoxide dismutase from bovine liver (Sigma) was used. The value described by the manufacturer was used as the amount of the enzyme (U).
[0240] As a result of the one-pot reaction, the yield of 2S,4R-Monatin was enhanced in cases of using the produced 10136 and ID166 mutant enzymes compared with the wild enzyme (Table 10).
TABLE-US-00014 TABLE 10 Yield of 2S,4R-Monatin in one-pot reaction using 100 mM Trp as substrate Yield of 2S,4R-Monatin in one-pot reaction (vs. yield of Trp (%)) ID Mutants 14 hr 18 hr 22 hr WT WT 23 30 30 136 S259G/I289A 68 77 72 166 T288G 84 83 85
Example 20
One-Pot Synthesis Reaction of 2S,4R-Monatin from 100 mM Trp (10166 on Scale of 400 mL)
[0241] A reaction was performed for 6 hours using purified AJ1616 LAT-ID166 under the following condition. The reaction was performed in a volume of 400 mL using a 1 liter volume jar. Sampling was appropriately performed, the sample was diluted with TE buffer, which was then ultrafiltrated using an Amicon Ultra-0.5 mL centrifugation type filter 10 kDa, and a filtrate was analyzed. HPLC and capillary electrophoresis were used for the analysis.
[0242] Reaction condition: 100 mM L-Trp, 50 mM PA-Na, 300 mM L-Asp-Na, 1 mM MgCl2, 50 μM PLP, 20 mM KPB (pH 7.6), pH<7.6 (1 M H2SO4), 40% Ps_aad broth, 10% SpAld broth, 5 U/mL of PpODC, 4 U/mL of AJ1616 LAT-ID166 (vs 10 mM 4R-IHOG) and 100 U/mL of SOD at 25° C. at 500 rpm, and with air at 20 mL/min (1/20 vvm).
[0243] pTB2/E. coli JM109ΔaspC broth was used as Ps_aad broth. The thermally treated broth described in Example 19 was used as SpAld broth. The purified enzyme described in Example 18 was used as PpODC. Superoxide dismutase from bovine liver (Sigma) was used as SOD.
[0244] As a result, the accumulation of 86 mM 2S,4R-Monatin was confirmed after 6 hours (FIG. 4). The yield relative to L-Trp calculated after calibrating the solution amount was 89%.
Example 21
One-Pot Synthesis Reaction of 2S,4R-Motatin from 150 mM L-Trp (ID189 on Scale of 80 mL)
[0245] A reaction was performed for 27 hours using purified AJ1616 LAT-ID189 under the following condition. The reaction was performed in a volume of 80 mL using a 250 mL volume jar. Sampling was appropriately performed, the sample was diluted with TE buffer, which was then ultrafiltrated using the Amicon Ultra-0.5 mL centrifugation type filter 10 kDa, and a filtrate was analyzed. HPLC and capillary electrophoresis were used for the analysis.
[0246] Reaction condition: 150 mM L-Trp, 50 mM PA-Na, 400 mM L-Asp-Na, 1 mM MgCl2, 50 μM PLP, 20 mM KPB (pH 7.6), pH<7.6 (1 M H2SO4), 40% Ps_aad broth, 10% SpAld broth, 5 U/mL of PpODC, 4 U/mL of AJ1616 LAT-ID189 (vs 10 mM 4R-IHOG) and 100 U/mL of SOD at 25° C. (380 rpm), and with air at 4 mL/min (1/20 vvm).
[0247] pTB2/E. coli JM109ΔaspC broth was used as the Ps_aad broth. The thermally treated broth described in Example 19 was used as the SpAld broth. The purified enzyme described in Example 18 was used as PpODC. Superoxide dismutase from bovine liver (Sigma) was used as SOD.
[0248] As a result, the accumulation of 105 mM 2S,4R-Monatin was confirmed after 27 hours (FIG. 5). The yield relative to L-Trp calculated after calibrating the solution amount was 78% (FIG. 5).
Example 22
Isolation of 2S,4R-Monatin
[0249] 2.59 g Of ZN charcoal was added to 435.66 g of a permeated solution obtained by treating 435.45 g of the enzyme reaction solution in Example 20 (lot 101213 J4) with UF (MWCO: 3000), and the mixture was stirred at room temperature (about 26° C.) for one hour. The activated charcoal was filtrated with a Kiriyama filter (5C), and the resulting filtrate was transferred to a 1 liter four-necked flask. The flask was immersed in an incubator at 5° C., the solution was neutralized with 35% hydrochloric acid to adjust pH to 3.5, and stirred using a mechanical stirrer (120 rpm). Then, 48 mg of seed crystals were added, and 1 N hydrochloric acid was sequentially added using a pH controller and a peristaltic pump to keep a target pH because the pH value elevated when the crystals began to precipitate. A slurry solution obtained by stirring for 24 hours was filtrated, the crystals were washed with 10 mL of water, and the wet crystals were dried under reduced pressure at 40° C. to yield 6.81 g of 2S,4R-Monatin. The quality of the obtained crystals was confirmed by HPLC and 1H-NMR analysis.
[0250] HPLC area purity (210 nm): 98.4%
[0251] 1H-NMR (in D2O+K2CO3) 2.08-2.14 (1H, dd), 2.35-2.39 (1H, dd), 3.09-3.17 (2H, dd), 3.85-3.88 (1H, dd), 7.04-7.15 (3H, m), 7.39-7.41 (1H, m), 7.64-7.66 (1H, d).
Example 23
Synthesis of 2R,4R-Monatin
[0252] 3.10 g (10.4 mmol) Of 2S,4R-Monatin obtained in Example 22 and 1.165 g (10.4 mmol) of 50% KOH were dissolved in 3.27 g of water, and further 1.3 g of EtOH, 0.0869 g (0.052 mmol) of 5-nitrosalicylaldehyde, and 0.187 g (3.12 mmol) of acetic acid were added thereto. After 25 hours, 20.5 g of EtOH and 10 mg of seed crystals (2R,4R-Monatin) were added, and the mixture was stirred for additional 46.5 hours. The resulting slurry solution was cooled to room temperature, and then filtrated. The crystals were washed with 4 g of 85% EtOH-water, and the wet crystals were dried under reduced pressure at 40° C. to yield 2.3 g of crude 2R,4R-Monatin. 2.1 g of the resulting crude 2R,4R-Monatin was dissolved in 6 mL of water, 0.2 g of BA charcoal was added, and the mixture was stirred at room temperature (around 25° C.) for one hour and then filtrated with a 0.45 μm membrane filter. The filtrate was concentrated to 6.38 g under reduced pressure. 12 g Of EtOH was dripped to the concentrated filtrate at 45° C., which was then stirred for one hour. Further, 13.5 g of EtOH was quantitatively dripped over one hour, which was then stirred at 45° C. for 16 hours and subsequently cooled to 25° C. The resulting slurry solution was filtrated, the crystals were washed with 3 g of 85% EtOH-water, and the wet crystals were dried under reduced pressure at 40° C. to yield 1.9 g (5.46 mmol) of 2R,4R-Monatin. The obtained crystals, the mother solution, and the washing solution were analyzed by HPLC to analyze yield and quality.
[0253] HPLC area purity (210 nm): 99.9%
[0254] 1H-NMR (in D2O) 1.93-2.00 (1H, dd), 2.57-2.61 (1H, dd), 2.99-3.02 (1H, d), 3.19-3.22 (1H, d), 3.55-3.56 (1H, dd), 7.04-7.15 (3H, m), 7.39-7.41 (1H, m), 7.64-7.66 (1H, d).
TABLE-US-00015 TABLE 11 HPLC analysis condition DEGASSER DGU-20A3 [SHIMAZU] PUMP LC-20AD [SHIMAZU, Two units] Column oven CTO-20AC [SHIMAZU] DIODE ARRAY SPD-M20A [SHIMAZU] DETECTOR Auto sampler SIL-20ACHT [SHIMAZU] COMMUNICATION CBM-20A BUS MODULE System LC solution [SHIMAZU] Column CAPCELL PAC C18 Type MG II 5 μm 3.0 mm Φ × 250 mm [supplied from Shiseido] Column temperature 40° C. Detection wavelength 210 nm Flow rate 0.35 ml/min Composition of mobile Solution A: 20 mM KH2PO4/CH3CN = 100/5 solution Solution B: 20 mM KH2PO4/CH3CN = 30/70 Injection amount 5 μl Autosampler solution CH3CN/H2O = 30/70 Solution A Solution B Time (min) (%) (%) Gradient pattern 0 100 0 15 100 0 40 46 54 45 46 54 45.1 100 0 60 100 0
Example 24
One-Pot Synthesis Reaction of 2S,4R-Motatin from 150 mM L-Trp (ID296 on Scale of 80 mL)
[0255] A reaction was performed for 51 hours using purified AJ1616 LAT-ID296 under the following condition. The reaction was performed in a volume of 80 mL using a 250 mL volume jar. Sampling was appropriately performed, the sample was diluted with TE buffer, which was then ultrafiltrated using the Amicon Ultra-0.5 mL centrifugation type filter 10 kDa, and a filtrate was analyzed. HPLC was used for the analysis.
[0256] Reaction condition: 150 mM L-Trp, 50 mM PA-Na, 400 mM L-Asp-Na, 1 mM MgCl2, 50 μM PLP, 20 mM KPB (pH 7.6), pH<7.6 (1 M H2SO4), 40% Ps_aad broth, 10% SpAld broth, 5 U/mL of PpODC, 4 U/mL of AJ1616 LAT-ID296 (vs 10 mM 4R-IHOG) and 100 U/mL of SOD at 25° C. (380 rpm), and with air at 4 mL/min (1/20 vvm).
[0257] pTB2/E. coli JM109ΔaspC broth was used as the Ps_aad broth. The thermally treated broth described in Example 19 was used as the SpAld broth. The purified enzyme described in Example 18 was used as PpODC. Superoxide dismutase from bovine liver (Sigma) was used as SOD.
[0258] As a result, the accumulation of 113 mM 2S,4R-Monatin was confirmed after 39 hours (FIG. 6). The yield relative to L-Trp calculated after calibrating the solution amount was 86% (FIG. 6).
Example 25
Purification of Aminotransferase Derived from Rhizobium radiobacter AJ3976
[0259] An aminotransferase that forms 2S,4R-Monatin was purified from a soluble fraction of Rhizobium radiobacter AJ3976 as follows. The reaction was performed in 100 mM L-Asp-Na-laq, 10 mM 4R-IHOG (containing 4S-IHOG in a trace amount), 50 μM PLP, and 100 mM Tris-HCl (pH 8.0) at 25° C. The formed 2S,4R-Monatin was quantified by UPLC analysis.
TABLE-US-00016 TABLE 12-1 UPLC Column: ACQUITY UPLC HSS T3 2.1 × 50 mm Column Temp.: 40° C. Sample Temp.: 4° C. Detection: UV 210 nm Injection vol.: 5 μl Mobile Phase A: 20 mM KH2PO4 (Filt.) Mobile Phase B: ACN Flow rate: 0.5 ml/min Method: 20 mM KH2PO4_05_HSS Time (min) A (%) B (%) 0 96 4 1.9 96 4 2.0 60 40 2.2 60 40 2.3 96 4 3.0 96 4
(1) Preparation of Soluble Fraction
[0260] Microbial cells of Rhizobium radiobacter AJ3976 were spread on an LB agar medium and cultured at 30° C. for two days.
[0261] One loopful of the obtained microbial cells was inoculated to 160 mL of an enzyme production medium (10 g/L of yeast extract, 10 g/L of trypton, 1 g/L of glucose, 3 g/L of dipotassium hydrogen phosphate, 1 g/L of potassium dihydrogen phosphate, 0.1 g/L of magnesium sulfate heptahydrate, and 5 g/L of ammonium sulfate) in a 500 mL Sakaguchi flask, and cultured at 30° C. for 20 hours with shaking. The microbial cells were collected from about 1920 mL of the resulting cultured medium by centrifugation, washed with and suspended in 20 mM Tris-HCl (pH 7.6), and sonicated at 4° C. for 30 minutes. Microbial cell debris was removed from the sonicated cell suspension by the centrifugation, and the resulting supernatant was used as a soluble fraction.
(2) Anion Exchange Chromatography
[0262] The above soluble fraction was applied onto an anion exchange chromatographic column HiLoad 26/10 Q Sepharose HP (supplied from GE Healthcare Bioscience, CV=53 mL) equilibrated with 20 mM Tris-HCl (pH 7.6) and absorbed to the carrier. Proteins that had not been absorbed to the carrier (unabsorbed protein) were washed out with 20 mM Tris-HCl (pH 7.6). Subsequently, proteins that had been absorbed to the carrier were eluted by linearly changing a concentration of NaCl from 0 mM to 500 mM at a flow rate of 2 mL/minute. A 2S,4R Monatin-forming activity was measured in each eluted fraction, and the 2S,4R-Monatin-forming activity was detected in fractions corresponding to about 250 mM NaCl.
(3) Hydrophobic Chromatography
[0263] The fractions in which the 2S,4R-Monatin-forming activity had been detected were combined, and ammonium sulfate and Tris-HCl (pH 7.6) were added thereto so that the concentrations of ammonium sulfate and Tris-HCl (pH 7.6) were 1.0 M and 20 mM, respectively. The resulting solution was applied onto a hydrophobic chromatographic column HiLoad 16/10 Phenyl Sepharose HP (supplied from GE Healthcare Bioscience, CV=20 mL) equilibrated with 1.0 M ammonium sulfate and 20 mM Tris-HCl (pH 7.6), and absorbed to the carrier. Unabsorbed proteins that had not been absorbed to the carrier were washed out using 1.0 M ammonium sulfate and 20 mM Tris-HCl (pH 7.6). Subsequently, a 2S,4R-Monatin-forming enzyme was eluted by linearly changing the concentration of ammonium sulfate from 1.0 M to 0 M at a flow rate of 3 mL/minute. The 2S,4R-Monatin-forming activity was measured in each obtained fraction, and the 2S,4R-Monatin-forming activity was detected in fractions corresponding to about 0.9 M of ammonium sulfate.
(4) Gel Filtration Chromatography
[0264] The fractions in which the 2S,4R-Monatin-forming activity had been detected were combined, and concentrated using Amicon Ultra-15 10k (Millipore). The resulting concentrated solution was diluted with 20 mM Tris-HCl (pH 7.6) and 150 mM NaCl. The resulting solution was applied onto a gel filtration column HiLoad 16/60 Superdex 200 pg (supplied from GE Healthcare Bioscience, CV=120 mL) equilibrated with 20 mM Tris-HCl (pH 7.6) and 150 mM NaCl, and proteins were eluted at a flow rate of 1 mL/minute. This manipulation confirmed the 2S,4R-Monatin-forming activity at a position in which a molecular weight was estimated to be about 100 kDa.
(5) SDS-PAGE
[0265] The resulting fraction was subjected to SDS-PAGE, and a single band derived from the active fraction was detected near 47 kDa. This band was subjected to analysis of an N-terminal amino acid sequence as a candidate of the aminotransferase that forms 2S,4R-Monatin.
Example 26
Determination of N-Terminal Amino Acid Sequence of Aminotransferase Derived from Rhizobium radiobacter AJ3976
[0266] The purified enzyme solution obtained in Example 25 was subjected to the analysis of the N-terminal amino acid sequence, and the N-terminal amino acid sequence of AFLADILSRVKPSATIAVTQ (SEQ ID NO:44) was obtained. The N-terminal amino acid sequence showed a high homology to that of aspartate aminotransferase (AAK87940) derived from Agrobacterium tumefaciens str. C58.
Example 27
Cloning of Aminotransferase Gene Derived from Rhizobium radiobacter AJ3976
[0267] The microbial cells of Rhizobium radiobacter AJ3976 were cultured in the same manner as in Example 25. The microbial cells were collected from the cultured medium by centrifugation, and genomic DNA was extracted therefrom.
[0268] A DNA fragment including the aminotransferase gene was amplified by PCR using the obtained genomic DNA as a template. Primers were designed from DNA sequences of upstream 100 bp and downstream 100 bp of the aminotransferase gene with reference to the genomic DNA sequence of Agrobacterium tumefaciens str. C58. The primer Ag-u100-f (5'-ctggtgcagataagccggcttttgacc-3': SEQ ID NO:45) and the primer Ag-d100-r (5'-ccaccttcatcatgctgctgtttctcg-3': SEQ ID NO:46) were used. PCR was performed using KOD-plus-ver. 2 (Toyobo) under the following condition.
TABLE-US-00017 1 cycle at 94° C. for 2 minutes 25 cycles at 98° C. for 10 seconds 55° C. for 10 seconds and 68° C. for 60 seconds 1 cycle at 68° C. for 60 seconds, and 4° C.
[0269] The nucleotide sequence of the amplified DNA fragment of about 1400 bp was determined, and was shown to be the nucleotide sequence including 1203 bp of ORF (SEQ ID NOs: 47 and 48), which had the high homology to the aspartate aminotransferase gene (Atu2196) derived from Agrobacterium tumefaciens str. C58. The homology was 92% in their DNA sequences and 97% in their amino acid sequences.
[0270] This amino acid sequence was consistent with the N-terminal amino acid sequence obtained in Example 26. Thus, it has been thought that the aminotransferase gene having the 2S,4R-Monatin-forming activity could be acquired.
Example 28
Expression of Aminotransferase Derived from Rhizobium radiobacter AJ3976 in E. coli
[0271] (1) Construction of Expression Plasmid for Aminotransferase Derived from Rhizobium radiobacter AJ3976
[0272] A DNA fragment including the aminotransferase gene derived from Rhizobium radiobacter AJ3976 was amplified by PCR with the genomic DNA of Rhizobium radiobacter AJ3976 as the template. The primer 3976AT-Nde-f (5'-ggaattccatATGGCCTTCCTTGCCGACATTCTCT-3': SEQ ID NO:49) and the primer 3976-xho-r (5'-actccgctcgagACGGCAATCGGCGCAGAAACGCTGA-3': SEQ ID NO:50) were used. PCR was performed using KOD-plus-ver. 2 (Toyobo) under the following condition.
TABLE-US-00018 1 cycle at 94° C. for 2 minutes 25 cycles at 98° C. for 10 seconds 55° C. for 10 seconds and 68° C. for 60 seconds 1 cycle at 68° C. for 60 seconds, and 4° C.
[0273] The resulting DNA fragment was treated with restriction enzymes NdeI and XhoI, and ligated to pET-22b (Novagen) likewise treated with NdeI and XhoI. E. coli JM109 was transformed with this ligation solution, an objective plasmid was selected from ampicillin resistant colonies, and this plasmid was designated as pET-22-3976AT-His. In this plasmid, the aminotransferase derived from Rhizobium radiobacter AJ3976 which having a His-tag added to a C-terminus end (3976AT-His) is expressed.
(2) Purification of 3976AT-His from E. coli Strain Expressing 3976AT-His
[0274] The constructed expression plasmid pET-22-3976AT-His was introduced into E. coli BL21 (DE3), and one loopful of the transformant was inoculated to 160 mL of Overnight Express Instant TB Medium (Novagen) containing 100 mg/L of ampicillin in a 500 mL Sakaguchi flask, and the Sakaguchi flask was shaken at 37° C. for 16 hours. After completion of the cultivation, microbial cells were collected from about 1000 mL of the cultured medium by centrifugation, washed with and suspended in 20 mM Tris-HCl (pH 7.6), 100 mM NaCl and 20 mM imidazole, and sonicated at 4° C. for 30 minutes. Microbial cell debris was removed from the sonicated cell suspension by centrifugation, and the resulting supernatant was used as a soluble fraction.
[0275] The obtained soluble fraction was applied onto a His-tag protein purification column HisPrep FF 16/10 (supplied from Pharmacia (GE Healthcare Bioscience), CV=20 mL) equilibrated with 20 mM Tris-HCl (pH 7.6), 100 mM NaCl and 20 mM imidazole to absorb proteins to the carrier. Proteins that had not been absorbed to the carrier (unabsorbed protein) were washed out with 20 mM Tris-HCl (pH 7.6), 100 mM NaCl and 20 mM imidazole. Subsequently, the absorbed proteins were eluted by linearly changing the concentration of imidazole from 20 mM to 250 mM at a flow rate of 3 mL/minute.
[0276] The obtained fractions were combined and concentrated using Amicon Ultra-15 30k (Millipore). The concentrated solution was diluted with 20 mM Tris-HCl (pH 7.6), and then applied onto an anion exchange chromatographic column HiLoad 16/10 Q Sepharose HP (supplied from GE Healthcare Bioscience, CV=20 mL) equilibrated with 20 mM Tris-HCl (pH 7.6) to absorb proteins to the carrier. Proteins that had not been absorbed to the carrier (unabsorbed protein) were washed out with 20 mM Tris-HCl (pH 7.6). Subsequently, the proteins that had been absorbed to the carrier were eluted by linearly changing the concentration of NaCl from 0 mM to 500 mM at a flow rate of 3 mL/minute.
[0277] The 2S,4R Monatin-forming activity was measured in each eluted fraction, and the fractions in which the 2S,4R Monatin-forming activity had been detected were combined, and concentrated using Amicon Ultra-15 30k (Millipore).
[0278] The concentrated solution was diluted with 20 mM Tris-HCl (pH 7.6), and used as a 3976AT-His solution.
Example 29
Results of Measuring Specific Activity of AJ3976LAT for Various Keto Acids
(1) Measurement of Activity for L-Asp/α-KG, L-Asp/PA and L-Asp/(±)-MHOG by Colorimetric Method
[0279] The activity of AJ3976LAT for various substrates was measured. The specific activities for 10 mM various keto acids were measured by a colorimetric method, using 100 mM L-Asp as an amino donor substrate for a transamination reaction.
[0280] Activity for L-Asp/α-KG: 100 mM L-Asp-Na-laq, 10 mM α-KG-2Na, 50 μM PLP, 100 mM Tris-HCl (pH 8.0), 0.25 mM NADH and 2 U/mL of MDH at 25° C. The activity was calculated from the reduction of the absorbance measured at 340 nm. Malic dehydrogenase from porcine heart (Sigma) was used as MDH. The activity for L-Asp/α-KG is shown in the column "α-KG" of the aminotransferase activity in Table 13.
[0281] Activity for L-Asp/PA: 100 mM L-Asp-Na-laq, 10 mM PA-Na, 50 μM PLP, 100 mM Tris-HCl (pH 8.0), 0.25 mM NADH, and 2 U/mL of MDH (same as above) at 25° C. The activity was calculated from the reduction of the absorbance measured at 340 nm. The activity for L-Asp/PA is shown in the column "PA" of the aminotransferase activity in Table 13.
[0282] Activity for L-ASP/(±)-MHOG: 100 mM L-Asp-Na-laq, 10 mM (±)-MHOG, 50 μM PLP, 100 mM Tris-HCl (pH 8.0), 0.25 mM NADH, 2 U/mL of MDH (same as above), and 10 U/mL of LDH at 25° C. The activity was calculated from the reduction of the absorbance measured at 340 nm. D-Lactate dehydrogenase from Leuconostoc mesenteroides (Oriental Yeast) was used as LDH. LDH was added in order to remove PA in a trace amount existed in (±)-MHOG. The activity for L-Asp/(±)-MHOG is shown in the column "(±)-MHOG" of the aminotransferase activity in Table 13.
(2) Measurement of Activity for L-Asp/4R-IHOG, L-Asp/(±)-IHOG and L-Asp/IPA
[0283] The activity to form the 2S,4R-Monatin from 4R-IHOG, the activity to form the 2S,4R-Monatin and 2S,4S-Monatin from (±)-IHOG, which are objective activities, and the activity to form L-Trp as a by-product from IPA were measured individually. The transamination reaction to 10 mM keto acid was performed using 100 mM L-Asp as the amino donor substrate, and the amount of the formed amino acid was quantified by UPLC to calculate the specific activity.
[0284] Activity for L-Asp/4R-IHOG: 100 mM L-Asp-Na-laq, 10 mM 4R-IHOG (containing 4S-IHOG in a trace amount), 50 μM PLP, and 100 mM Tris-HCl (pH 8.0) at 25° C. The formed 2S,4R-Monatin and 2S,4S-Monatin were quantified by UPLC analysis. A solution of 200 mM sodium citrate (pH 4.5) was used as a solution for stopping the reaction. The activity for L-Asp/4R-IHOG is shown in the column "4R-IHOG" of the aminotransferase activity in Table 13.
[0285] Activity for L-Asp/(±)-IHOG: 100 mM L-Asp-Na-laq, 10 mM (±)-IHOG, 50 μM PLP, and 100 mM Tris-HCl (pH 8.0) at 25° C. The formed 2S,4R-Monatin and 2S,4S-Monatin were quantified by the UPLC analysis. A solution of 200 mM sodium citrate (pH 4.5) was used as a solution for stopping the reaction. The activity for L-Asp/4R-IHOG is shown in the column "(±)-IHOG" of the aminotransferase activity in Table 13.
[0286] Activity for L-Asp/IPA: 100 mM L-Asp-Na-laq, 10 mM IPA, 50 μM PLP, and 100 mM Tris-HCl (pH 8.0) (pH was adjusted to 8.0 with 1 N NaOH after preparing the reaction solution) at 25° C. Formed Trp was quantified by the UPCL analysis. A solution of 200 mM sodium citrate (pH 4.5) was used as a solution for stopping the reaction. The activity for L-Asp/IPA is shown in the column "IPA" of the aminotransferase activity in Table 13.
[0287] The formed Monatin and Trp were quantified using ACQUITY UPLC system supplied from Waters. The condition for the measurement is shown below. 0.2 mL of the reaction solution was reacted for 15 minutes, then the reaction was stopped. The reaction solution after stopping the reaction was centrifuged, and about 0.2 mL of the supernatant was subjected to the UPLC analysis.
TABLE-US-00019 TABLE 12-2 UPLC Column: ACQUITY UPLC HSS T3 2.1 × 50 mm Column Temp.: 40° C. Sample Temp.: 4° C. Detection: UV 210 nm Injection vol.: 5 μl Mobile Phase A: 20 mM KH2PO4 (Filt.) Mobile Phase B: ACN Flow rate: 0.5 ml/min Method: 20 mM KH2PO4_05_HSS Time (min) A (%) B (%) 0 96 4 1.9 96 4 2.0 60 40 2.2 60 40 2.3 96 4 3.0 96 4
[0288] The 2S,4R-Monatin, the 2S,4S-Monatin and Trp can be quantified distinctively at 1.1 minutes, 1.5 minutes and 1.3 minutes, respectively.
(3) Results of Measuring Specific Activity of AJ3976LAT for Various Keto Acids
[0289] The results of measuring the specific activity for 10 mM keto acid when 3976-AT-His was used and L-Asp was used as the amino donor are shown in Table 13.
TABLE-US-00020 TABLE 13 Specific activity of AJ3976LAT for various keto acids Aminotransferase activity (U/mg) 4R-IHOG ±IHOG α-KG PA ±MHOG SR SS SR SS IPA 106 4.0 48 0.58 1.5 0.052 3.7 0.012
Example 30
Reaction for Synthesis of 2S,4R-Monatin Using pET-22-3976AT-His/E. coli BL21 (DE3)
[0290] One loopful of microbial cells of pET-22-3976AT-His/E. coli BL21 (DE3) prepared in Example 28 was inoculated to 3 mL of Overnight Express Instant TB medium (Novagen) containing 100 mg/L of ampicillin in a test tube, and the test tube was then shaken at 37° C. for 16 hours. After the completion of the cultivation, the microbial cells were collected from 1 mL of the cultured medium by centrifugation, and suspended in 1 mL of BugBuster Master Mix (Novagen). The resulting suspension was incubated at room temperature for 15 minutes to lyse the microbial cells. Microbial cell debris was removed by centrifugation, and the resulting supernatant was used as a soluble fraction.
[0291] The reaction for the synthesis of the 2S,4R-Monatin from 4R-IHOG was carried out using the obtained soluble fraction. To 0.1 mL of a reaction solution [100 mM L-Asp-Na-laq, 10 mM 4R-IHOG (containing 4S-IHOG in a trace amount), 50 μM PLP, and 100 mM Tris-HCl (pH 8.0)], 0.05 mL of the above soluble fraction was added, and the mixture was reacted at 25° C. for one hour. After the completion of the reaction, the formed 2S,4R-Monatin was quantified to be 0.84 mM. The 2S,4R-Monatin was quantified by the UPLC analysis. The condition for the analysis is the same in Example 29.
Example 31
Purification of Aminotransferase Derived from Rhizobium sp. AJ12469
[0292] Aminotransferase that forms 2S,4R-Monatin was purified from the soluble fraction of Rhizobium sp. AJ12469 as follows. The synthetic reaction and quantification of 2S,4R-Monatin was carried out in the same manner as in Example 25.
(1) Preparation of Soluble Fraction
[0293] Microbial cells of Rhizobium sp. AJ12469 were spread on the LB agar medium, and cultured at 30° C. for two days.
[0294] One loopful of the resulting microbial cells was inoculated to 160 mL of an enzyme production medium (10 g/L of yeast extract, 10 g/L of trypton, 1 g/L of glucose, 3 g/L of dipotassium hydrogen phosphate, 1 g/L of potassium dihydrogen phosphate, 0.1 g/L of magnesium sulfate heptahydrate, and 5 g/L of ammonium sulfate) in a 500 mL Sakaguchi flask, and cultured at 30° C. for 16 hours with shaking. The microbial cells were collected from about 1920 mL of the resulting cultured medium by centrifugation, washed with and suspended in 20 mM Tris-HCl (pH 7.6), and sonicated at 4° C. for 30 minutes. The microbial cell debris was removed from the sonicated cell suspension by centrifugation, and the resulting supernatant was used as a soluble fraction.
(2) Anion Exchange Chromatography
[0295] The above soluble fraction was applied onto an anion exchange chromatographic column HiLoad 26/10 Q Sepharose HP (supplied from GE Healthcare Bioscience, CV=53 mL) equilibrated with 20 mM Tris-HCl (pH 7.6) to be absorbed to the carrier. Proteins that had not been absorbed to the carrier (unabsorbed protein) were washed out with 20 mM Tris-HCl (pH 7.6). Subsequently, proteins that had been absorbed to the carrier were eluted by linearly changing the concentration of NaCl from 0 mM to 500 mM at a flow rate of 8 mL/minute. The 2S,4R Monatin-forming activity was measured in each eluted fraction, and the 2S,4R-Monatin-forming activity was detected in fractions corresponding to about 200 mM NaCl.
(3) Hydrophobic Chromatography
[0296] The fractions in which the 2S,4R-Monatin-forming activity had been detected were combined, and ammonium sulfate and Tris-HCl (pH 7.6) were added thereto so that the concentrations of ammonium sulfate and Tris-HCl (pH 7.6) were 1.5 M and 20 mM, respectively. The resulting solution was applied onto the hydrophobic chromatographic column HiLoad 16/10 Phenyl Sepharose HP (supplied from GE Healthcare Bioscience, CV=20 mL) equilibrated with 1.5 M ammonium sulfate and 20 mM Tris-HCl (pH 7.6) to be absorbed to the carrier. Unabsorbed proteins that had not been absorbed to the carrier were washed out using 1.5 M ammonium sulfate and 20 mM Tris-HCl (pH 7.6). Subsequently, the 2S,4R-Monatin-forming enzyme was eluted by linearly changing the concentration of ammonium sulfate from 1.5 M to 0 M at a flow rate of 3 mL/minute. The 2S,4R-Monatin-forming activity was measured in obtained each fraction, and the 2S,4R-Monatin-forming activity was detected in fractions corresponding to about 0.8 M ammonium sulfate.
(4) Gel Filtration Chromatography
[0297] The fractions in which the 2S,4R-Monatin-forming activity had been detected were combined, and concentrated using Amicon Ultra-15 10k (Millipore). The resulting concentrated solution was diluted with 20 mM Tris-HCl (pH 7.6) and 150 mM NaCl. The resulting solution was applied onto a gel filtration column HiLoad 16/60 Superdex 200 pg (supplied from GE Healthcare Bioscience, CV=120 mL) equilibrated with 20 mM Tris-HCl (pH 7.6) and 150 mM NaCl, and proteins were eluted at a flow rate of 1 mL/minute. This manipulation confirmed the 2S,4R-Monatin-forming activity at a position at which the molecular weight was estimated to be about 100 kDa.
(5) Anion Exchange Chromatography
[0298] The fractions in which the 2S,4R-Monatin-forming activity had been detected were combined, and the resulting solution was applied onto an anion exchange chromatographic column Mono Q 5/5 (supplied from Pharmacia (GE Healthcare Bioscience, CV=1 mL) to absorb proteins to the carrier. Proteins that had not been absorbed to the carrier (unabsorbed proteins) were washed out with 20 mM Tris-HCl (pH 7.6). Subsequently, the absorbed proteins were eluted by linearly changing the concentration of NaCl from 0 mM to 500 mM at a flow rate of 0.5 mL/minute. The 2S,4R-Monatin-forming activity was measured in each fraction, and the 2S,4R-Monatin-forming activity was detected in the fractions corresponding to about 300 mM NaCl.
(6) SDS-PAGE
[0299] The obtained fractions were subjected to SDS-PAGE, and a band derived from the active fraction was detected near 47 kDa. This band was subjected to the analysis of the N-terminal amino acid sequence as the candidate for the aminotransferase that forms 2S,4R-Monatin.
Example 32
Determination of N-Terminal Amino Acid Sequence of Aminotransferase Derived from Rhizobium sp. AJ12469
[0300] The purified enzyme solution obtained in Example 31 was subjected to the analysis of the N-terminal amino acid sequence, and an N-terminal amino acid sequence of AFLADILSRVKPSATIAVTQ (SEQ ID NO:51) was obtained. The N-terminal amino acid sequence showed the high homology to aspartate aminotransferase (AAK87940) derived from Agrobacterium tumefaciens str. C58.
Example 33
Cloning of Aminotransferase Gene Derived from Rhizobium sp. AJ12469
[0301] The microbial cells of Rhizobium radiobacter AJ3976 were cultured in the same manner as in Example 31. The microbial cells were collected from the cultured medium by centrifugation, and genomic DNA was extracted therefrom.
[0302] A DNA fragment containing the aminotransferase gene was amplified by PCR with the obtained genomic DNA as the template. Primers were designed from DNA sequences of upstream 100 bp and downstream 100 bp of the aminotransferase gene with reference to the genomic DNA sequence of Agrobacterium tumefaciens str. C58. The primer Ag-u100-f (5'-ctggtgcagataagccggcttttgacc-3': SEQ ID NO:45) and the primer Ag-d100-r (5'-ccaccttcatcatgctgctgtttctcg-3': SEQ ID NO:46) were used. PCR was performed using KOD-plus-ver. 2 (Toyobo) under the following condition.
TABLE-US-00021 1 cycle at 94° C. for 2 minutes 25 cycles at 98° C. for 10 seconds 55° C. for 10 seconds and 68° C. for 60 seconds 1 cycle at 68° C. for 60 seconds, and 4° C.
[0303] A nucleotide sequence of the amplified DNA fragment of about 1400 bp was determined, and was shown to be the nucleotide sequence including 1203 bp of ORF (SEQ ID NOs:52 and 53), which had the high homology to the aspartate aminotransferase gene (Atu2196) derived from Agrobacterium tumefaciens str. C58. The homology was 97% in their DNA sequences and 99% in their amino acid sequences.
[0304] This amino acid sequence was consistent with the N-terminal amino acid sequence obtained in Example 32. Thus, it has been thought that the aminotransferase gene having the 2S,4R-Monatin-forming activity could be acquired.
Example 34
Expression of Aminotransferase Derived from Rhizobium sp. AJ12469 in E. coli
[0305] (1) Construction of Expression Plasmid for Aminotransferase Derived from Rhizobium sp. AJ12469
[0306] A DNA fragment containing an aminotransferase gene derived from Rhizobium sp. AJ12469 was amplified by PCR with the genomic DNA of Rhizobium sp. AJ12469 as the template. The primer 12469AT-Nde-f (5'-ggaattccatATGGCCTTCCTTGCCGACATTCTCT-3': SEQ ID NO:54) and the primer 12469-xho-r (5'-actccgctcgagGCGGCAATCGGCGCAGAAACGCTGA-3': SEQ ID NO:55) were used. PCR was performed using KOD-plus-ver. 2 (Toyobo) under the following condition.
TABLE-US-00022 1 cycle at 94° C. for 2 minutes 25 cycles at 98° C. for 10 seconds 55° C. for 10 seconds and 68° C. for 60 seconds 1 cycle at 68° C. for 60 seconds, and 4° C.
[0307] The resulting DNA fragment was treated with restriction enzymes NdeI and XhoI, and ligated to pET-22b (Novagen) likewise treated with NdeI and XhoI. E. coli JM109 was transformed with this ligation solution, an objective plasmid was selected from ampicillin resistant colonies, and this plasmid was designated as pET-22-12469AT-His. In this plasmid, the aminotransferase derived from Rhizobium sp. AJ12469 which has a His-tag added to the C-terminus end (12469AT-His) is expressed.
(2) Purification of 12469AT-His from E. coli Strain Expressing 12469AT-His
[0308] The constructed expression plasmid pET-22-12469AT-His was introduced into E. coli BL21 (DE3), and one loopful of the transformant was inoculated to 160 mL of Overnight Express Instant TB Medium (Novagen) containing 100 mg/L of ampicillin in a 500 mL Sakaguchi flask, and the Sakaguchi flask was shaken at 37° C. for 16 hours. After the completion of the cultivation, microbial cells were collected from about 1000 mL of the cultured medium by centrifugation, washed with and suspended in 20 mM Tris-HCl (pH 7.6), 100 mM NaCl and 20 mM imidazole, and sonicated at 4° C. for 30 minutes. The microbial cell debris was removed from the sonicated cell suspension by centrifugation, and the resulting supernatant was used as a soluble fraction.
[0309] The obtained soluble fraction was applied onto the His-tag protein purification column HisPrep FF 16/10 (supplied from Pharmacia (GE Healthcare Bioscience), CV=20 mL) equilibrated with 20 mM Tris-HCl (pH 7.6), 100 mM NaCl and 20 mM imidazole to absorb proteins to the carrier. Proteins that had not been absorbed to the carrier (unabsorbed protein) were washed out with 20 mM Tris-HCl (pH 7.6), 100 mM NaCl and 20 mM imidazole. Subsequently, absorbed proteins were eluted by linearly changing the concentration of imidazole from 20 mM to 250 mM at a flow rate of 3 mL/minute.
[0310] The obtained fractions were combined and concentrated using Amicon Ultra-15 30k (Millipore). The concentrated solution was diluted with 20 mM Tris-HCl (pH 7.6), and then applied onto the anion exchange chromatographic column HiLoad 16/10 Q Sepharose HP (supplied from GE Healthcare Bioscience, CV=20 mL) equilibrated with 20 mM Tris-HCl (pH 7.6) to absorb proteins to the carrier. Proteins that had not been absorbed to the carrier (unabsorbed protein) were washed out with 20 mM Tris-HCl (pH 7.6). Subsequently, absorbed proteins were eluted by linearly changing the concentration of NaCl from 0 mM to 500 mM at a flow rate of 3 mL/minute.
[0311] The 2S,4R-Monatin-forming activity was measured in each eluted fraction. The fractions in which the 2S,4R-Monatin-forming activity had been detected were combined, and concentrated using Amicon Ultra-15 30k (Millipore). The concentrated solution was diluted with 20 mM Tris-HCl (pH 7.6) to use as a 12469AT-His solution.
Example 35
Results of Measuring Specific Activity of AJ12469LAT for Various Keto Acids
(1) Measurement of Activity for L-Asp/α-KG, L-Asp/PA and L-Asp/(±)-MHOG by Colorimetric Method
[0312] The activity of AJ12469LAT for various substrates was measured. The specific activity for 10 mM keto acid was measured by the colorimetric method, using 100 mM L-Asp as the amino donor substrate for the transamination reaction.
[0313] Activity for L-Asp/α-KG: 100 mM L-Asp-Na-laq, 10 mM α-KG-2Na, 50 μM PLP, 100 mM Tris-HCl (pH 8.0), 0.25 mM NADH and 2 U/mL of MDH at 25° C. The activity was calculated from the reduction of the absorbance measured at 340 nm. Malic dehydrogenase from porcine heart (Sigma) was used as MDH. The activity for L-Asp/α-KG is shown in the column "α-KG" of the aminotransferase activity in Table 15.
[0314] Activity for L-Asp/PA: 100 mM L-Asp-Na-laq, 10 mM PA-Na, 50 μM PLP, 100 mM Tris-HCl (pH 8.0), 0.25 mM NADH, and 2 U/mL of MDH (same as above) at 25° C. The activity was calculated from the reduction of the absorbance measured at 340 nm. The activity for L-Asp/PA is shown in the column "PA" of the aminotransferase activity in Table 15.
[0315] Activity for L-ASP/(±)-MHOG: 100 mM L-Asp-Na-laq, 10 mM (±)-MHOG, 50 μM PLP, 100 mM Tris-HCl (pH 8.0), 0.25 mM NADH, 2 U/mL of MDH (same as above), and 10 U/mL of LDH at 25° C. The activity was calculated from the reduction of the absorbance measured at 340 nm. D-Lactate dehydrogenase from Leuconostoc mesenteroides (Oriental Yeast) was used as LDH. LDH was added in order to remove PA in a trace amount contaminated in (±)-MHOG. The activity for L-Asp/(±)-MHOG is shown in the column "(±)-MHOG" of the aminotransferase activity in Table 15.
(2) Measurement of Activity for L-Asp/4R-IHOG, L-Asp/(±)-IHOG and L-Asp/IPA
[0316] The activity to form 2S,4R-Monatin from 4R-IHOG, the activity to form 2S,4R-Monatin and 2S,4S-Monatin from (±)-IHOG, which are the objective activities, and the activity to form L-Trp as a by-product from IPA were measured individually. The transamination reaction to 10 mM keto acid was performed using 100 mM L-Asp as the amino donor substrate for the transamination reaction, and the amount of the formed amino acid was quantified by UPLC to calculate the specific activity.
[0317] Activity for L-Asp/4R-IHOG: 100 mM L-Asp-Na-laq, 10 mM 4R-IHOG (containing 4S-IHOG in a trace amount), 50 μM PLP, and 100 mM Tris-HCl (pH 8.0) at 25° C. Formed 2S,4R-Monatin and 2S,4S-Monatin were quantified by the UPLC analysis. A solution of 200 mM sodium citrate (pH 4.5) was used as a solution for stopping the reaction. The activity for L-Asp/4R-IHOG is shown in the column "4R-IHOG" of the aminotransferase activity in Table 15.
[0318] Activity for L-Asp/(±)-IHOG: 100 mM L-Asp-Na-laq, 10 mM (±)-IHOG, 50 μM PLP, and 100 mM Tris-HCl (pH 8.0) at 25° C. The formed 2S,4R-Monatin and 2S,4S-Monatin were quantified by the UPLC analysis. A solution of 200 mM sodium citrate (pH 4.5) was used as a solution for stopping the reaction. The activity for L-Asp/(±)-IHOG is shown in the column "(±)-IHOG" of the aminotransferase activity in Table 13.
[0319] Activity for L-Asp/IPA: 100 mM L-Asp-Na-laq, 10 mM IPA, 50 μM PLP, and 100 mM Tris-HCl (pH 8.0) (pH was adjusted to 8.0 with 1 N NaOH after preparing the reaction solution) at 25° C. Formed Trp was quantified by the UPCL analysis. A solution of 200 mM sodium citrate (pH 4.5) was used as a solution for stopping the reaction. The activity for L-Asp/IPA is shown in the column "IPA" of the aminotransferase activity in Table 15.
[0320] The formed Monatin and Trp were quantified using ACQUITY UPLC system supplied from Waters. The condition for the measurement is shown below. 0.2 mL of the reaction solution was reacted for 15 minutes, then the reaction was stopped. The reaction solution after stopping the reaction was centrifuged, and about 0.2 mL of the supernatant was subjected to the UPLC analysis.
TABLE-US-00023 TABLE 14 UPLC Column: ACQUITY UPLC HSS T3 2.1 × 50 mm Column Temp.: 40° C. Sample Temp.: 4° C. Detection: UV 210 nm Injection vol.: 5 μl Mobile Phase A: 20 mM KH2PO4 (Filt.) Mobile Phase B: ACN Flow rate: 0.5 ml/min Method: 20 mM KH2PO4_05_HSS Time (min) A (%) B (%) 0 96 4 1.9 96 4 2.0 60 40 2.2 60 40 2.3 96 4 3.0 96 4
[0321] The 2S,4R-Monatin, the 2S,4S-Monatin and Trp can be quantified distinctively at 1.1 minutes, 1.5 minutes and 1.3 minutes, respectively.
(3) Results of Measuring Specific Activity of AJ12469LAT for Various Keto Acids
[0322] The results of measuring the specific activity for 10 mM keto acid when 12469-AT-His was used and 100 mM L-Asp was used as the amino donor are shown in Table 15.
TABLE-US-00024 TABLE 15 Specific activity of AJ12469LAT for various keto acids Aminotransferase acitivity (U/mg) 4R-IHOG ±IHOG α-KG PA ±MHOG SR SS SR SS IPA 96 4.8 44 0.56 1.6 0.066 3.3 0.016
Example 36
Reaction for Synthesis of 2S,4R-Monatin Using pET-22-12469AT-His/E. coli BL21 (DE3)
[0323] One loopful of microbial cells of pET-22-12469AT-His/E. coli BL21 (DE3) prepared in Example 34 was inoculated to 3 mL of Overnight Express Instant TB medium (Novagen) containing 100 mg/L of ampicillin in a test tube, and the test tube was then shaken at 37° C. for 16 hours. After the completion of the cultivation, the microbial cells were collected from 1 mL of the cultured medium by centrifugation, and suspended in 1 mL of BugBuster Master Mix (Novagen). The resulting suspension was incubated at room temperature for 15 minutes to lyse the microbial cells. The microbial cell debris was removed by centrifugation, and the resulting supernatant was used as a soluble fraction.
[0324] The reaction for the synthesis of the 2S,4R-Monatin from 4R-IHOG was carried out using the obtained soluble fraction. To 0.1 mL of a reaction solution [100 mM L-Asp-Na-lag, 10 mM 4R-IHOG (containing 4S-IHOG in a trace amount), 50 μM PLP, and 100 mM Tris-HCl (pH 8.0)], 0.05 mL of the above soluble fraction was added, and the mixture was reacted at 25° C. for one hour. After the completion of the reaction, the amount of the formed 2S,4R-Monatin was quantified to be 0.87 mM. The 2S,4R-Monatin was quantified by the UPLC analysis. The condition for the analysis is the same as that in Example 29.
Example 37
Purification of Aminotransferase Derived from Corynebacterium ammoniagenes AJ1444
[0325] Aminotransferase that formed the 2S,4R-Monatin was purified from a soluble fraction from Corynebacterium ammoniagenes AJ1444 as follows. The synthetic reaction and quantification of the 2S,4R-Monatin were carried out in the same manner as in Example 25.
(1) Preparation of Soluble Fraction
[0326] Microbial cells of Corynebacterium ammonia genes AJ1444 were spread on the LB agar medium and cultured at 30° C. for two days.
[0327] One loopful of the obtained microbial cells was inoculated to 160 mL of the enzyme production medium (10 g/L of yeast extract, 10 g/L of trypton, 1 g/L of glucose, 3 g/L of dipotassium hydrogen phosphate, 1 g/L of potassium dihydrogen phosphate, 0.1 g/L of magnesium sulfate heptahydrate, and 5 g/L of ammonium sulfate) in a 500 mL Sakaguchi flask, and cultured at 30° C. for 16 hours with shaking. The microbial cells were collected from about 1760 mL of the cultured medium by centrifugation, washed with and suspended in 20 mM Tris-HCl (pH 7.6), and disrupted by adding glass beads and using a multibead shocker (Yasui Kikai Corporation). The microbial cell debris was removed from the disrupted cell suspension by centrifugation, and the resulting supernatant was used as a soluble fraction.
(2) Ammonium Sulfate Precipitation
[0328] Ammonium sulfate was added to the above soluble fraction so that a final concentration of ammonium sulfate was 90% (w/w), and an ammonium sulfate precipitate was obtained by centrifugation.
(3) Hydrophobic Chromatography
[0329] The above ammonium sulfate precipitate was dissolved in 1.0 M ammonium sulfate and 20 mM Tris-HCl (pH 7.6). This solution was applied onto the hydrophobic chromatographic column HiLoad 26/10 Phenyl Sepharose HP (supplied from GE Healthcare Bioscience, CV=53 mL) equilibrated with 1.0 M ammonium sulfate and 20 mM Tris-HCl (pH 7.6) to absorb proteins to the carrier. Unabsorbed proteins that had not been absorbed to the carrier were washed out with 1.0 M ammonium sulfate and 20 mM Tris-HCl (pH 7.6). Subsequently, the 2S,4R-Monatin-forming enzyme was eluted by linearly changing the concentration of ammonium sulfate from 1.0 M to 0 M at a flow rate of 3 mL/minute. The 2S,4R-Monatin-forming activity was measured in each eluted fraction, and detected in fractions corresponding to about 0.2 M ammonium sulfate.
(4) Anion Exchange Chromatography
[0330] The fractions in which the 2S,4R-Monatin-forming activity had been detected were combined, and dialyzed against 20 mM Tris-HCl (pH 7.6) overnight. The resulting solution was applied onto the anion exchange chromatographic column HiLoad 16/10 Q Sepharose HP (supplied from GE Healthcare Bioscience, CV=20 mL) equilibrated with 20 mM Tris-HCl (pH 7.6) to absorb proteins to the carrier. Proteins that had not been absorbed to the carrier (unabsorbed proteins) were washed out with 20 mM Tris-HCl (pH 7.6). Subsequently, absorbed proteins were eluted by linearly changing the concentration of NaCl from 0 mM to 500 mM at a flow rate of 2.25 mL/minute. The 2S,4R-Monatin-forming activity was measured in each eluted fraction, and detected in the fractions corresponding to about 400 mM NaCl.
(5) Gel Filtration Chromatography
[0331] The fractions in which the 2S,4R-Monatin-forming activity had been detected were combined, and concentrated using Amicon Ultra-15 10k (Millipore). The resulting concentrated solution was diluted with 20 mM Tris-HCl (pH 7.6) and 150 mM NaCl. The resulting solution was applied onto the gel filtration column HiLoad 16/60 Superdex 200 pg (supplied from GE Healthcare Bioscience, CV=120 mL) equilibrated with 20 mM Tris-HCl (pH 7.6) and 150 mM NaCl, and proteins were eluted at a flow rate of 1.2 mL/minute. This manipulation confirmed the 2S,4R-Monatin-forming activity at a position at which the molecular weight was estimated to be about 85 kDa.
(6) Anion Exchange Chromatography
[0332] The fractions in which the 2S,4R-Monatin-forming activity had been detected were combined, and the resulting solution was applied onto the anion exchange chromatographic column Mono Q 5/5 (supplied from Pharmacia (GE Healthcare Bioscience), CV=1 mL) to absorb proteins to the carrier. Proteins that had not been absorbed to the carrier (unabsorbed proteins) were washed out with 20 mM Tris-HCl (pH 7.6). Subsequently, absorbed proteins were eluted by linearly changing the concentration of NaCl from 0 mM to 500 mM at a flow rate of 1 mL/minute. The 2S,4R-Monatin-forming activity was measured in each fraction, and the 2S,4R-Monatin-forming activity was detected in the fractions corresponding to about 400 mM NaCl.
(7) SDS-PAGE
[0333] The obtained fractions were subjected to SDS-PAGE, and a band derived from the active fraction was detected near 43 kDa. This band was subjected to the analysis of the N-terminal amino acid sequence as the candidate for the aminotransferase that forms the 2S,4R-Monatin.
Example 38
Determination of N-Terminal Amino Acid Sequence of Aminotransferase Derived from Corynebacterium ammoniagenes AJ1444
[0334] The purified enzyme solution obtained in Example 37 was subjected to the analysis of the N-terminal amino acid sequence, and the N-terminal amino acid sequence of MSXIAQXILDQ (SEQ ID NO:112) was obtained. This N-terminal amino acid sequence showed the high homology to aspartate aminotransferase (ZP--03935516) derived from Corynebacterium striatum ATCC6940 and aspartate aminotransferase (ZP--06838515) derived from Corynebacterium ammoniagenes DSM20306.
Example 39
Cloning of Aminotransferase Gene Derived from Corynebacterium ammoniagenes AJ1444
[0335] Microbial cells of Corynebacterium ammoniagenes AJ1444 were cultured in the same manner as in Example 37. The microbial cells were collected from the resulting cultured medium by centrifugation, and genomic DNA was extracted therefrom.
[0336] A DNA fragment including the aminotransferase gene was amplified by PCR with the obtained genomic DNA as the template. The primer Co-d50-r (5'-cttccttggaacaagtcgaggaagac-3': SEQ ID NO:56) designed from the DNA sequence of downstream 50 bp of the aminotransferase gene with reference to the genomic DNA sequence of Corynebacterium ammoniagenes DSM20306, and the primer Co-800-f (5'-gctatcgcacaattccaccgcacctt-3': SEQ ID NO:57) designed with reference to partial sequences that had the high homology between the aspartate aminotransferase (ZP--03935516) derived from Corynebacterium striatum ATCC6940 and the aspartate aminotransferase (ZP--06838515) derived from Corynebacterium ammoniagenes DSM20306 were used. PCR was performed using KOD-plus-ver. 2 (Toyobo) under the following condition.
TABLE-US-00025 1 cycle at 94° C. for 2 minutes 25 cycles at 98° C. for 10 seconds 55° C. for 10 seconds and 68° C. for 60 seconds 1 cycle at 68° C. for 60 seconds, and 4° C.
[0337] A nucleotide sequence of about 400 bp of the amplified DNA fragment was determined, and the primer Co-890-r (5'-acatcgttaagcaagcgaaccaccag-3': SEQ ID NO:58) and the primer Co-1060-r (5'-gaaagacaagcgaatgtggtgctcg-3': SEQ ID NO:59 were designed based on that nucleotide sequence. PCR was performed using LA PCR in vitro Cloning Kit (Takara). PCR was performed using KOD-plus-ver. 2 (Toyobo) under the following condition.
TABLE-US-00026 1 cycle at 94° C. for 2 minutes 25 cycles at 98° C. for 10 seconds 55° C. for 10 seconds and 68° C. for 60 seconds 1 cycle at 68° C. for 60 seconds, and 4° C.
[0338] As a result, the nucleotide sequence including 1134 bp of ORF (SEQ ID NOs: 60 and 61), which has the high homology to the aspartate aminotransferase gene (HMPREF0281--02480) derived from Corynebacterium ammoniagenes DSM20306 was determined. The homology was 76% in their DNA sequences and 82% in their amino acid sequences.
[0339] This amino acid sequence was consistent with the N-terminal amino acid sequence obtained in Example 38. Thus, it has been thought that the aminotransferase gene having the 2S,4R-Monatin-forming activity could be acquired.
Example 40
Expression of Aminotransferase Derived from Corynebacterium ammoniagenes AJ1444 in E. coli
[0340] (1) Construction of Expression Vector for Aminotransferase Derived from Corynebacterium ammoniagenes AJ1444
[0341] A DNA fragment including the aminotransferase gene derived from Corynebacterium ammoniagenes AJ1444 was amplified by PCR with the genomic DNA of Corynebacterium ammoniagenes AJ1444 as the template. The primer 1444AT-Nde-f (5'-ggaattccatATGAGCCACATCGCTCAACGCATCC-3': SEQ ID NO:62) and a primer 1444-xho-r (5'-actccgctcgagGGACTTTTCGAAGTATTGGCGAATG-3': SEQ ID NO:63) were used. PCR was performed using KOD-plus-ver. 2 (Toyobo) under the following condition.
TABLE-US-00027 1 cycle at 94° C. for 2 minutes 25 cycles at 98° C. for 10 seconds 55° C. for 10 seconds and 68° C. for 60 seconds 1 cycle at 68° C. for 60 seconds, and 4° C.
[0342] The resulting DNA fragment was treated with the restriction enzymes NdeI and XhoI, and ligated to pET-22b (Novagen) likewise treated with NdeI and XhoI. E. coli JM109 was transformed with this ligation solution, an objective plasmid was selected from ampicillin resistant E. coli colonies, and this plasmid was designated as pET-22-1444AT-His. In this plasmid, the aminotransferase derived from Corynebacterium ammoniagenes AJ1444 which has the His-tag added to the C-terminus end (1444AT-His) is expressed.
(2) Purification of 1444AT-His from E. coli Strain Expressing 1444AT-His
[0343] The constructed expression plasmid pET-22-1444AT-His was introduced into E. coli BL21 (DE3), and one loopful of the transformant was inoculated to 160 mL of Overnight Express Instant TB Medium (Novagen) containing 100 mg/L of ampicillin in a 500 mL Sakaguchi flask, and the Sakaguchi flask was shaken at 37° C. for 16 hours. After completion of the cultivation, microbial cells were collected from about 1000 mL of the cultured medium by centrifugation, washed with and suspended in 20 mM Tris-HCl (pH 7.6), 300 mM NaCl and 10 mM imidazole, and sonicated at 4° C. for 30 minutes. The microbial cell debris was removed from the sonicated cell suspension by centrifugation, and the resulting supernatant was used as a soluble fraction.
[0344] The obtained soluble fraction was applied onto a His-tag protein purification column His TALON superflow 5 mL Centrifuge (Clontech) equilibrated with 20 mM Tris-HCl (pH 7.6), 300 mM NaCl and 10 mM imidazole to absorb proteins to the carrier. Proteins that had not been absorbed to the carrier (unabsorbed protein) were washed out with 20 mM Tris-HCl (pH 7.6), 300 mM NaCl and 10 mM imidazole. Subsequently, absorbed proteins were eluted using 20 mM Tris-HCl (pH 7.6), 300 mM NaCl and 150 mM imidazole at a flow rate of 5 mL/minute.
[0345] The obtained fractions were combined and concentrated using Amicon Ultra-15 30k (Millipore). The concentrated solution was diluted with 20 mM Tris-HCl (pH 7.6), and then applied onto the anion exchange chromatographic column HiLoad 16/10 Q Sepharose HP (supplied from GE Healthcare Bioscience, CV=20 mL) equilibrated with 20 mM Tris-HCl (pH 7.6) to absorb proteins to the carrier. Proteins that had not been absorbed to the carrier (unabsorbed protein) were washed out with 20 mM Tris-HCl (pH 7.6). Subsequently, unabsorbed proteins were eluted by linearly changing the concentration of NaCl from 0 mM to 500 mM at a flow rate of 3 mL/minute.
[0346] The 2S,4R-Monatin-forming activity was measured in each eluted fraction. The fractions in which the 2S,4R-Monatin-forming activity had been detected were combined, and concentrated using Amicon Ultra-15 30k (Millipore). The concentrated solution was diluted with 20 mM Tris-HCl (pH 7.6) to use as a 1444AT-His solution.
Example 41
Results of Measuring Specific Activity of AJ1444LAT for Various Keto Acids
(1) Measurement of Activity for L-Asp/α-KG, L-Asp/PA, L-Asp/(±)-MHOG, L-Glu/PA and L-Glu/(±)-MHOG by Colorimetric Method
[0347] The activity of AJ1444LAT for various substrates was measured. The specific activity for 10 mM keto acid was measured by colorimetric method, using 100 mM L-Asp or L-Glu as the amino donor substrate for the transamination reaction.
[0348] Activity for L-Asp/α-KG: 100 mM L-Asp-Na-laq, 10 mM α-KG-2Na, 50 μM PLP, 100 mM Tris-HCl (pH 8.0), 0.25 mM NADH and 2 U/mL of MDH at 25° C. The activity was calculated from the reduction of the absorbance measured at 340 nm. Malic dehydrogenase from porcine heart (Sigma) was used as MDH. The activity for L-Asp/α-KG is shown in the column "α-KG" of the aminotransferase activity in Table 17.
[0349] Activity for L-Asp/PA: 100 mM L-Asp-Na-laq, 10 mM PA-Na, 50 μM PLP, 100 mM Tris-HCl (pH 8.0), 0.25 mM NADH, and 2 U/mL of MDH (same as above) at 25° C. The activity was calculated from the reduction of the absorbance measured at 340 nm. The activity for L-Asp/PA is shown in the column "PA" of the aminotransferase activity in Table 17.
[0350] Activity for L-Asp/(±)-MHOG: 100 mM L-Asp-Na-laq, 10 mM (±)-MHOG, 50 μM PLP, 100 mM Tris-HCl (pH 8.0), 0.25 mM NADH, 2 U/mL of MDH (same as above), and 10 U/mL of LDH at 25° C. The activity was calculated from the reduction of the absorbance measured at 340 nm. D-Lactate dehydrogenase from Leuconostoc mesenteroides (Oriental Yeast) was used as LDH. LDH was added in order to remove PA in a trace amount contaminated in (±)-MHOG. The activity for L-Asp/(±)-MHOG is shown in the column "(±)-MHOG" of the aminotransferase activity in Table 17.
[0351] Activity for L-Glu/PA: 100 mM L-Glu-Na, 10 mM PA, 50 μM PLP, 100 mM Tris-HCl (pH 8.0), 100 mM NH4Cl, 0.25 mM NADH and 10 U/mL of GDH at 25° C. The activity was calculated from the reduction of the absorbance measured at 340 nm. L-Glutamic dehydrogenase from bovine liver (Sigma) was used as GDH. The activity for L-Glu/PA is shown in the column "PA" of the aminotransferase activity in Table 17.
[0352] Activity for L-Glu/(±)-MHOG: 100 mM L-Glu-Na, 10 mM (±)-MHOG, 50 μM PLP, 100 mM Tris-HCl (pH 8.0), 100 mM NH4Cl, 0.25 mM NADH, and 10 U/mL of GDH at 25° C. The activity was calculated from the reduction of the absorbance measured at 340 nm. The activity for L-Glu/(±)-MHOG is shown in the column "(±)-MHOG" of the aminotransferase activity in Table 17.
(2) Measurement of Activity for L-Asp/4R-IHOG, L-Asp/(±)-IHOG, L-Asp/IPA, L-Glu/4R-IHOG and L-Glu/IPA
[0353] The activity to form 2S,4R-Monatin from 4R-IHOG, the activity to form 2S,4R-Monatin and 2S,4S-Monatin from (±)-IHOG, which are the objective activities, and the activity to form L-Trp as the by-product from IPA were measured individually. The transamination reaction to 10 mM keto acid was performed using 100 mM L-Asp or L-Glu as the amino donor substrate of the transamination reaction, and the amount of the formed amino acid was quantified by UPLC to calculate the specific activity.
[0354] Activity for L-Asp/4R-IHOG: 100 mM L-Asp-Na-laq, 10 mM 4R-IHOG (containing 4S-IHOG in a trace amount), 50 μM PLP, and 100 mM Tris-HCl (pH 8.0) at 25° C. The formed 2S,4R-Monatin and 2S,4S-Monatin were quantified by UPLC analysis. A solution of 200 mM sodium citrate (pH 4.5) was used as a solution for stopping the reaction. The activity for L-Asp/4R-IHOG is shown in the column "4R-IHOG" of the aminotransferase activity in Table 17.
[0355] Activity for L-Asp/(±)-IHOG: 100 mM L-Asp-Na-laq, 10 mM (±)-IHOG, 50 μM PLP, and 100 mM Tris-HCl (pH 8.0) at 25° C. The formed 2S,4R-Monatin and 2S,4S-Monatin were quantified by UPLC analysis. A solution of 200 mM sodium citrate (pH 4.5) was used as a solution for stopping the reaction. The activity for L-Asp/(±)-IHOG is shown in the column "(±)-IHOG" of the aminotransferase activity in Table 17.
[0356] Activity for L-Asp/IPA: 100 mM L-Asp-Na-laq, 10 mM IPA, 50 μM PLP, and 100 mM Tris-HCl (pH 8.0) (pH was adjusted to 8.0 with 1 N NaOH after preparing the reaction solution) at 25° C. The formed Trp was quantified by UPCL analysis. A solution of 200 mM sodium citrate (pH 4.5) was used as a solution for stopping the reaction. The activity for L-Asp/IPA is shown in the column "IPA" of the aminotransferase activity in Table 17.
[0357] Activity for L-Glu/4R-IHOG: 100 mM L-Glu-Na, 10 mM 4R-IHOG (containing 4S-IHOG in a trace amount), 50 μM PLP, and 100 mM Tris-HCl (pH 8.0) at 25° C. The formed 2S,4R-Monatin and 2S,4S-Monatin were quantified by UPLC analysis. A solution of 200 mM sodium citrate (pH 4.5) was used as a solution for stopping the reaction. The activity for L-Glu/4R-IHOG is shown in the column "4R-IHOG" of the aminotransferase activity in Table 17.
[0358] Activity for L-Glu/IPA: 100 mM L-Glu-Na, 10 mM IPA, 50 PLP, and 100 mM Tris-HCl (pH 8.0) (pH was adjusted to 8.0 with 1 N NaOH after preparing the reaction solution) at 25° C. The formed Trp was quantified by UPCL analysis. A solution of 200 mM sodium citrate (pH 4.5) was used as a solution for stopping the reaction. The activity for L-Glu/IPA is shown in the column "IPA" of the aminotransferase activity in Table 17.
[0359] The formed Monatin and Trp were quantified using ACQUITY UPLC system supplied from Waters. The condition for the measurement is shown below. 0.2 mL of the reaction solution was reacted for 15 minutes, then the reaction was stopped. The reaction solution after stopping the reaction was centrifuged, and about 0.2 mL of the supernatant was subjected to UPLC analysis.
TABLE-US-00028 TABLE 16 UPLC Column: ACQUITY UPLC HSS T3 2.1 × 50 mm Column Temp.: 40° C. Sample Temp.: 4° C. Detection: UV 210 nm Injection vol.: 5 μl Mobile Phase A: 20 mM KH2PO4 (Filt.) Mobile Phase B: ACN Flow rate: 0.5 ml/min Method: 20 mM KH2PO4_05_HSS Time (min) A (%) B (%) 0 96 4 1.9 96 4 2.0 60 40 2.2 60 40 2.3 96 4 3.0 96 4
[0360] The 2S,4R-Monatin, 2S,4S-Monatin and Trp can be quantified distinctively at 1.1 minutes, 1.5 minutes and 1.3 minutes, respectively.
(3) Results of Measuring Specific Activity of AJ1444LAT for Various Keto Acids
[0361] The results of measuring the specific activity for 10 mM keto acid when 1444-AT-His was used and L-Asp was used as the amino donor are shown in Table 17.
TABLE-US-00029 TABLE 17 Specific activity of AJ1444LAT for various keto acids Aminotransferase activity (U/mg) 4R-IHOG ±IHOG α-KG PA ±MHOG SR SS SR SS IPA L-Asp 4.0 1.7 2.7 2.4 0.26 0.91 1.7 0.085 L-Glu -- 8.7 145 200 15 -- -- 0.21
Example 42
Reaction for Synthesis of 2S,4R-Monatin Using pET-22-1444AT-His/E. coli BL21 (DE3)
[0362] One loopful of microbial cells of pET-22-1444AT-His/E. coli BL21 (DE3) prepared in Example 40 was inoculated to 3 mL of Overnight Express Instant TB medium (Novagen) containing 100 mg/L of ampicillin in a test tube, and the test tube was then shaken at 37° C. for 16 hours. After the completion of the cultivation, the microbial cells were collected from 1 mL of the cultured medium by centrifugation, and suspended in 1 mL of BugBuster Master Mix (Novagen). The resulting suspension was left stand at room temperature for 15 minutes to lyse the microbial cells. The microbial cell debris was removed by centrifugation, and the resulting supernatant was used as a soluble fraction.
[0363] The reaction for the synthesis of 2S,4R-Monatin from 4R-IHOG was carried out using the obtained soluble fraction. To 0.1 mL of the reaction solution [100 mM L-Asp-Na-laq, 10 mM 4R-IHOG (containing 4S-IHOG in a trace amount), 50 μM PLP, and 100 mM Tris-HCl (pH 8.0)], 0.05 mL of the above soluble fraction was added, and the mixture was reacted at 25° C. for one hour. After the completion of the reaction, the amount of the formed 2S,4R-Monatin was quantified to be 0.13 mM. The 2S,4R-Monatin was quantified by the UPLC analysis. The condition for the analysis is the same as that in Example 29.
Example 43
One-Pot Reaction for Synthesis of 2S,4R-Monatin from 20 mM L-Trp (AJ3976LAT, AJ12469LAT, AJ1444LAT)
[0364] A reaction was performed under the following condition for 12 hours using purified 3976AT-His, 12469AT-His and 1444AT-His. The reaction was performed in 1 mL using a test tube. The reaction solution was appropriately sampled, the sample was diluted with TE buffer, ultrafiltrated using an Amicon Ultra-0.5 mL centrifugal filter 10 kDa (Millipore), and the resulting filtrate was analyzed. HPLC and capillary electrophoresis were used for the analysis.
[0365] Reaction condition: 20 mM L-Trp, 40 mM PA-Na, 160 mM L-Asp-Na-laq, 1 mM MgCl2, 50 μM PLP, 100 mM Tris-HCl, 20 mM KPB (pH 7.0), 20% Ps_aad broth, 30 U/mL of purified SpAld enzyme, 10 U/mL of commercially available OAA DCase enzyme, 2 U/mL of purified LAT enzyme (vs 10 mM 4R-IHOG), and 200 U/mL of commercially available SOD enzyme at 25° C. at 120 rpm.
[0366] The methods for preparing the enzymes subjected to the reaction are shown below.
[0367] Ps_aad broth: it was prepared according to the method described in Example 17.
[0368] Purified SpAld enzyme: it was prepared according to the method described in Example 19.
[0369] AJ3976LAT, AJ12469LAT and AJ1444LAT: they are prepared according to the methods described in Examples 28, 34 and 40.
[0370] OAA DCase: oxaloacetate decarboxylase from Pseudomonas sp. (Sigma) was used. A value described by the manufacturer was used as an enzyme amount (U).
[0371] SOD: superoxide dismutase from bovine liver (Sigma) was used. A value described by the manufacturer was used as an enzyme amount (U).
[0372] As a result of the one-pot reactions, 12 mM, 11 mM and 13 mM 2S,4R-Monatin were formed after 4 hours using AJ3976LAT, AJ12469LAT and AJ1444LAT, respectively, and their yields from L-Trp were 58%, 53% and 64%, respectively.
Example 44
One-Pot Reaction for Synthesis of 2S,4R-Monatin from 50 mM Trp (AJ3976 on Scale of 80 mL)
[0373] A reaction was performed for 12 hours using purified 3976AT-HIs under the following condition. The reaction was performed in a volume of 80 mL using a 250 mL volume mini-jar. The reaction solution was appropriately sampled, the sample was diluted with TE buffer, which was then ultrafiltrated using the Amicon Ultra-0.5 mL centrifugal filter 10 kDa (Millipore), and the resulting filtrate was analyzed. HPLC and capillary electrophoresis were used for the analysis.
[0374] Reaction condition: 50 mM L-Trp, 50 mM PA-Na, 200 mM L-Asp-Na-laq, 1 mM MgCl2, 50 μM PLP, 100 mM Tris-HCl (pH 7.6), 20 mM KPB (pH 7.6), 0.0025% GD113K, pH<7.6 (1 M H2SO4), 20% Ps_aad broth, 30 U/mL of purified SpAld enzyme, 10 U/mL of commercially available OAA DCase enzyme, 2 U/mL of purified LAT enzyme (vs 10 mM 4R-IHOG), and 200 U/mL of commercially available SOD enzyme at 25° C. at 350-400 rpm with air at 8 mL/minute (1/10 vvm).
[0375] The methods for preparing the enzymes subjected to the reaction are shown below.
[0376] Ps_aad broth: it was prepared according to the method described in Example 17.
[0377] Purified SpAld enzyme: it was prepared according to the method described in Example 19.
[0378] AJ3976LAT: it was prepared according to the methods described in Examples 2B.
[0379] OAA DCase: oxaloacetate decarboxylase from Pseudomonas sp. (Sigma) was used. The value described by the manufacturer was used as the enzyme amount (U).
[0380] SOD: superoxide dismutase from bovine liver (Sigma) was used. The value described by the manufacturer was used as the enzyme amount (U).
[0381] As a result of the one-pot reaction, 27 mM 2S,4R-Monatin was confirmed to be accumulated after 8 hours, and the yield from L-Trp which was calculated after calibrating the solution amounts was 56%.
Example 45
Expression of in Silico Selected Aminotransferase in E. coli
(1) Construction of Expression Plasmid for in Silico Selected Aminotransferase
[0382] A DNA sequence obtained by conferring a NdeI recognition sequence and a XhoI recognition sequence to the 5'-end and 3'-end of the genetic sequence of the aminotransferase selected in silico was subjected to Optimum Gene Codon Optimization Analysis supplied by GenScript to obtain synthesized DNA, an expression efficiency of which had been optimized in E. coli. Types of the aminotransferase are as follows.
[0383] Putative aminotransferase derived from Deinococcus Geothermalis DSM 11300 (Dge, ABF45244) (SEQ ID NOs: 64 and 65), hypothetical protein derived from Corynebacterium glutamicum R (Cgl, BAF53276) (SEQ ID NOs: 66 and 67), Lysn, alpha-aminoadipate aminotransferase derived from Thermus thermophilus HB27 (TtHB, AAS80391) (SEQ ID NOs: 68 and 69), aminotransferase (Putative) derived from Thermotoga Maritima (Tma1, AAD36207) (SEQ ID NOs: 70 and 71), human kynurenine aminotransferase II Homologue derived from Pyrococcus Horikoshii Ot3 (PhoH, 1X0M) (SEQ ID NOs: 72 and 73), aspartate aminotransferase derived from Phormidium Lapideum (Pla, BAB86290) (SEQ ID NOs: 74 and 75), aspartate aminotransferase derived from Thermus Thermophilus (Tth, BAD69869) (SEQ ID NOs: 76 and 77), aromatic aminotransferase derived from Pyrococcus Horikoshii Ot3 (PhoA, 1DJU) (SEQ ID NOs: 78 and 79), Mj0684 derived from Methanococcus jannaschii (Mja, AAB98679) (SEQ ID NOs: 80 and 81), aspartate aminotransferase derived from Thermotoga Maritima (Tma2, AAD36764) (SEQ ID NOs: 82 and 83), aspartate aminotransferase derived from Saccharomyces cerevisiae (Sce, CAY81265) (SEQ ID NOs: 84 and 85), aspartate aminotransferase derived from Eubacterium rectale (Ere, ACR74350) (SEQ ID NOs: 86 and 87), aspartate aminotransferase derived from Bacillus pumilus SAFR-032 (Bpu, ABV62783) (SEQ ID NOs: 88 and 89), putative transcriptional regulator (GntR family) derived from Bacillus cellulosilyticus DSM 2522 (Bce, ADU30616) (SEQ ID NOs: 90 and 91), aspartate aminotransferase aspC derived from Bacillus species (strain YM-2) (Bsp, AAA22250) (SEQ ID NOs: 92 and 93), aspartate aminotransferase aatB derived from Sinorhizobium meliloti 1021 (SmeB, CAC47870) (SEQ ID NOs: 94 and 95), branched-chain amino-acid aminotransferase derived from Methanothermobacter thermautotrophicus str. Delta H (Mth, AAB85907) (SEQ ID NOs: 96 and 97), aspartate aminotransferase derived from Lactobacillus acidophilus (Lba, AAV43507) (SEQ ID NOs: 98 and 99), aspartate aminotransferase aatA derived from Sinorhizobium meliloti 1021 (SmeA, CAC46904) (SEQ ID NOs: 100 and 101), hypothetical serine aminotransferase derived from Pyrococcus horikoshi OT3 (PhoS, BAA30413) (SEQ ID NOs: 102 and 103), PLP-dependent aminotransferases derived from Thermoanaerobacter tengcongensis MB4 (Tte, AAM24436) (SEQ ID NOs: 104 and 105), putative transcriptional regulator (GntR family) derived from Clostridium cellulolyticum H10 (Cce, ACL75101) (SEQ ID NOs: 106 and 107), aspartate aminotransferase AspT derived from Rhodococcus erythropolis PR4 (Rer, BAH31070) (SEQ ID NOs: 108 and 109), and transcriptional regulator derived from Saccharophagus degradans 2-40 (Sde, ABD82545) (SEQ ID NOs: 110 and 111).
TABLE-US-00030 TABLE 18 Comparison of percent identities of amino acid sequences Amino acid Amino acid sequence identity sequence identity ID Abbreviation (%) to AJ1616LAT (%) to AJ3976LAT 1 Dge 46 23 2 Cgl 46 30 3 TtHB 20 22 4 Tmal 21 20 5 PhoH 20 22 6 Pla 18 45 7 Tth 17 47 8 PhoA 16 39 9 Mja 17 33 10 Tma2 15 27 11 Sce 20 19 12 Ere 30 26 13 Bpu 93 23 14 Bce 67 22 15 Bsp 17 45 16 SmeB 20 58 17 Mth 17 16 18 Lba 20 24 19 SmeA 21 89 20 PhoS 19 15 21 Tte 17 48 22 Cce 61 24 23 Rer 49 16 24 Sde 49 26
[0384] The synthesized DNA was treated with the restriction enzymes NdeI and XhoI, and ligated to pET-22b (Novagen) likewise treated with NdeI and XhoI. E. coli JM109 was transformed with this ligation solution, the objective plasmids were selected from ampicillin resistant colonies, and these plasmid were designated as pET-22-AT-His. In these plasmids, the aminotransferases having the His-tag added to the C terminus end (AT-His) are expressed.
(2) Purification of AT-His from E. coli Strains Expressing AT-His
[0385] Each of the constructed plasmids pET-22-AT-His was introduced into E. coli BL21 (DE3), and one loopful of the transformant was inoculated to 100 mL of Overnight Express Instant TB Medium (Novagen) containing 100 mg/L of ampicillin in a 500 mL Sakaguchi flask, and the Sakaguchi flask was shaken for 16 hours. The shaking was performed for Lba at 25° C., for Dge, Pla, Tth, Tma2, Sce, Ere, Bpu, Bce, Bsp, SmeA, PhoS, Rer and Sde at 30° C., for Cgl, TtHB, PhoH, PhoA, SmeB, Tte and Cce at 37° C., and for Tma1, Mja and Mth at 42° C. After the completion of the cultivation, microbial cells were collected from the cultured medium by the centrifugation, washed with and suspended in 20 mM Tris-HCl (pH 7.6), 300 mM NaCl and 10 mM imidazole, and sonicated. The microbial cell debris was removed from the sonicated cell suspension by the centrifugation, and the resulting supernatant was used as a soluble fraction.
[0386] The obtained soluble fraction was applied onto the His-tag protein purification column His TALON superflow 5 mL Centrifuge (Clontech) equilibrated with 20 mM Tris-HCl (pH 7.6), 300 mM NaCl and 10 mM imidazole to absorb proteins to the carrier. Proteins that had not been absorbed to the carrier (unabsorbed protein) were washed out with 20 mM Tris-HCl (pH 7.6), 300 mM NaCl and 10 mM imidazole. Subsequently, absorbed proteins were eluted using 20 mM Tris-HCl (pH 7.6), 300 mM NaCl and 150 mM imidazole at a flow rate of 5 mL/minute. The obtained fractions were combined and concentrated using Amicon Ultra-15 10k (Millipore). The concentrated solution was diluted with 20 mM Tris-HCl (pH 7.6) to use as a LAT solution. If necessary, further purification was carried out by increasing the amount of the medium to be cultured and the number of His TALON columns to be linked.
Example 46
One-Pot Reaction for Synthesis of 2S,4R-Monatin from 20 mM L-Trp
[0387] Each reaction was performed under the following condition for 15 hours using purified various AT-His. The reaction was performed in a volume of 1 mL using a test tube. After the completion of the reaction, each sample was diluted with TE buffer, ultrafiltrated using the Amicon Ultra-0.5 mL centrifugal filter 10 kDa (Millipore), and the resulting filtrate was analyzed. HPLC and capillary electrophoresis were used for the analysis.
[0388] Reaction condition: 20 mM L-Trp, 40 mM PA-Na, 160 mM L-Asp-Na-laq, 1 mM MgCl2, 50 μM PLP, 100 mM Tris-HCl, 20 mM KPB (pH 7.0), 20% Ps_aad broth, 30 U/mL of purified SpAld enzyme, 10 U/mL of commercially available OAA DCase enzyme, 1 mg/mL of purified LAT enzyme, and 200 U/mL of commercially available SOD enzyme at 25° C. at 120 rpm.
[0389] The methods for preparing the enzymes subjected to the reaction were shown below.
[0390] Ps_aad broth: it was prepared according to the method described in Example 17.
[0391] Purified SpAld enzyme: it was prepared according to the method described in Example 19.
[0392] Various LAT: they were prepared according to the method described in Example 45.
[0393] OAA DCase: oxaloacetate decarboxylase from Pseudomonas
Example 47
One-pot reaction for synthesis of 2S,4R-Monatin from 20 mM L-Trp (Tth, Bpu, SmeA and Sde)
[0394] Reactions were performed under the following condition for 15 hours using purified various AT-His. The reaction was performed in a volume of 1 mL using a test tube. After the completion of the reaction, the sample was diluted with TE buffer, ultrafiltrated using the Amicon Ultra-0.5 mL centrifugal filter 10 kDa (Millipore), and the resulting filtrate was analyzed. HPLC and capillary electrophoresis were used for the analysis.
[0395] Reaction condition: 20 mM L-Trp, 40 mM PA-Na, 160 mM L-Asp-Na-laq, 1 mM MgCl2, 50 μM PLP, 100 mM Tris-HCl, 20 mM KPB (pH 7.0), 20% Ps_aad broth, 30 U/mL of purified SpAld enzyme, 10 U/mL of commercially available OAA DCase enzyme, 3 mg/mL of purified LAT enzyme (12 mg/mL of Tth, 1 mg/mL of Bpu), and 200 U/mL of commercially available SOD enzyme at 25° C. at 120 rpm.
[0396] The methods for preparing the enzymes subjected to the reaction are shown below.
[0397] Ps_aad broth: it was prepared according to the method described in Example 17.
[0398] Purified SpAld enzyme: it was prepared according to the method described in Example 19.
[0399] Various LAT: they were prepared according to the method described in Example 45.
[0400] OAA DCase: oxaloacetate decarboxylase from Pseudomonas sp. (Sigma) was used. The value described by the manufacturer was used as the enzyme amount (U).
[0401] SOD: superoxide dismutase from bovine liver (Sigma) was used. The value described by the manufacturer was used as the enzyme amount (U).
[0402] The results of the one-pot reactions are shown in Table 20. The 2S,4R-Monatin at 18 mM, 17 mM, 11 mM and 12 mM were formed using Tth, Bpu, SmeA and Sde, respectively, and their yields from L-Trp were 92%, 87%, 54% and 61%, respectively.
TABLE-US-00031 TABLE 20 Yields of 2S,4R-Monatin in one-pot reaction using 20 mM Trp as substrate Abbreviation Yield from Trp (%) Tth 92 Bpu 87 SmeA 54 Sde 61
Example 48
One-Pot Reaction for Synthesis of 2S,4R-Monatin from 100 mM L-Trp (Tth, Bpu, SmeA and Sde)
[0403] Reactions were performed under the following condition for 18 hours using purified various AT-His, Tth, Bpu, SmeA and Sde. The reaction was performed in a volume of 1 mL using a test tube. After the completion of the reaction, the sample was diluted with TE buffer, ultrafiltrated using the Amicon Ultra-0.5 mL centrifugal filter 10 kDa (Millipore), and the resulting filtrate was analyzed. HPLC and capillary electrophoresis were used for the analysis.
[0404] Reaction condition: 100 mM L-Trp, 50 mM PA-Na, 300 mM L-Asp-Na-laq, 1 mM MgCl2, 50 μM PLP, 100 mM Tris-HCl, 20 mM KPB (pH 7.0), 40% Ps_aad broth, 60 U/mL of purified SpAld enzyme, 10 U/mL of commercially available OAA DCase enzyme, 3 mg/mL of purified LAT enzyme (12 mg/mL for Tth), and 200 U/mL of commercially available SOD enzyme at 25° C. at 150 rpm.
[0405] The methods for preparing the enzymes subjected to the reaction are shown below.
[0406] Ps_aad broth: it was prepared according to the method described in Example 17.
[0407] Purified SpAld enzyme: it was prepared according to the method described in Example 19.
[0408] Various LAT: they were prepared according to the method described in Example 45.
[0409] OAA DCase: oxaloacetate decarboxylase from Pseudomonas sp. (Sigma) was used. The value described by the manufacturer was used as the enzyme amount (U).
[0410] SOD: superoxide dismutase from bovine liver (Sigma) was used. The value described by the manufacturer was used as the enzyme amount (U).
[0411] The results of the one-pot reactions are shown in Table 21. The 2S,4R-Monatin at 72 mM, 46 mM, 6.4 mM and 20 mM were formed using Tth, Bpu, SmeA and Sde, respectively, and their yields from L-Trp were 72%, 46%, 6.4% and 20%, respectively.
TABLE-US-00032 TABLE 21 Yields of 2S,4R-MOnatin in one pot reaction using 100 mM Trp as substrate Yield from Abbreviation Trp (%) Tth 72 Bpu 46 SmeA 6.4 Sde 20
(Information on Microorganisms)
[0412] The microorganisms specified by deposit numbers which are described herein can be available from certain deposit authority. The microorganisms described in Table 22 have been deposited to National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary (1-1-1 Central No. 6 Higashi, Tsukuba-shi, Ibaraki Prefecture, Japan) on the following dates, and the following deposit numbers have been conferred to them. As described in Table 22, these microorganisms are currently classified in the following ways as a result of reidentification, although different names were previously conferred to them.
TABLE-US-00033 TABLE 22 Current names for microorganims (Previous names for Deposit microorganims) number Deposited date Bacillus altitudinis AJ1616 FERM-BP 11429 Oct. 4, (Bacillus sp. AJ1616) 2011 Stenotrophomonas sp. AJ3447 FERM-BP 11422 Sep. 30, (Xanthomonas oryzae AJ3447) 2011 Stenotrophomonas sp. AJ11634 FERM-BP 11423 Sep. 30, (Xanthomonas albilineans 2011 AJ11634) Ochrobactrum pseudogrignonense FERM-BP 11432 Oct. 4, AJ3735 2011 (Pseudomonas betainovorans AJ3735) Stenotrophomonas sp. AJ1591 FERM-BP 11419 Sep. 27, (Pseudomonas putrefaciens 2011 AJ1591) Stenotrophomonas sp. AJ3839 FERM-BP 11416 Sep. 15, (Pseudomonas peptidolytica 2011 AJ3839) Brevundimonas diminuta AJ3958 FERM-BP 11425 Sep. 30, (Pseudomonas hydrogenovora 2011 AJ3958) Rhizobium sp. AJ12469 FERM-BP 11430 Oct. 4, (Alcaligenes faecalis AJ12469) 2011 Carnimonas sp. AJ3230 FERM-BP 11431 Oct. 4, (Achromobacter brunificans 2011 AJ3230) Pseudomonas sp. AJ1594 FERM-BP 11424 Sep. 30, (Pseudomonas ovalis AJ1594) 2011
[0413] In addition, the microorganisms described in Table 23 are currently classified in the following ways as a result of reidentification, although different names were previously conferred to them. The bacterial strain, Stenotrophomonas sp. AJ13127 is identical to the known bacterial strain specified by the deposit number FERM-BP 5568.
TABLE-US-00034 TABLE 23 Current names for microorganims (Previous names for microorganims) Rhizobium radiobacter LAT1 (Rhizobium sp. LAT1) Rhizobium radiobacter AJ11568 (Pseudomonas umorosa AJ11568) Dietzia maris AJ2788 (Pseudomonas tabaci AJ2788) Stenotrophomonas sp. AJ13127 (Stenotrophomonas sp. AJ13127) Arthrobacter sp. IAM1390 (Arthrobacter ureafaciens IAM1390) Burkholderia sp. AJ3084 (Pseudomonas multivorans AJ3084) Rhizobium radiobacter AJ2557 (Alcaligenes metalcaligenes AJ2557) Pseudomonas sp. LMG2833 (Achromobacter butyri LMG2833)
INDUSTRIAL APPLICABILITY
[0414] As described above, the methods of the present invention are useful for producing the Monatin which can be used as the sweetener.
Sequence Listing Free Text
[0415] SEQ ID NO:1: Nucleotide sequence of aminotransferase gene derived from Bacillus altitudinis SEQ ID NO:2: Amino acid sequence of aminotransferase derived from Bacillus altitudinis SEQ ID NO:3: Nucleotide sequence of aminotransferase gene (nucleotide numbers 231-1538) and the upstream and downstream regions thereof which are derived from Bacillus altitudinis SEQ ID NO:4: Amino acid sequence of a fragment of aminotransferase derived from Bacillus altitudinis SEQ ID NO:5: Amino acid sequence of a fragment of aminotransferase derived from Bacillus altitudinis SEQ ID NO:6: Forward primer for amplifying DNA fragment containing aminotransferase gene derived from Bacillus altitudinis (Bp-u200-f) SEQ ID NO:7: Reverse primer for amplifying DNA fragment containing aminotransferase gene derived from Bacillus altitudinis (Bp-d200-r) SEQ ID NO:8: Forward primer for amplifying DNA fragment containing aminotransferase gene derived from Bacillus altitudinis (1616AT-Nde-f) SEQ ID NO:9: Reverse primer for amplifying DNA fragment containing aminotransferase gene derived from Bacillus altitudinis (1616-xho-r) SEQ ID NO:10: Forward primer for converting DNA sequence recognized by NdeI, which is found on aminotransferase gene derived from Bacillus altitudinis (1616-delNde-f) SEQ ID NO:11: Reverse primer for converting DNA sequence recognized by NdeI, which is found on aminotransferase gene derived from Bacillus altitudinis (1616-delNde-r) SEQ ID NO:12: Forward primer for amplifying DNA fragment containing SpAld gene (SpAld-f-NdeI) SEQ ID NO:13: Reverse primer for amplifying DNA fragment containing SpAld gene (SpAld-r-HindIII) SEQ ID NO:14: Forward primer for converting rare codon 6L in SpAld gene (6L-f) SEQ ID NO:15: Reverse primer for converting rare codon 6L in SpAld gene (6L-r) SEQ ID NO:16: Forward primer for converting rare codon 13L in SpAld gene (13L-f) SEQ ID NO:17: Reverse primer for converting rare codon 13L in SpAld gene (13L-r) SEQ ID NO:18: Forward primer for converting rare codon 18P in SpAld gene (18P-f) SEQ ID NO:19: Reverse primer for converting rare codon 18P in SpAld gene (18P-r) SEQ ID NO:20: Forward primer for converting rare codon 38P in SpAld gene (38P-f) SEQ ID NO:21: Reverse primer for converting rare codon 38P in SpAld gene (38P-r) SEQ ID NO:22: Forward primer for converting rare codon 50P in SpAld gene (50P-f) SEQ ID NO:23: Reverse primer for converting rare codon 50P in SpAld gene (50P-r) SEQ ID NO:24: Forward primer for converting rare codons 77P, 81P and 84R in SpAld gene (77P-81P-84R-f) SEQ ID NO:25: Reverse primer for converting rare codons 77P, 81P and B4R in SpAld gene (77P-81P-84R-r) SEQ ID NO:26: Forward primer for preparing the aminotransferase mutant K39R derived from Bacillus altitudinis AJ1616 (K39R_FW) SEQ ID NO:27: Reverse primer for preparing the aminotransferase mutant K39R derived from Bacillus altitudinis AJ1616 (K39R_RV) SEQ ID NO:28: Forward primer for preparing the aminotransferase mutant S258G derived from Bacillus altitudinis AJ1616 (S258G_FW) SEQ ID NO:29: Reverse primer for preparing the aminotransferase mutant S258G derived from Bacillus altitudinis AJ1616 (S258G_RV) SEQ ID NO:30: Forward primer for preparing the aminotransferase mutant T288G derived from Bacillus altitudinis AJ1616 (T288G_FW) SEQ ID NO:31: Reverse primer for preparing the aminotransferase mutant T288G derived from Bacillus altitudinis AJ1616 (T288G_RV) SEQ ID NO:32: Forward primer for preparing the aminotransferase mutant I289A derived from Bacillus altitudinis AJ1616 (I289A_FW) SEQ ID NO:33: Reverse primer for preparing the aminotransferase mutant I289A derived from Bacillus altitudinis AJ1616 (I289A_RV) SEQ ID NO:34: Forward primer for preparing the aminotransferase mutant Q287E/T288G derived from Bacillus altitudinis AJ1616 (Q287E/T288G_FW) SEQ ID NO:35: Reverse primer for preparing the aminotransferase mutant Q287E/T288G derived from Bacillus altitudinis AJ1616 (Q287E/T288G_RV) SEQ ID NO:36: Primer for preparing a DNA fragment for destroying aspC gene (aspC-L1) SEQ ID NO:37: Primer for preparing a DNA fragment for destroying aspC gene (aspC-R1) SEQ ID NO:38: Primer for confirming the insertion of attL-cat-attR in the region of aspC gene (aspC-up) SEQ ID NO:39: Primer for confirming the insertion of attL-cat-attR in the region of aspC gene (attL-1) SEQ ID NO:40: Primer for confirming the insertion of attL-cat-attR in the region of aspC gene (aspC-down) SEQ ID NO:41: Primer for confirming the insertion of attL-cat-attR in the region of aspC gene (attR-1) SEQ ID NO:42: Nucleotide sequence of oxaloacetate decarboxylase gene derived from Pseudomonas putida SEQ ID NO:43: Amino acid sequence of oxaloacetate decarboxylase derived from Pseudomonas putida SEQ ID NO:44: Amino acid sequence of a fragment of aminotransferase derived from Rhizobium radiobacter SEQ ID NO:45: Forward primer which is designed based on the genomic DNA sequence from Agrobacterium tumefaciens str. C58 (Ag-u100-f) SEQ ID NO:46: Reverse primer which is designed based on the genomic DNA sequence from Agrobacterium tumefaciens str. C58 (Ag-d100-r) SEQ ID NO:47: Nucleotide sequence of aminotransferase gene derived from Rhizobium radiobacter SEQ ID NO:48: Amino acid sequence of aminotransferase derived from Rhizobium radiobacter SEQ ID NO:49: Forward primer for amplifying DNA fragment containing aminotransferase gene derived from Rhizobium radiobacter (3976AT-Nde-f) SEQ ID NO:50: Reverse primer for amplifying DNA fragment containing aminotransferase gene derived from Rhizobium radiobacter (3976-xho-r) SEQ ID NO:51: Amino acid sequence of a fragment of aminotransferase derived from Rhizobium sp. SEQ ID NO:52: Nucleotide sequence of aminotransferase gene derived from Rhizobium sp. SEQ ID NO:53: Amino acid sequence of aminotransferase derived from Rhizobium sp. SEQ ID NO:54: Forward primer for amplifying DNA fragment containing aminotransferase gene derived from Rhizobium sp. (12469AT-Nde-f) SEQ ID NO:55: Reverse primer for amplifying DNA fragment containing aminotransferase gene derived from Rhizobium sp. (12469-xho-r) SEQ ID NO:56: Forward primer which is designed based on the genomic DNA sequence from Corynebacterium ammonia genes DSM20306 (Co-d50-r) SEQ ID NO:57: Reverse primer which is designed based on a homologus region between the genomic DNA sequences corresponding to the aspartate aminotransferases from Corynebacterium striatum ATCC6940 (ZP--03935516) and from Corynebacterium ammonia genes DSM20306 SEQ ID NO:58: Forward primer for amplifying DNA fragment containing aminotransferase gene derived from Corynebacterium ammoniagenes (Co-890-r) SEQ ID NO:59: Reverse primer for amplifying DNA fragment containing aminotransferase gene derived from Corynebacterium ammoniagenes (Co-1060-r) SEQ ID NO:60: Nucleotide sequence of aminotransferase gene derived from Corynebacterium ammoniagenes SEQ ID NO:61: Amino acid sequence of aminotransferase derived from Corynebacterium ammoniagenes SEQ ID NO:62: Forward primer for amplifying DNA fragment containing aminotransferase gene derived from Corynebacterium ammoniagenes (1444AT-Nde-f) SEQ ID NO:63: Reverse primer for amplifying DNA fragment containing aminotransferase gene derived from Corynebacterium ammoniagenes (1444-xho-r) SEQ ID NO:64: Nucleotide sequence of aminotransferase gene derived from Deinococcus geothermalis SEQ ID NO:65: Amino acid sequence of aminotransferase derived from Deinococcus geothermalis SEQ ID NO:66: Nucleotide sequence of aminotransferase gene derived from Corynebacterium glutamicum SEQ ID NO:67: Amino acid sequence of aminotransferase derived from Corynebacterium glutamicum SEQ ID NO:68: Nucleotide sequence of aminotransferase gene derived from Thermus thermophilus SEQ ID NO:69: Amino acid sequence of aminotransferase derived from Thermus thermophilus SEQ ID NO:70: Nucleotide sequence of aminotransferase gene derived from Thermotoga maritima SEQ ID NO:71: Amino acid sequence of aminotransferase derived from Thermotoga maritima SEQ ID NO:72: Nucleotide sequence of aminotransferase gene derived from Pyrococcus horikoshii SEQ ID NO:73: Amino acid sequence of aminotransferase derived from Pyrococcus horikoshii SEQ ID NO:74: Nucleotide sequence of aminotransferase gene derived from Phormidium lapideum SEQ ID NO:75: Amino acid sequence of aminotransferase derived from Phormidium lapideum SEQ ID NO:76: Nucleotide sequence of aminotransferase gene derived from Thermos thermophilus SEQ ID NO:77: Amino acid sequence of aminotransferase derived from Thermos thermophilus SEQ ID NO:78: Nucleotide sequence of aminotransferase gene derived from Pyrococcus horikoshii SEQ ID NO:79: Amino acid sequence of aminotransferase derived from Pyrococcus horikoshii SEQ ID NO:80: Nucleotide sequence of aminotransferase gene derived from Methanococcus jannaschii SEQ ID NO:81: Amino acid sequence of aminotransferase derived from Methanococcus jannaschii SEQ ID NO:82: Nucleotide sequence of aminotransferase gene derived from Thermotoga maritima SEQ ID NO:83: Amino acid sequence of aminotransferase derived from Thermotoga maritima SEQ ID NO:84: Nucleotide sequence of aminotransferase gene derived from Saccharomyces cerevisiae SEQ ID NO:85: Amino acid sequence of aminotransferase derived from Saccharomyces cerevisiae SEQ ID NO:86: Nucleotide sequence of aminotransferase gene derived from Eubacterium rectale SEQ ID NO:87: Amino acid sequence of aminotransferase derived from Eubacterium rectale SEQ ID NO:88: Nucleotide sequence of aminotransferase gene derived from Bacillus pumilus SEQ ID NO:89: Amino acid sequence of aminotransferase derived from Bacillus pumilus SEQ ID NO:90: Nucleotide sequence of aminotransferase gene derived from Bacillus cellulosilyticus SEQ ID NO:91: Amino acid sequence of aminotransferase derived from Bacillus cellulosilyticus SEQ ID NO:92: Nucleotide sequence of aminotransferase gene derived from Bacillus sp. SEQ ID NO:93: Amino acid sequence of aminotransferase derived from Bacillus sp. SEQ ID NO:94: Nucleotide sequence of aminotransferase gene derived from Sinorhizobium meliloti SEQ ID NO:95: Amino acid sequence of aminotransferase derived from Sinorhizobium meliloti SEQ ID NO:96: Nucleotide sequence of aminotransferase gene derived from Methanothermobacter thermautotrophicus SEQ ID NO:97: Amino acid sequence of aminotransferase derived from Methanothermobacter thermautotrophicus SEQ ID NO:98: Nucleotide sequence of aminotransferase gene derived from Lactobacillus acidophilus SEQ ID NO:99: Amino acid sequence of aminotransferase derived from Lactobacillus acidophilus SEQ ID NO:100: Nucleotide sequence of aminotransferase gene derived from Sinorhizobium meliloti SEQ ID NO:101: Amino acid sequence of aminotransferase derived from Sinorhizobium meliloti SEQ ID NO:102: Nucleotide sequence of aminotransferase gene derived from Pyrococcus horikoshii SEQ ID NO:103: Amino acid sequence of aminotransferase derived from Pyrococcus horikoshii SEQ ID NO:104: Nucleotide sequence of aminotransferase gene derived from Thermoanaerobacter tengcongensis SEQ ID NO:105: Amino acid sequence of aminotransferase derived from Thermoanaerobacter tengcongensis SEQ ID NO:106: Nucleotide sequence of aminotransferase gene derived from Clostridium cellulolyticum SEQ ID NO:107: Amino acid sequence of aminotransferase derived from Clostridium cellulolyticum SEQ ID NO:108: Nucleotide sequence of aminotransferase gene derived from Rhodococcus erythropolis SEQ ID NO:109: Amino acid sequence of aminotransferase derived from Rhodococcus erythropolis SEQ ID NO:110: Nucleotide sequence of aminotransferase gene derived from Saccharophagus degradans SEQ ID NO:111: Amino acid sequence of aminotransferase derived from Saccharophagus degradans SEQ ID NO:112: Amino acid sequence of a fragment of aminotransferase derived from Corynebacterium ammoniagenes
Sequence CWU
1
1
11211308DNABacillus altitudinisCDS(1)..(1308) 1atg agc ggt ttt aca gcg tta
agt gaa gca gaa tta aat gac cta tat 48Met Ser Gly Phe Thr Ala Leu
Ser Glu Ala Glu Leu Asn Asp Leu Tyr 1 5
10 15 gca gca cga caa aaa gag tat
gaa acg tac aaa agt aaa aac tta cac 96Ala Ala Arg Gln Lys Glu Tyr
Glu Thr Tyr Lys Ser Lys Asn Leu His 20
25 30 tta gac atg tct aga ggg aaa
cct tca cca aaa cag ctc gat tta tct 144Leu Asp Met Ser Arg Gly Lys
Pro Ser Pro Lys Gln Leu Asp Leu Ser 35
40 45 atg ggc atg ctc gat gtc gtg
aca tca aag gat gca atg aca gct gag 192Met Gly Met Leu Asp Val Val
Thr Ser Lys Asp Ala Met Thr Ala Glu 50 55
60 gat ggt aca gat gtg cga aac
tat ggc ggc ttg aca ggc ctt cct gaa 240Asp Gly Thr Asp Val Arg Asn
Tyr Gly Gly Leu Thr Gly Leu Pro Glu 65 70
75 80 aca aag aaa ttt ttt gca agt
gtg ctc aat ctg aag cca gaa caa atc 288Thr Lys Lys Phe Phe Ala Ser
Val Leu Asn Leu Lys Pro Glu Gln Ile 85
90 95 atc att ggc ggt aat tct agc
cta aat atg atg cat gac aca att gcc 336Ile Ile Gly Gly Asn Ser Ser
Leu Asn Met Met His Asp Thr Ile Ala 100
105 110 cgt gct atg act cac ggc gta
tat ggc agc aaa aca cct tgg gga gag 384Arg Ala Met Thr His Gly Val
Tyr Gly Ser Lys Thr Pro Trp Gly Glu 115
120 125 ctt cca aag gta aaa ttc ctt
gca cca agc cca ggg tat gat cgt cat 432Leu Pro Lys Val Lys Phe Leu
Ala Pro Ser Pro Gly Tyr Asp Arg His 130 135
140 ttt gcc att tgt gag cat ttt
aac ata gag atg att acg gta gat atg 480Phe Ala Ile Cys Glu His Phe
Asn Ile Glu Met Ile Thr Val Asp Met 145 150
155 160 aag tcg gat gga cct gac atg
gat cag gtg gaa aaa ttg gtt gca gaa 528Lys Ser Asp Gly Pro Asp Met
Asp Gln Val Glu Lys Leu Val Ala Glu 165
170 175 gat gaa gcc atc aaa ggg att
tgg tgt gta cca aaa tat agc aac cct 576Asp Glu Ala Ile Lys Gly Ile
Trp Cys Val Pro Lys Tyr Ser Asn Pro 180
185 190 gac ggc att acg tat tca gat
gag gtt gtc gac cgt ctt gct tcc atg 624Asp Gly Ile Thr Tyr Ser Asp
Glu Val Val Asp Arg Leu Ala Ser Met 195
200 205 cag aca aaa gca gac gac ttc
cgt att ttt tgg gat gat gcc tat gca 672Gln Thr Lys Ala Asp Asp Phe
Arg Ile Phe Trp Asp Asp Ala Tyr Ala 210 215
220 gtc cac cat cta aca gat acg
cct gat acg tta aaa gat att ttt caa 720Val His His Leu Thr Asp Thr
Pro Asp Thr Leu Lys Asp Ile Phe Gln 225 230
235 240 gca gta gac aaa gca ggg cat
gca aac cgt gtg ttt atg ttc gcc tct 768Ala Val Asp Lys Ala Gly His
Ala Asn Arg Val Phe Met Phe Ala Ser 245
250 255 act tct aaa att acg ttc cca
ggc tca ggt gtt gca ctg atg gca tct 816Thr Ser Lys Ile Thr Phe Pro
Gly Ser Gly Val Ala Leu Met Ala Ser 260
265 270 agt cag gac aac gtc agc ttt
att caa aaa cag cta tca gtt caa acc 864Ser Gln Asp Asn Val Ser Phe
Ile Gln Lys Gln Leu Ser Val Gln Thr 275
280 285 att ggg cca gat aaa atc aat
caa tta aga cac ctt cgt ttc ttc aag 912Ile Gly Pro Asp Lys Ile Asn
Gln Leu Arg His Leu Arg Phe Phe Lys 290 295
300 aat cca gaa gga ttg aag gaa
cat atg aaa aag cat gca gcg att att 960Asn Pro Glu Gly Leu Lys Glu
His Met Lys Lys His Ala Ala Ile Ile 305 310
315 320 aag ccg aaa ttt gac ctc gtt
ctt tcg atc ctt gat gaa aag ctt ggt 1008Lys Pro Lys Phe Asp Leu Val
Leu Ser Ile Leu Asp Glu Lys Leu Gly 325
330 335 gga aca ggc atc gct gag tgg
cac aaa cca aat ggc gga tat ttt att 1056Gly Thr Gly Ile Ala Glu Trp
His Lys Pro Asn Gly Gly Tyr Phe Ile 340
345 350 agc tta aat aca ctc gat cat
tgt gca aaa gct gtt gtg caa aaa gcg 1104Ser Leu Asn Thr Leu Asp His
Cys Ala Lys Ala Val Val Gln Lys Ala 355
360 365 aaa gaa gcc ggt gtg aca cta
aca ggt gca ggg gcg aca tat cct tat 1152Lys Glu Ala Gly Val Thr Leu
Thr Gly Ala Gly Ala Thr Tyr Pro Tyr 370 375
380 gga aac gac ccg ctt gat cgt
aac atc cgt att gcg cca acg ttc cca 1200Gly Asn Asp Pro Leu Asp Arg
Asn Ile Arg Ile Ala Pro Thr Phe Pro 385 390
395 400 acg ctt gaa gaa cta gag cag
gcg att gat atc ttt acg tta tgc gtt 1248Thr Leu Glu Glu Leu Glu Gln
Ala Ile Asp Ile Phe Thr Leu Cys Val 405
410 415 cag ctt gtc agc att gaa aag
ctg ctg tct gag aaa agt caa tca gca 1296Gln Leu Val Ser Ile Glu Lys
Leu Leu Ser Glu Lys Ser Gln Ser Ala 420
425 430 cca acg gta taa
1308Pro Thr Val
435
2435PRTBacillus altitudinis
2Met Ser Gly Phe Thr Ala Leu Ser Glu Ala Glu Leu Asn Asp Leu Tyr 1
5 10 15 Ala Ala Arg Gln
Lys Glu Tyr Glu Thr Tyr Lys Ser Lys Asn Leu His 20
25 30 Leu Asp Met Ser Arg Gly Lys Pro Ser
Pro Lys Gln Leu Asp Leu Ser 35 40
45 Met Gly Met Leu Asp Val Val Thr Ser Lys Asp Ala Met Thr
Ala Glu 50 55 60
Asp Gly Thr Asp Val Arg Asn Tyr Gly Gly Leu Thr Gly Leu Pro Glu 65
70 75 80 Thr Lys Lys Phe Phe
Ala Ser Val Leu Asn Leu Lys Pro Glu Gln Ile 85
90 95 Ile Ile Gly Gly Asn Ser Ser Leu Asn Met
Met His Asp Thr Ile Ala 100 105
110 Arg Ala Met Thr His Gly Val Tyr Gly Ser Lys Thr Pro Trp Gly
Glu 115 120 125 Leu
Pro Lys Val Lys Phe Leu Ala Pro Ser Pro Gly Tyr Asp Arg His 130
135 140 Phe Ala Ile Cys Glu His
Phe Asn Ile Glu Met Ile Thr Val Asp Met 145 150
155 160 Lys Ser Asp Gly Pro Asp Met Asp Gln Val Glu
Lys Leu Val Ala Glu 165 170
175 Asp Glu Ala Ile Lys Gly Ile Trp Cys Val Pro Lys Tyr Ser Asn Pro
180 185 190 Asp Gly
Ile Thr Tyr Ser Asp Glu Val Val Asp Arg Leu Ala Ser Met 195
200 205 Gln Thr Lys Ala Asp Asp Phe
Arg Ile Phe Trp Asp Asp Ala Tyr Ala 210 215
220 Val His His Leu Thr Asp Thr Pro Asp Thr Leu Lys
Asp Ile Phe Gln 225 230 235
240 Ala Val Asp Lys Ala Gly His Ala Asn Arg Val Phe Met Phe Ala Ser
245 250 255 Thr Ser Lys
Ile Thr Phe Pro Gly Ser Gly Val Ala Leu Met Ala Ser 260
265 270 Ser Gln Asp Asn Val Ser Phe Ile
Gln Lys Gln Leu Ser Val Gln Thr 275 280
285 Ile Gly Pro Asp Lys Ile Asn Gln Leu Arg His Leu Arg
Phe Phe Lys 290 295 300
Asn Pro Glu Gly Leu Lys Glu His Met Lys Lys His Ala Ala Ile Ile 305
310 315 320 Lys Pro Lys Phe
Asp Leu Val Leu Ser Ile Leu Asp Glu Lys Leu Gly 325
330 335 Gly Thr Gly Ile Ala Glu Trp His Lys
Pro Asn Gly Gly Tyr Phe Ile 340 345
350 Ser Leu Asn Thr Leu Asp His Cys Ala Lys Ala Val Val Gln
Lys Ala 355 360 365
Lys Glu Ala Gly Val Thr Leu Thr Gly Ala Gly Ala Thr Tyr Pro Tyr 370
375 380 Gly Asn Asp Pro Leu
Asp Arg Asn Ile Arg Ile Ala Pro Thr Phe Pro 385 390
395 400 Thr Leu Glu Glu Leu Glu Gln Ala Ile Asp
Ile Phe Thr Leu Cys Val 405 410
415 Gln Leu Val Ser Ile Glu Lys Leu Leu Ser Glu Lys Ser Gln Ser
Ala 420 425 430 Pro
Thr Val 435 3 1678DNABacillus altitudinis 3gaaacaatta
ctcaaagaag cccgcgatat attcccgctg actgaagctg cacatgattt 60cttttctttt
cctattgacc gtaccctgta aaaattggat gaatcgtcaa aaatagttgt 120gtgatttttt
tgatatttat gatgacgctc tttttcaggt agtggtaaaa tggtgaagaa 180aaaacaaatg
acgaattaca ctatgaagaa tacgggaggc acaatcaaag atgagcggtt 240ttacagcgtt
aagtgaagca gaattaaatg acctatatgc agcacgacaa aaagagtatg 300aaacgtacaa
aagtaaaaac ttacacttag acatgtctag agggaaacct tcaccaaaac 360agctcgattt
atctatgggc atgctcgatg tcgtgacatc aaaggatgca atgacagctg 420aggatggtac
agatgtgcga aactatggcg gcttgacagg ccttcctgaa acaaagaaat 480tttttgcaag
tgtgctcaat ctgaagccag aacaaatcat cattggcggt aattctagcc 540taaatatgat
gcatgacaca attgcccgtg ctatgactca cggcgtatat ggcagcaaaa 600caccttgggg
agagcttcca aaggtaaaat tccttgcacc aagcccaggg tatgatcgtc 660attttgccat
ttgtgagcat tttaacatag agatgattac ggtagatatg aagtcggatg 720gacctgacat
ggatcaggtg gaaaaattgg ttgcagaaga tgaagccatc aaagggattt 780ggtgtgtacc
aaaatatagc aaccctgacg gcattacgta ttcagatgag gttgtcgacc 840gtcttgcttc
catgcagaca aaagcagacg acttccgtat tttttgggat gatgcctatg 900cagtccacca
tctaacagat acgcctgata cgttaaaaga tatttttcaa gcagtagaca 960aagcagggca
tgcaaaccgt gtgtttatgt tcgcctctac ttctaaaatt acgttcccag 1020gctcaggtgt
tgcactgatg gcatctagtc aggacaacgt cagctttatt caaaaacagc 1080tatcagttca
aaccattggg ccagataaaa tcaatcaatt aagacacctt cgtttcttca 1140agaatccaga
aggattgaag gaacatatga aaaagcatgc agcgattatt aagccgaaat 1200ttgacctcgt
tctttcgatc cttgatgaaa agcttggtgg aacaggcatc gctgagtggc 1260acaaaccaaa
tggcggatat tttattagct taaatacact cgatcattgt gcaaaagctg 1320ttgtgcaaaa
agcgaaagaa gccggtgtga cactaacagg tgcaggggcg acatatcctt 1380atggaaacga
cccgcttgat cgtaacatcc gtattgcgcc aacgttccca acgcttgaag 1440aactagagca
ggcgattgat atctttacgt tatgcgttca gcttgtcagc attgaaaagc 1500tgctgtctga
gaaaagtcaa tcagcaccaa cggtataacg aaaaaactcc ttgactgatg 1560tccggtcaag
gagtttttgt ttttagttag ctgtttgata ataagtggca ggctgttttg 1620ctgcacacca
tacatcataa atcgcaagct gcacaatatg tggttcatcg tgatgaat
1678415PRTBacillus altitudinis 4Ser Gly Phe Thr Ala Leu Ser Glu Ala Glu
Leu Asn Asp Leu Tyr 1 5 10
15 512PRTBacillus altitudinis 5Gln Leu Asp Leu Ser Met Gly Met Leu Asp
Val Val 1 5 10 630DNAArtificial
SequenceForward primer for amplifying DNA fragment containing
aminotransferase gene derived from Bacillus altitudinis (Bp-u200-f)
6ctcaggaagc aggcgcaaaa agattaattt
30730DNAArtificial SequenceReverse primer for amplifying DNA fragment
containing aminotransferase gene derived from Bacillus altitudinis
(Bp-d200-r) 7ggatgctgtc tttgtcatcc caaagtggat
30830DNAArtificial SequenceForward primer for amplifying DNA
fragment containing aminotransferase gene derived from Bacillus
altitudinis (1616AT-Nde-f) 8ggaattccat atgagcggtt ttacagcgtt
30940DNAArtificial SequenceReverse primer
for amplifying DNA fragment containing aminotransferase gene derived
from Bacillus altitudinis (1616-xho-r) 9gtcaaggagt ttttctcgag
taccgttggt gctgattgac 401029DNAArtificial
SequenceForward primer for converting DNA sequence recognized by
NdeI, which is found on aminotransferase gene derived from Bacillus
altitudinis (1616-delNde-f) 10ggattgaagg aacacatgaa aaagcatgc
291129DNAArtificial SequenceReverse primer for
converting DNA sequence recognized by NdeI, which is found on
aminotransferase gene derived from Bacillus altitudinis
(1616-delNde-r) 11gcatgctttt tcatgtgttc cttcaatcc
291230DNAArtificial SequenceForward primer for amplifying
DNA fragment containing SpAld gene (SpAld-f-NdeI) 12ggaattccat
atgacccaga cgcgcctcaa
301330DNAArtificial SequenceReverse primer for amplifying DNA fragment
containing SpAld gene (SpAld-r-HindIII) 13gcccaagctt tcagtacccc
gccagttcgc 301429DNAArtificial
SequenceForward primer for converting rare codon 6L in SpAld gene
(6L-f) 14acccagacgc gcctgaacgg catcatccg
291529DNAArtificial SequenceReverse primer for converting rare codon
6L in SpAld gene (6L-r) 15cggatgatgc cgttcaggcg cgtctgggt
291629DNAArtificial SequenceForward primer for
converting rare codon 13L in SpAld gene (13L-f) 16atcatccgcg
ctctggaagc cggcaagcc
291729DNAArtificial SequenceReverse primer for converting rare codon 13L
in SpAld gene (13L-r) 17ggcttgccgg cttccagagc gcggatgat
291829DNAArtificial SequenceForward primer for
converting rare codon 18P in SpAld gene (18P-f) 18gaagccggca
agccggcttt cacctgctt
291929DNAArtificial SequenceReverse primer for converting rare codon 18P
in SpAld gene (18P-r) 19aagcaggtga aagccggctt gccggcttc
292029DNAArtificial SequenceForward primer for
converting rare codon 38P in SpAld gene (38P-f) 20ctgaccgatg
ccccgtatga cggcgtggt
292129DNAArtificial SequenceReverse primer for converting rare codon 38P
in SpAld gene (38P-r) 21accacgccgt catacggggc atcggtcag
292229DNAArtificial SequenceForward primer for
converting rare codon 50P in SpAld gene (50P-f) 22atggagcaca
acccgtacga tgtcgcggc
292329DNAArtificial SequenceReverse primer for converting rare codon 50P
in SpAld gene (50P-r) 23gccgcgacat cgtacgggtt gtgctccat
292442DNAArtificial SequenceForward primer for
converting rare codons 77P, 81P and 84R in SpAld gene
(77P-81P-84R-f) 24cggtcgcgcc gtcggtcacc ccgatcgcgc gcatcccggc ca
422542DNAArtificial SequenceReverse primer for converting
rare codons 77P, 81P and 84R in SpAld gene (77P-81P-84R-r)
25tggccgggat gcgcgcgatc ggggtgaccg acggcgcgac cg
422633DNAArtificial SequenceForward primer for preparing the
aminotransferase mutant K39R derived from Bacillus altitudinis
AJ1616 (K39R_FW) 26gacatgtcta gagggcgtcc ttcaccaaaa cag
332733DNAArtificial SequenceReverse primer for preparing
the aminotransferase mutant K39R derived from Bacillus altitudinis
AJ1616 (K39R_RV) 27ctgttttggt gaaggacgcc ctctagacat gtc
332830DNAArtificial SequenceForward primer for
preparing the aminotransferase mutant S258G derived from Bacillus
altitudinis AJ1616 (S258G_FW) 28gttcgcctct actggtaaaa ttacgttccc
302930DNAArtificial SequenceReverse
primer for preparing the aminotransferase mutant S258G derived from
Bacillus altitudinis AJ1616 (S258G_RV) 29gggaacgtaa ttttaccagt
agaggcgaac 303036DNAArtificial
SequenceForward primer for preparing the aminotransferase mutant
T288G derived from Bacillus altitudinis AJ1616 (T288G_FW)
30cagctatcag ttcaaggcat tgggccagat aaaatc
363136DNAArtificial SequenceReverse primer for preparing the
aminotransferase mutant T288G derived from Bacillus altitudinis
AJ1616 (T288G_RV) 31gattttatct ggcccaatgc cttgaactga tagctg
363233DNAArtificial SequenceForward primer for preparing
the aminotransferase mutant I289A derived from Bacillus altitudinis
AJ1616 (I289A_FW) 32ctatcagttc aaaccgctgg gccagataaa atc
333333DNAArtificial SequenceReverse primer for
preparing the aminotransferase mutant I289A derived from Bacillus
altitudinis AJ1616 (I289A_RV) 33gattttatct ggcccagcgg tttgaactga tag
333428DNAArtificial SequenceForward
primer for preparing the aminotransferase mutant Q287E/T288G derived
from Bacillus altitudinis AJ1616 (Q287E/T288G_FW) 34cagctatcag
ttgaaggcat tgggccag
283528DNAArtificial SequenceReverse primer for preparing the
aminotransferase mutant Q287E/T288G derived from Bacillus
altitudinis AJ1616 (Q287E/T288G_RV) 35ctggcccaat gccttcaact gatagctg
283660DNAArtificial SequencePrimer for
preparing a DNA fragment for destroying aspC gene (aspC-L1)
36tttgagaaca ttaccgccgc tcctgccgac ccgattctgg gctgaagcct gcttttttat
603760DNAArtificial SequencePrimer for preparing a DNA fragment for
destroying aspC gene (aspC-R1) 37cagcactgcc acaatcgctt cgcacagcgg
agccatgtta tccgctcaag ttagtataaa 603830DNAArtificial SequencePrimer
for confirming the insertion of attL-cat-attR in the region of aspC
gene (aspC-up) 38aacctcttgg caacggtaaa aaagctgaac
303918DNAArtificial SequencePrimer for confirming the
insertion of attL-cat-attR in the region of aspC gene (attL-1)
39tagtgacctg ttcgttgc
184030DNAArtificial SequencePrimer for confirming the insertion of
attL-cat-attR in the region of aspC gene (aspC-down) 40gcctgcgcaa
agtcgtatgt ttggtctgga
304118DNAArtificial SequencePrimer for confirming the insertion of
attL-cat-attR in the region of aspC gene (attR-1) 41ttacgtttct cgttcagc
1842882DNAPseudomonas
putidaCDS(1)..(882) 42atg att atg ccg aaa gcc tcc cat cag gat ctg cgt ttt
gcg ttc cgt 48Met Ile Met Pro Lys Ala Ser His Gln Asp Leu Arg Phe
Ala Phe Arg 1 5 10
15 gaa ctg ctg gcc tct ggt agc tgt ttc cac acc gcg tca
gtt ttt gat 96Glu Leu Leu Ala Ser Gly Ser Cys Phe His Thr Ala Ser
Val Phe Asp 20 25
30 ccg atg agc gca cgt att gcg gcc gac ctg ggc ttc gaa
gtc ggt atc 144Pro Met Ser Ala Arg Ile Ala Ala Asp Leu Gly Phe Glu
Val Gly Ile 35 40 45
ctg ggc ggt agt gtc gcg tcc ctg caa gtg ctg gca gct
ccg gat ttt 192Leu Gly Gly Ser Val Ala Ser Leu Gln Val Leu Ala Ala
Pro Asp Phe 50 55 60
gcc ctg att acg ctg tct gaa ttc gtg gaa cag gca acc
cgt atc ggt 240Ala Leu Ile Thr Leu Ser Glu Phe Val Glu Gln Ala Thr
Arg Ile Gly 65 70 75
80 cgt gtt gct caa ctg ccg gtc ctg gca gat gca gac cat
ggt tat ggt 288Arg Val Ala Gln Leu Pro Val Leu Ala Asp Ala Asp His
Gly Tyr Gly 85 90
95 aac gca ctg aat gtt atg cgt acc gtc att gaa ctg gaa
cgt gct ggt 336Asn Ala Leu Asn Val Met Arg Thr Val Ile Glu Leu Glu
Arg Ala Gly 100 105
110 gtg gca gca ctg acc atc gaa gat acg ctg ctg ccg gcg
cag ttt ggt 384Val Ala Ala Leu Thr Ile Glu Asp Thr Leu Leu Pro Ala
Gln Phe Gly 115 120 125
cgc aaa agt acc gac ctg att ccg gtg gaa gaa ggc gtt
ggt aaa atc 432Arg Lys Ser Thr Asp Leu Ile Pro Val Glu Glu Gly Val
Gly Lys Ile 130 135 140
cgt gca gct ctg gaa gcc cgc gtt gat agc tct ctg tcc
att atc gcg 480Arg Ala Ala Leu Glu Ala Arg Val Asp Ser Ser Leu Ser
Ile Ile Ala 145 150 155
160 cgt acc aac gcc ggt gtc ctg agc acg gaa gaa att atc
gtg cgc acc 528Arg Thr Asn Ala Gly Val Leu Ser Thr Glu Glu Ile Ile
Val Arg Thr 165 170
175 cag tct tat caa aaa gca ggc gct gat ggt att tgc atg
gtc ggc gtg 576Gln Ser Tyr Gln Lys Ala Gly Ala Asp Gly Ile Cys Met
Val Gly Val 180 185
190 aaa gac ttt gaa cag ctg gaa caa atc gcg gaa cat ctg
acg gtg ccg 624Lys Asp Phe Glu Gln Leu Glu Gln Ile Ala Glu His Leu
Thr Val Pro 195 200 205
ctg atg ctg gtt acc tac ggt aac ccg aat ctg cgt gat
gac gaa cgt 672Leu Met Leu Val Thr Tyr Gly Asn Pro Asn Leu Arg Asp
Asp Glu Arg 210 215 220
ctg gca cgt ctg ggt gtt cgt att gtg gtt gat ggt cac
gcg gcc tat 720Leu Ala Arg Leu Gly Val Arg Ile Val Val Asp Gly His
Ala Ala Tyr 225 230 235
240 ttc gca gct atc aaa gcc acg tac gac tgt ctg cgt ctg
caa cgt ggc 768Phe Ala Ala Ile Lys Ala Thr Tyr Asp Cys Leu Arg Leu
Gln Arg Gly 245 250
255 cgc caa aac aaa tca gaa aat ctg tcg gca acc gaa ctg
agc cac acc 816Arg Gln Asn Lys Ser Glu Asn Leu Ser Ala Thr Glu Leu
Ser His Thr 260 265
270 tac acc cag ccg gaa gac tac att cgt tgg gca aaa gaa
tac atg agc 864Tyr Thr Gln Pro Glu Asp Tyr Ile Arg Trp Ala Lys Glu
Tyr Met Ser 275 280 285
gtt gaa gaa ctc gag tga
882Val Glu Glu Leu Glu
290
43293PRTPseudomonas putida 43Met Ile Met Pro Lys Ala
Ser His Gln Asp Leu Arg Phe Ala Phe Arg 1 5
10 15 Glu Leu Leu Ala Ser Gly Ser Cys Phe His Thr
Ala Ser Val Phe Asp 20 25
30 Pro Met Ser Ala Arg Ile Ala Ala Asp Leu Gly Phe Glu Val Gly
Ile 35 40 45 Leu
Gly Gly Ser Val Ala Ser Leu Gln Val Leu Ala Ala Pro Asp Phe 50
55 60 Ala Leu Ile Thr Leu Ser
Glu Phe Val Glu Gln Ala Thr Arg Ile Gly 65 70
75 80 Arg Val Ala Gln Leu Pro Val Leu Ala Asp Ala
Asp His Gly Tyr Gly 85 90
95 Asn Ala Leu Asn Val Met Arg Thr Val Ile Glu Leu Glu Arg Ala Gly
100 105 110 Val Ala
Ala Leu Thr Ile Glu Asp Thr Leu Leu Pro Ala Gln Phe Gly 115
120 125 Arg Lys Ser Thr Asp Leu Ile
Pro Val Glu Glu Gly Val Gly Lys Ile 130 135
140 Arg Ala Ala Leu Glu Ala Arg Val Asp Ser Ser Leu
Ser Ile Ile Ala 145 150 155
160 Arg Thr Asn Ala Gly Val Leu Ser Thr Glu Glu Ile Ile Val Arg Thr
165 170 175 Gln Ser Tyr
Gln Lys Ala Gly Ala Asp Gly Ile Cys Met Val Gly Val 180
185 190 Lys Asp Phe Glu Gln Leu Glu Gln
Ile Ala Glu His Leu Thr Val Pro 195 200
205 Leu Met Leu Val Thr Tyr Gly Asn Pro Asn Leu Arg Asp
Asp Glu Arg 210 215 220
Leu Ala Arg Leu Gly Val Arg Ile Val Val Asp Gly His Ala Ala Tyr 225
230 235 240 Phe Ala Ala Ile
Lys Ala Thr Tyr Asp Cys Leu Arg Leu Gln Arg Gly 245
250 255 Arg Gln Asn Lys Ser Glu Asn Leu Ser
Ala Thr Glu Leu Ser His Thr 260 265
270 Tyr Thr Gln Pro Glu Asp Tyr Ile Arg Trp Ala Lys Glu Tyr
Met Ser 275 280 285
Val Glu Glu Leu Glu 290 4420PRTRhizobium radiobacter
44Ala Phe Leu Ala Asp Ile Leu Ser Arg Val Lys Pro Ser Ala Thr Ile 1
5 10 15 Ala Val Thr Gln
20 4527DNAArtificial SequenceForward primer which is designed
based on the genomic DNA sequence from Agrobacterium tumefaciens
str. C58 (Ag-u100-f) 45ctggtgcaga taagccggct tttgacc
274627DNAArtificial SequenceReverse primer which
is designed based on the genomic DNA sequence from Agrobacterium
tumefaciens str. C58 (Ag-d100-r) 46ccaccttcat catgctgctg tttctcg
27471203DNARhizobium
radiobacterCDS(1)..(1203) 47atg gcc ttc ctt gcc gac att ctc tcc cgc gta
aag cca tcc gcc acc 48Met Ala Phe Leu Ala Asp Ile Leu Ser Arg Val
Lys Pro Ser Ala Thr 1 5 10
15 atc gcc gtt acc cag aaa gcc cgt gag ctg aaa
gcg aag ggc cgc gat 96Ile Ala Val Thr Gln Lys Ala Arg Glu Leu Lys
Ala Lys Gly Arg Asp 20 25
30 gtg atc agc ctt ggc gcc ggc gag ccg gat ttc
gat acg ccc gat aat 144Val Ile Ser Leu Gly Ala Gly Glu Pro Asp Phe
Asp Thr Pro Asp Asn 35 40
45 atc aag gaa gcg gcc atc gac gcc atc aag cgc
ggc gaa acg aaa tac 192Ile Lys Glu Ala Ala Ile Asp Ala Ile Lys Arg
Gly Glu Thr Lys Tyr 50 55
60 acg ccc gtt tcc ggc att ccg gaa ctg cgc aag
gcc att gct gac aag 240Thr Pro Val Ser Gly Ile Pro Glu Leu Arg Lys
Ala Ile Ala Asp Lys 65 70 75
80 ttc aag cgc gaa aac ggc ctc gac tac aag ccg
gag cag acg att gtc 288Phe Lys Arg Glu Asn Gly Leu Asp Tyr Lys Pro
Glu Gln Thr Ile Val 85 90
95 ggc acc ggc ggc aag cag ata ctt ttc aac gcc
ttc atg gcc acc ctc 336Gly Thr Gly Gly Lys Gln Ile Leu Phe Asn Ala
Phe Met Ala Thr Leu 100 105
110 aac ccg ggt gac gaa gtc gtc att ccc gcg cct
tac tgg gtc agc tac 384Asn Pro Gly Asp Glu Val Val Ile Pro Ala Pro
Tyr Trp Val Ser Tyr 115 120
125 ccg gaa atg gtg gcg atc tgc ggc ggc acg cct
gta ttc gtc gac acc 432Pro Glu Met Val Ala Ile Cys Gly Gly Thr Pro
Val Phe Val Asp Thr 130 135
140 acg ctt gaa gac aat ttc aag ctg acg ccg gaa
gcg ctg gaa aag gcg 480Thr Leu Glu Asp Asn Phe Lys Leu Thr Pro Glu
Ala Leu Glu Lys Ala 145 150 155
160 atc aca ccg aag aca aag tgg ttc gtc ttc aac
tcg cct tca aac ccc 528Ile Thr Pro Lys Thr Lys Trp Phe Val Phe Asn
Ser Pro Ser Asn Pro 165 170
175 tcg ggt gcc gcc tat tcg cat gac gaa ctg aag
gcg ctg acg gac gtg 576Ser Gly Ala Ala Tyr Ser His Asp Glu Leu Lys
Ala Leu Thr Asp Val 180 185
190 ctg gtc aag cat ccg caa gtc tgg gtg ctg acg
gac gac atg tac gag 624Leu Val Lys His Pro Gln Val Trp Val Leu Thr
Asp Asp Met Tyr Glu 195 200
205 cac ctc acc tat ggc gat ttc aaa ttc gtc acc
ccg gtt gag gtt gag 672His Leu Thr Tyr Gly Asp Phe Lys Phe Val Thr
Pro Val Glu Val Glu 210 215
220 cct gcg ctc tat gat cgc acg ctg acg atg aac
ggc gtc tcc aag gcc 720Pro Ala Leu Tyr Asp Arg Thr Leu Thr Met Asn
Gly Val Ser Lys Ala 225 230 235
240 tat gcc atg acc ggc tgg cgt atc ggt tac gcg
gcc ggc ccg ctg ccg 768Tyr Ala Met Thr Gly Trp Arg Ile Gly Tyr Ala
Ala Gly Pro Leu Pro 245 250
255 ctg atc aag gcc atg gac atg atc cag ggc cag
cag acc tcg ggc gcc 816Leu Ile Lys Ala Met Asp Met Ile Gln Gly Gln
Gln Thr Ser Gly Ala 260 265
270 agc tcg atc gcg caa tgg gcg gct gtt gaa gcg
ctg aac ggc acg cag 864Ser Ser Ile Ala Gln Trp Ala Ala Val Glu Ala
Leu Asn Gly Thr Gln 275 280
285 gat ttc att ccg acc aac aag aaa atc ttc gaa
ggt cgc cgt gat ctc 912Asp Phe Ile Pro Thr Asn Lys Lys Ile Phe Glu
Gly Arg Arg Asp Leu 290 295
300 gtc gtc tcc atg ctc aac cag gcc aag ggc atc
aat tgc ccg tca ccg 960Val Val Ser Met Leu Asn Gln Ala Lys Gly Ile
Asn Cys Pro Ser Pro 305 310 315
320 gaa ggc gca ttc tac gtc tac ccg tcc tgc gcc
ggc ctg att ggc aag 1008Glu Gly Ala Phe Tyr Val Tyr Pro Ser Cys Ala
Gly Leu Ile Gly Lys 325 330
335 acc gcg cca tct ggc aag gtc atc gag tcg gat
gtg gac ttc gtc tcc 1056Thr Ala Pro Ser Gly Lys Val Ile Glu Ser Asp
Val Asp Phe Val Ser 340 345
350 gag ctt ctg gaa gcc gaa ggc gtc gcc gtc gtg
cag gga tcg gct ttc 1104Glu Leu Leu Glu Ala Glu Gly Val Ala Val Val
Gln Gly Ser Ala Phe 355 360
365 ggc ctc ggc ccg aac ttc cgc att tcc tac gcc
acg tcg gaa agc ctg 1152Gly Leu Gly Pro Asn Phe Arg Ile Ser Tyr Ala
Thr Ser Glu Ser Leu 370 375
380 ctg gaa gaa gcc tgc aag cgc att cag cgt ttc
tgc gcc gat tgc cgt 1200Leu Glu Glu Ala Cys Lys Arg Ile Gln Arg Phe
Cys Ala Asp Cys Arg 385 390 395
400 tga
120348400PRTRhizobium radiobacter 48Met Ala Phe
Leu Ala Asp Ile Leu Ser Arg Val Lys Pro Ser Ala Thr 1 5
10 15 Ile Ala Val Thr Gln Lys Ala Arg
Glu Leu Lys Ala Lys Gly Arg Asp 20 25
30 Val Ile Ser Leu Gly Ala Gly Glu Pro Asp Phe Asp Thr
Pro Asp Asn 35 40 45
Ile Lys Glu Ala Ala Ile Asp Ala Ile Lys Arg Gly Glu Thr Lys Tyr 50
55 60 Thr Pro Val Ser
Gly Ile Pro Glu Leu Arg Lys Ala Ile Ala Asp Lys 65 70
75 80 Phe Lys Arg Glu Asn Gly Leu Asp Tyr
Lys Pro Glu Gln Thr Ile Val 85 90
95 Gly Thr Gly Gly Lys Gln Ile Leu Phe Asn Ala Phe Met Ala
Thr Leu 100 105 110
Asn Pro Gly Asp Glu Val Val Ile Pro Ala Pro Tyr Trp Val Ser Tyr
115 120 125 Pro Glu Met Val
Ala Ile Cys Gly Gly Thr Pro Val Phe Val Asp Thr 130
135 140 Thr Leu Glu Asp Asn Phe Lys Leu
Thr Pro Glu Ala Leu Glu Lys Ala 145 150
155 160 Ile Thr Pro Lys Thr Lys Trp Phe Val Phe Asn Ser
Pro Ser Asn Pro 165 170
175 Ser Gly Ala Ala Tyr Ser His Asp Glu Leu Lys Ala Leu Thr Asp Val
180 185 190 Leu Val Lys
His Pro Gln Val Trp Val Leu Thr Asp Asp Met Tyr Glu 195
200 205 His Leu Thr Tyr Gly Asp Phe Lys
Phe Val Thr Pro Val Glu Val Glu 210 215
220 Pro Ala Leu Tyr Asp Arg Thr Leu Thr Met Asn Gly Val
Ser Lys Ala 225 230 235
240 Tyr Ala Met Thr Gly Trp Arg Ile Gly Tyr Ala Ala Gly Pro Leu Pro
245 250 255 Leu Ile Lys Ala
Met Asp Met Ile Gln Gly Gln Gln Thr Ser Gly Ala 260
265 270 Ser Ser Ile Ala Gln Trp Ala Ala Val
Glu Ala Leu Asn Gly Thr Gln 275 280
285 Asp Phe Ile Pro Thr Asn Lys Lys Ile Phe Glu Gly Arg Arg
Asp Leu 290 295 300
Val Val Ser Met Leu Asn Gln Ala Lys Gly Ile Asn Cys Pro Ser Pro 305
310 315 320 Glu Gly Ala Phe Tyr
Val Tyr Pro Ser Cys Ala Gly Leu Ile Gly Lys 325
330 335 Thr Ala Pro Ser Gly Lys Val Ile Glu Ser
Asp Val Asp Phe Val Ser 340 345
350 Glu Leu Leu Glu Ala Glu Gly Val Ala Val Val Gln Gly Ser Ala
Phe 355 360 365 Gly
Leu Gly Pro Asn Phe Arg Ile Ser Tyr Ala Thr Ser Glu Ser Leu 370
375 380 Leu Glu Glu Ala Cys Lys
Arg Ile Gln Arg Phe Cys Ala Asp Cys Arg 385 390
395 400 4935DNAArtificial SequenceForward primer
for amplifying DNA fragment containing aminotransferase gene derived
from Rhizobium radiobacter (3976AT-Nde-f) 49ggaattccat atggccttcc
ttgccgacat tctct 355037DNAArtificial
SequenceReverse primer for amplifying DNA fragment containing
aminotransferase gene derived from Rhizobium radiobacter
(3976-xho-r) 50actccgctcg agacggcaat cggcgcagaa acgctga
375120PRTRhizobium sp. 51Ala Phe Leu Ala Asp Ile Leu Ser Arg
Val Lys Pro Ser Ala Thr Ile 1 5 10
15 Ala Val Thr Gln 20 521203DNARhizobium
sp.CDS(1)..(1203) 52atg gcc ttc ctt gcc gac att ctc tcc cgc gta aag cca
tcc gcc acc 48Met Ala Phe Leu Ala Asp Ile Leu Ser Arg Val Lys Pro
Ser Ala Thr 1 5 10
15 atc gcc gtt acc cag aaa gcc cgt gag ctg aaa gcc aag
ggt cgc gat 96Ile Ala Val Thr Gln Lys Ala Arg Glu Leu Lys Ala Lys
Gly Arg Asp 20 25
30 gtg att agc ctt ggc gcc ggc gag ccg gat ttc gat acg
ccc gat aat 144Val Ile Ser Leu Gly Ala Gly Glu Pro Asp Phe Asp Thr
Pro Asp Asn 35 40 45
atc aag gaa gcg gcc att gac gcc atc aag cgc ggc gaa
acc aaa tac 192Ile Lys Glu Ala Ala Ile Asp Ala Ile Lys Arg Gly Glu
Thr Lys Tyr 50 55 60
acg ccg gtt tcc ggc att cct gaa ctg cgc aag gcg att
gcc gac aag 240Thr Pro Val Ser Gly Ile Pro Glu Leu Arg Lys Ala Ile
Ala Asp Lys 65 70 75
80 ttc aag cgt gaa aac ggc ctc gac tac aag ccg gaa cag
acc atc gtc 288Phe Lys Arg Glu Asn Gly Leu Asp Tyr Lys Pro Glu Gln
Thr Ile Val 85 90
95 ggc acc ggc ggc aag cag atc ctc ttc aac gcc ttc atg
gcg acg ctg 336Gly Thr Gly Gly Lys Gln Ile Leu Phe Asn Ala Phe Met
Ala Thr Leu 100 105
110 aac ccc ggt gat gag gtc gtc att ccc gcg cct tac tgg
gtc agc tac 384Asn Pro Gly Asp Glu Val Val Ile Pro Ala Pro Tyr Trp
Val Ser Tyr 115 120 125
ccg gaa atg gtg gcg atc tgc ggc ggt acg ccg gtt ttc
gtc aac gcc 432Pro Glu Met Val Ala Ile Cys Gly Gly Thr Pro Val Phe
Val Asn Ala 130 135 140
acg ctc gaa gac aat ttc aag ctg aag ccg gaa gcg ctg
gaa aag gct 480Thr Leu Glu Asp Asn Phe Lys Leu Lys Pro Glu Ala Leu
Glu Lys Ala 145 150 155
160 atc acg ccg aag aca aag tgg ttc gtc ttc aac tcg cct
tcc aac ccc 528Ile Thr Pro Lys Thr Lys Trp Phe Val Phe Asn Ser Pro
Ser Asn Pro 165 170
175 tcg ggt gcg gcc tat tcg cat gag gag ttg aag gcg ctg
acg gac gtg 576Ser Gly Ala Ala Tyr Ser His Glu Glu Leu Lys Ala Leu
Thr Asp Val 180 185
190 ctg gtc aag cat ccg cat gtc tgg gtg ctg acg gac gac
atg tat gag 624Leu Val Lys His Pro His Val Trp Val Leu Thr Asp Asp
Met Tyr Glu 195 200 205
cac ctg acc tat ggc gat ttc aaa ttc gtc acc cct gtg
gaa gtc gag 672His Leu Thr Tyr Gly Asp Phe Lys Phe Val Thr Pro Val
Glu Val Glu 210 215 220
cct tcg ctc tat gac cgg acg ttg acg atg aac ggc gtc
tcc aag gcc 720Pro Ser Leu Tyr Asp Arg Thr Leu Thr Met Asn Gly Val
Ser Lys Ala 225 230 235
240 tat gcc atg acc ggc tgg cgt atc ggt tac gct gcc ggc
ccg ctg ccg 768Tyr Ala Met Thr Gly Trp Arg Ile Gly Tyr Ala Ala Gly
Pro Leu Pro 245 250
255 ctg atc aag gcc atg gac atg atc cag ggc cag cag acc
tcg ggc gca 816Leu Ile Lys Ala Met Asp Met Ile Gln Gly Gln Gln Thr
Ser Gly Ala 260 265
270 agc tcg atc gca cag tgg gcc gct gtc gaa gct ctg aac
ggc acg cag 864Ser Ser Ile Ala Gln Trp Ala Ala Val Glu Ala Leu Asn
Gly Thr Gln 275 280 285
gat ttc att ccg gcg aac aag aag atc ttc gaa ggc cgt
cgc gat ctc 912Asp Phe Ile Pro Ala Asn Lys Lys Ile Phe Glu Gly Arg
Arg Asp Leu 290 295 300
gtc gtt tcc atg ctc aac cag gcc aag ggc atc agc tgc
ccg tca ccg 960Val Val Ser Met Leu Asn Gln Ala Lys Gly Ile Ser Cys
Pro Ser Pro 305 310 315
320 gaa ggt gca ttc tac gtc tac ccg tcc tgc gcc ggc ttg
atc ggc aag 1008Glu Gly Ala Phe Tyr Val Tyr Pro Ser Cys Ala Gly Leu
Ile Gly Lys 325 330
335 acc gcg cct tcg ggc aag gtc atc gag acg gat acg gat
ttc gtt tcc 1056Thr Ala Pro Ser Gly Lys Val Ile Glu Thr Asp Thr Asp
Phe Val Ser 340 345
350 gag ctt ctg gaa gcc gaa ggc gtt gcc gtc gtg cag gga
tcg gct ttc 1104Glu Leu Leu Glu Ala Glu Gly Val Ala Val Val Gln Gly
Ser Ala Phe 355 360 365
ggc ctt ggc ccg aac ttc cgc atc tcc tac gcc acg tcg
gaa act ctt 1152Gly Leu Gly Pro Asn Phe Arg Ile Ser Tyr Ala Thr Ser
Glu Thr Leu 370 375 380
ctc gaa gag gcc tgc aag cgc att cag cgt ttc tgc gcc
gat tgc cgc 1200Leu Glu Glu Ala Cys Lys Arg Ile Gln Arg Phe Cys Ala
Asp Cys Arg 385 390 395
400 taa
120353400PRTRhizobium sp. 53Met Ala Phe Leu Ala Asp Ile
Leu Ser Arg Val Lys Pro Ser Ala Thr 1 5
10 15 Ile Ala Val Thr Gln Lys Ala Arg Glu Leu Lys
Ala Lys Gly Arg Asp 20 25
30 Val Ile Ser Leu Gly Ala Gly Glu Pro Asp Phe Asp Thr Pro Asp
Asn 35 40 45 Ile
Lys Glu Ala Ala Ile Asp Ala Ile Lys Arg Gly Glu Thr Lys Tyr 50
55 60 Thr Pro Val Ser Gly Ile
Pro Glu Leu Arg Lys Ala Ile Ala Asp Lys 65 70
75 80 Phe Lys Arg Glu Asn Gly Leu Asp Tyr Lys Pro
Glu Gln Thr Ile Val 85 90
95 Gly Thr Gly Gly Lys Gln Ile Leu Phe Asn Ala Phe Met Ala Thr Leu
100 105 110 Asn Pro
Gly Asp Glu Val Val Ile Pro Ala Pro Tyr Trp Val Ser Tyr 115
120 125 Pro Glu Met Val Ala Ile Cys
Gly Gly Thr Pro Val Phe Val Asn Ala 130 135
140 Thr Leu Glu Asp Asn Phe Lys Leu Lys Pro Glu Ala
Leu Glu Lys Ala 145 150 155
160 Ile Thr Pro Lys Thr Lys Trp Phe Val Phe Asn Ser Pro Ser Asn Pro
165 170 175 Ser Gly Ala
Ala Tyr Ser His Glu Glu Leu Lys Ala Leu Thr Asp Val 180
185 190 Leu Val Lys His Pro His Val Trp
Val Leu Thr Asp Asp Met Tyr Glu 195 200
205 His Leu Thr Tyr Gly Asp Phe Lys Phe Val Thr Pro Val
Glu Val Glu 210 215 220
Pro Ser Leu Tyr Asp Arg Thr Leu Thr Met Asn Gly Val Ser Lys Ala 225
230 235 240 Tyr Ala Met Thr
Gly Trp Arg Ile Gly Tyr Ala Ala Gly Pro Leu Pro 245
250 255 Leu Ile Lys Ala Met Asp Met Ile Gln
Gly Gln Gln Thr Ser Gly Ala 260 265
270 Ser Ser Ile Ala Gln Trp Ala Ala Val Glu Ala Leu Asn Gly
Thr Gln 275 280 285
Asp Phe Ile Pro Ala Asn Lys Lys Ile Phe Glu Gly Arg Arg Asp Leu 290
295 300 Val Val Ser Met Leu
Asn Gln Ala Lys Gly Ile Ser Cys Pro Ser Pro 305 310
315 320 Glu Gly Ala Phe Tyr Val Tyr Pro Ser Cys
Ala Gly Leu Ile Gly Lys 325 330
335 Thr Ala Pro Ser Gly Lys Val Ile Glu Thr Asp Thr Asp Phe Val
Ser 340 345 350 Glu
Leu Leu Glu Ala Glu Gly Val Ala Val Val Gln Gly Ser Ala Phe 355
360 365 Gly Leu Gly Pro Asn Phe
Arg Ile Ser Tyr Ala Thr Ser Glu Thr Leu 370 375
380 Leu Glu Glu Ala Cys Lys Arg Ile Gln Arg Phe
Cys Ala Asp Cys Arg 385 390 395
400 5435DNAArtificial SequenceForward primer for amplifying DNA
fragment containing aminotransferase gene derived from Rhizobium sp.
(12469AT-Nde-f) 54ggaattccat atggccttcc ttgccgacat tctct
355537DNAArtificial SequenceReverse primer for
amplifying DNA fragment containing aminotransferase gene derived
from Rhizobium sp. (12469-xho-r) 55actccgctcg aggcggcaat cggcgcagaa
acgctga 375626DNAArtificial SequenceForward
primer which is designed based on the genomic DNA sequence from
Corynebacterium ammoniagenes DSM20306 (Co-d50-r) 56cttccttgga
acaagtcgag gaagac
265726DNAArtificial SequenceReverse primer which is designed based on a
homologus region between the genomic DNA sequences corresponding
to the aspartate aminotransferases from Corynebacterium striatum
ATCC6940 (ZP_03935516) and from Corynebacterium ammoniagenes
DSM20306 57gctatcgcac aattccaccg cacctt
265826DNAArtificial SequenceForward primer for amplifying DNA
fragment containing aminotransferase gene derived from
Corynebacterium ammoniagenes (Co-890-r) 58acatcgttaa gcaagcgaac
caccag 265925DNAArtificial
SequenceReverse primer for amplifying DNA fragment containing
aminotransferase gene derived from Corynebacterium ammoniagenes
(Co-1060-r) 59gaaagacaag cgaatgtggt gctcg
25601134DNACorynebacterium ammoniagenesCDS(1)..(1134) 60atg agc
cac atc gct caa cgc atc ctt gac cag cgt caa gca tct ctt 48Met Ser
His Ile Ala Gln Arg Ile Leu Asp Gln Arg Gln Ala Ser Leu 1
5 10 15 cgc ccg
ccg ctt ggg gtg gtg ccg ccc ggc gcg gtg tcg ttg gcg ttg 96Arg Pro
Pro Leu Gly Val Val Pro Pro Gly Ala Val Ser Leu Ala Leu
20 25 30 ggc gag
ccg gac ttt gcc cca ccg cag gcg gtt atc gat gcc acc acg 144Gly Glu
Pro Asp Phe Ala Pro Pro Gln Ala Val Ile Asp Ala Thr Thr
35 40 45 caa gca
gtc gcc caa ggc cgc acc aac tac acg gat cag cac ggt atc 192Gln Ala
Val Ala Gln Gly Arg Thr Asn Tyr Thr Asp Gln His Gly Ile 50
55 60 gct gag
ctt cgc gat gcc ctc ctc gcc gcc ctt ccc acc cgc ccc tct 240Ala Glu
Leu Arg Asp Ala Leu Leu Ala Ala Leu Pro Thr Arg Pro Ser 65
70 75 80 aac tgg
gac cgc gac aat att gtg gtg aca cac ggt gca acc gcg gga 288Asn Trp
Asp Arg Asp Asn Ile Val Val Thr His Gly Ala Thr Ala Gly
85 90 95 ctg ggt
gcg ctg ttt ttc gcg ctg att gaa ccc ggt gac aag gtc gtt 336Leu Gly
Ala Leu Phe Phe Ala Leu Ile Glu Pro Gly Asp Lys Val Val
100 105 110 atc cca
cag cct gcg tat tct ttg tac gcc gac cag gtg gtt tta gcc 384Ile Pro
Gln Pro Ala Tyr Ser Leu Tyr Ala Asp Gln Val Val Leu Ala
115 120 125 ggc ggc
acc gtg gaa ttt gtt ccc atg ggc aag gac ctc cac ttt gat 432Gly Gly
Thr Val Glu Phe Val Pro Met Gly Lys Asp Leu His Phe Asp 130
135 140 ttt gac
cag ctt gcc acg gcg ctt gac ggc gca aag atg gtg gtc ttt 480Phe Asp
Gln Leu Ala Thr Ala Leu Asp Gly Ala Lys Met Val Val Phe 145
150 155 160 tct aat
cct tct aat ccc aat gga atc gtg cat acc cgc gat gag ctg 528Ser Asn
Pro Ser Asn Pro Asn Gly Ile Val His Thr Arg Asp Glu Leu
165 170 175 gaa agg
ctt gca cag ctt ctc gat gcc acc gat acc ctc gtc gtt tcc 576Glu Arg
Leu Ala Gln Leu Leu Asp Ala Thr Asp Thr Leu Val Val Ser
180 185 190 gac gaa
gcc tac tcg gcc ctc acc tat acc gcc gaa cca ttc acc tcc 624Asp Glu
Ala Tyr Ser Ala Leu Thr Tyr Thr Ala Glu Pro Phe Thr Ser
195 200 205 gct tta
gag gtt ccc ggc ttg cag gag cgc acg tta tac gtg cag acc 672Ala Leu
Glu Val Pro Gly Leu Gln Glu Arg Thr Leu Tyr Val Gln Thr 210
215 220 ttt tcc
aag aaa tac tgc atg acg ggc ttc cgc gtc ggc tac gtc gcg 720Phe Ser
Lys Lys Tyr Cys Met Thr Gly Phe Arg Val Gly Tyr Val Ala 225
230 235 240 ggc gcg
aga gat ttg atc gct gcg att gcg cag atg cac cgc acc ttc 768Gly Ala
Arg Asp Leu Ile Ala Ala Ile Ala Gln Met His Arg Thr Phe
245 250 255 aac ggc
tca gtg tcg gag cag gcg cag ctg gca gcg ctc gcc gcg gta 816Asn Gly
Ser Val Ser Glu Gln Ala Gln Leu Ala Ala Leu Ala Ala Val
260 265 270 tct ctg
ccg gag tct gtg gtc aca ccg atg ttg gaa gaa tac gcc cag 864Ser Leu
Pro Glu Ser Val Val Thr Pro Met Leu Glu Glu Tyr Ala Gln
275 280 285 cgc cgc
gac ctg gtg gtt cgc ttg ctt aac gat gtc ccc cac gtc caa 912Arg Arg
Asp Leu Val Val Arg Leu Leu Asn Asp Val Pro His Val Gln 290
295 300 ctc ttc
gag ccc gaa ggc gcg ttt tat gca ttc ttc tct tat gac ttg 960Leu Phe
Glu Pro Glu Gly Ala Phe Tyr Ala Phe Phe Ser Tyr Asp Leu 305
310 315 320 gac aag
ccc tct tcc cag gta gca gcc gag ctg gcc gaa cgc ggt gta 1008Asp Lys
Pro Ser Ser Gln Val Ala Ala Glu Leu Ala Glu Arg Gly Val
325 330 335 cta gtg
cgc gct ggt gcc gaa tat ggc ccc gcc gcc gag cac cac att 1056Leu Val
Arg Ala Gly Ala Glu Tyr Gly Pro Ala Ala Glu His His Ile
340 345 350 cgc ttg
tct ttc gca gcc tcg caa gca gat atc gaa cgt ggc att gga 1104Arg Leu
Ser Phe Ala Ala Ser Gln Ala Asp Ile Glu Arg Gly Ile Gly
355 360 365 atc att
cgc caa tac ttc gaa aag tcc tag 1134Ile Ile
Arg Gln Tyr Phe Glu Lys Ser 370
375
61377PRTCorynebacterium ammoniagenes 61Met Ser His Ile Ala Gln Arg Ile
Leu Asp Gln Arg Gln Ala Ser Leu 1 5 10
15 Arg Pro Pro Leu Gly Val Val Pro Pro Gly Ala Val Ser
Leu Ala Leu 20 25 30
Gly Glu Pro Asp Phe Ala Pro Pro Gln Ala Val Ile Asp Ala Thr Thr
35 40 45 Gln Ala Val Ala
Gln Gly Arg Thr Asn Tyr Thr Asp Gln His Gly Ile 50
55 60 Ala Glu Leu Arg Asp Ala Leu Leu
Ala Ala Leu Pro Thr Arg Pro Ser 65 70
75 80 Asn Trp Asp Arg Asp Asn Ile Val Val Thr His Gly
Ala Thr Ala Gly 85 90
95 Leu Gly Ala Leu Phe Phe Ala Leu Ile Glu Pro Gly Asp Lys Val Val
100 105 110 Ile Pro Gln
Pro Ala Tyr Ser Leu Tyr Ala Asp Gln Val Val Leu Ala 115
120 125 Gly Gly Thr Val Glu Phe Val Pro
Met Gly Lys Asp Leu His Phe Asp 130 135
140 Phe Asp Gln Leu Ala Thr Ala Leu Asp Gly Ala Lys Met
Val Val Phe 145 150 155
160 Ser Asn Pro Ser Asn Pro Asn Gly Ile Val His Thr Arg Asp Glu Leu
165 170 175 Glu Arg Leu Ala
Gln Leu Leu Asp Ala Thr Asp Thr Leu Val Val Ser 180
185 190 Asp Glu Ala Tyr Ser Ala Leu Thr Tyr
Thr Ala Glu Pro Phe Thr Ser 195 200
205 Ala Leu Glu Val Pro Gly Leu Gln Glu Arg Thr Leu Tyr Val
Gln Thr 210 215 220
Phe Ser Lys Lys Tyr Cys Met Thr Gly Phe Arg Val Gly Tyr Val Ala 225
230 235 240 Gly Ala Arg Asp Leu
Ile Ala Ala Ile Ala Gln Met His Arg Thr Phe 245
250 255 Asn Gly Ser Val Ser Glu Gln Ala Gln Leu
Ala Ala Leu Ala Ala Val 260 265
270 Ser Leu Pro Glu Ser Val Val Thr Pro Met Leu Glu Glu Tyr Ala
Gln 275 280 285 Arg
Arg Asp Leu Val Val Arg Leu Leu Asn Asp Val Pro His Val Gln 290
295 300 Leu Phe Glu Pro Glu Gly
Ala Phe Tyr Ala Phe Phe Ser Tyr Asp Leu 305 310
315 320 Asp Lys Pro Ser Ser Gln Val Ala Ala Glu Leu
Ala Glu Arg Gly Val 325 330
335 Leu Val Arg Ala Gly Ala Glu Tyr Gly Pro Ala Ala Glu His His Ile
340 345 350 Arg Leu
Ser Phe Ala Ala Ser Gln Ala Asp Ile Glu Arg Gly Ile Gly 355
360 365 Ile Ile Arg Gln Tyr Phe Glu
Lys Ser 370 375 6235DNAArtificial
SequenceForward primer for amplifying DNA fragment containing
aminotransferase gene derived from Corynebacterium ammoniagenes
(1444AT-Nde-f) 62ggaattccat atgagccaca tcgctcaacg catcc
356337DNAArtificial SequenceReverse primer for amplifying
DNA fragment containing aminotransferase gene derived from
Corynebacterium ammoniagenes (1444-xho-r) 63actccgctcg agggactttt
cgaagtattg gcgaatg 37641275DNADeinococcus
geothermalisCDS(1)..(1275) 64atg acc aaa gaa gca tcc cgc ccg gca ctg gac
ctg gct cgt caa gcg 48Met Thr Lys Glu Ala Ser Arg Pro Ala Leu Asp
Leu Ala Arg Gln Ala 1 5 10
15 tat gaa gca ttt aaa gct cgt ggc ctg aat ctg
aat atg cag cgt ggt 96Tyr Glu Ala Phe Lys Ala Arg Gly Leu Asn Leu
Asn Met Gln Arg Gly 20 25
30 caa ccg gct gat gcg gac ttt gat ctg tct aac
ggc ctg ctg acc gtt 144Gln Pro Ala Asp Ala Asp Phe Asp Leu Ser Asn
Gly Leu Leu Thr Val 35 40
45 ctg ggt gcc gaa gac gtc cgt atg gac ggc ctg
gat ctg cgc aat tat 192Leu Gly Ala Glu Asp Val Arg Met Asp Gly Leu
Asp Leu Arg Asn Tyr 50 55
60 ccg ggc ggt gtg gca ggt ctg ccg agc gcc cgc
gca ctg ttt gcc ggt 240Pro Gly Gly Val Ala Gly Leu Pro Ser Ala Arg
Ala Leu Phe Ala Gly 65 70 75
80 tac ctg gat gtt aaa gca gaa aac gtt ctg gtc
tgg aac aat agc tct 288Tyr Leu Asp Val Lys Ala Glu Asn Val Leu Val
Trp Asn Asn Ser Ser 85 90
95 ctg gaa ctg caa ggt ctg gtt ctg acc ttc gcc
ctg ctg cat ggt gtc 336Leu Glu Leu Gln Gly Leu Val Leu Thr Phe Ala
Leu Leu His Gly Val 100 105
110 cgt ggt agc acg ggt ccg tgg ctg tct caa acc
ccg aaa atg att gtg 384Arg Gly Ser Thr Gly Pro Trp Leu Ser Gln Thr
Pro Lys Met Ile Val 115 120
125 acg gtt ccg ggc tat gat cgc cac ttt ctg ctg
ctg caa acc ctg ggt 432Thr Val Pro Gly Tyr Asp Arg His Phe Leu Leu
Leu Gln Thr Leu Gly 130 135
140 ttc gaa ctg ctg acg gtg gac atg caa agc gat
ggc ccg gac gtc gat 480Phe Glu Leu Leu Thr Val Asp Met Gln Ser Asp
Gly Pro Asp Val Asp 145 150 155
160 gcc gtg gaa cgt ctg gca ggc acc gat ccg tct
gtg aaa ggt att ctg 528Ala Val Glu Arg Leu Ala Gly Thr Asp Pro Ser
Val Lys Gly Ile Leu 165 170
175 ttt gtt ccg acc tac tca aac ccg ggc ggt gaa
acg atc tcg ctg gaa 576Phe Val Pro Thr Tyr Ser Asn Pro Gly Gly Glu
Thr Ile Ser Leu Glu 180 185
190 aaa gct cgt cgc ctg gca ggt ctg caa gcg gcc
gca ccg gac ttt acg 624Lys Ala Arg Arg Leu Ala Gly Leu Gln Ala Ala
Ala Pro Asp Phe Thr 195 200
205 att ttc gct gat gac gcg tat cgt gtc cat cac
ctg gtg gaa gaa gat 672Ile Phe Ala Asp Asp Ala Tyr Arg Val His His
Leu Val Glu Glu Asp 210 215
220 cgc gcc gaa ccg gtg aat ttc gtg gtt ctg gcc
cgt gac gca ggt tac 720Arg Ala Glu Pro Val Asn Phe Val Val Leu Ala
Arg Asp Ala Gly Tyr 225 230 235
240 ccg gat cgt gcc ttt gtt ttc gca tca acc tcg
aaa atc acg ttt gct 768Pro Asp Arg Ala Phe Val Phe Ala Ser Thr Ser
Lys Ile Thr Phe Ala 245 250
255 ggt gca ggt ctg ggt ttc gtg gcg agt tcc gaa
gat aac att cgt tgg 816Gly Ala Gly Leu Gly Phe Val Ala Ser Ser Glu
Asp Asn Ile Arg Trp 260 265
270 ctg agt aaa tat ctg ggc gcg cag tcc atc ggt
ccg aat aaa gtc gaa 864Leu Ser Lys Tyr Leu Gly Ala Gln Ser Ile Gly
Pro Asn Lys Val Glu 275 280
285 caa gcc cgt cat gtg aaa ttt ctg acc gaa tac
ccg ggc ggt ctg gaa 912Gln Ala Arg His Val Lys Phe Leu Thr Glu Tyr
Pro Gly Gly Leu Glu 290 295
300 ggt ctg atg cgc gac cac gct gcg att atc gct
ccg aaa ttc cgt gcg 960Gly Leu Met Arg Asp His Ala Ala Ile Ile Ala
Pro Lys Phe Arg Ala 305 310 315
320 gtt gat gaa gtc ctg cgc gct gaa ctg ggc gaa
ggc ggt gaa tat gca 1008Val Asp Glu Val Leu Arg Ala Glu Leu Gly Glu
Gly Gly Glu Tyr Ala 325 330
335 acc tgg acg ctg ccg aaa ggc ggt tac ttt atc
agt ctg gac acc gct 1056Thr Trp Thr Leu Pro Lys Gly Gly Tyr Phe Ile
Ser Leu Asp Thr Ala 340 345
350 gaa ccg gtg gcg gat cgc gtc gtg aaa ctg gcg
gaa gcc gca ggc gtt 1104Glu Pro Val Ala Asp Arg Val Val Lys Leu Ala
Glu Ala Ala Gly Val 355 360
365 agc ctg acc ccg gcg ggt gca acg tat ccg gca
ggt caa gat ccg cat 1152Ser Leu Thr Pro Ala Gly Ala Thr Tyr Pro Ala
Gly Gln Asp Pro His 370 375
380 aac cgt aat ctg cgt ctg gca ccg acc cgt ccg
ccg gtg gaa gaa gtt 1200Asn Arg Asn Leu Arg Leu Ala Pro Thr Arg Pro
Pro Val Glu Glu Val 385 390 395
400 cgc acg gca atg caa gtg gtc gcc gcg tgt atc
cgc ctg gca acc gaa 1248Arg Thr Ala Met Gln Val Val Ala Ala Cys Ile
Arg Leu Ala Thr Glu 405 410
415 gaa tat cgt gct ggt cat ctc gag tga
1275Glu Tyr Arg Ala Gly His Leu Glu
420
65424PRTDeinococcus geothermalis 65Met Thr
Lys Glu Ala Ser Arg Pro Ala Leu Asp Leu Ala Arg Gln Ala 1 5
10 15 Tyr Glu Ala Phe Lys Ala Arg
Gly Leu Asn Leu Asn Met Gln Arg Gly 20 25
30 Gln Pro Ala Asp Ala Asp Phe Asp Leu Ser Asn Gly
Leu Leu Thr Val 35 40 45
Leu Gly Ala Glu Asp Val Arg Met Asp Gly Leu Asp Leu Arg Asn Tyr
50 55 60 Pro Gly Gly
Val Ala Gly Leu Pro Ser Ala Arg Ala Leu Phe Ala Gly 65
70 75 80 Tyr Leu Asp Val Lys Ala Glu
Asn Val Leu Val Trp Asn Asn Ser Ser 85
90 95 Leu Glu Leu Gln Gly Leu Val Leu Thr Phe Ala
Leu Leu His Gly Val 100 105
110 Arg Gly Ser Thr Gly Pro Trp Leu Ser Gln Thr Pro Lys Met Ile
Val 115 120 125 Thr
Val Pro Gly Tyr Asp Arg His Phe Leu Leu Leu Gln Thr Leu Gly 130
135 140 Phe Glu Leu Leu Thr Val
Asp Met Gln Ser Asp Gly Pro Asp Val Asp 145 150
155 160 Ala Val Glu Arg Leu Ala Gly Thr Asp Pro Ser
Val Lys Gly Ile Leu 165 170
175 Phe Val Pro Thr Tyr Ser Asn Pro Gly Gly Glu Thr Ile Ser Leu Glu
180 185 190 Lys Ala
Arg Arg Leu Ala Gly Leu Gln Ala Ala Ala Pro Asp Phe Thr 195
200 205 Ile Phe Ala Asp Asp Ala Tyr
Arg Val His His Leu Val Glu Glu Asp 210 215
220 Arg Ala Glu Pro Val Asn Phe Val Val Leu Ala Arg
Asp Ala Gly Tyr 225 230 235
240 Pro Asp Arg Ala Phe Val Phe Ala Ser Thr Ser Lys Ile Thr Phe Ala
245 250 255 Gly Ala Gly
Leu Gly Phe Val Ala Ser Ser Glu Asp Asn Ile Arg Trp 260
265 270 Leu Ser Lys Tyr Leu Gly Ala Gln
Ser Ile Gly Pro Asn Lys Val Glu 275 280
285 Gln Ala Arg His Val Lys Phe Leu Thr Glu Tyr Pro Gly
Gly Leu Glu 290 295 300
Gly Leu Met Arg Asp His Ala Ala Ile Ile Ala Pro Lys Phe Arg Ala 305
310 315 320 Val Asp Glu Val
Leu Arg Ala Glu Leu Gly Glu Gly Gly Glu Tyr Ala 325
330 335 Thr Trp Thr Leu Pro Lys Gly Gly Tyr
Phe Ile Ser Leu Asp Thr Ala 340 345
350 Glu Pro Val Ala Asp Arg Val Val Lys Leu Ala Glu Ala Ala
Gly Val 355 360 365
Ser Leu Thr Pro Ala Gly Ala Thr Tyr Pro Ala Gly Gln Asp Pro His 370
375 380 Asn Arg Asn Leu Arg
Leu Ala Pro Thr Arg Pro Pro Val Glu Glu Val 385 390
395 400 Arg Thr Ala Met Gln Val Val Ala Ala Cys
Ile Arg Leu Ala Thr Glu 405 410
415 Glu Tyr Arg Ala Gly His Leu Glu 420
661287DNACorynebacterium glutamicumCDS(1)..(1287) 66atg agc tcg gtg
tcg ctg caa gac ttt gac gct gaa cgc att ggc ctg 48Met Ser Ser Val
Ser Leu Gln Asp Phe Asp Ala Glu Arg Ile Gly Leu 1
5 10 15 ttc cac gaa gat
att aaa cgt aaa ttt gat gaa ctg aaa tct aaa aac 96Phe His Glu Asp
Ile Lys Arg Lys Phe Asp Glu Leu Lys Ser Lys Asn 20
25 30 ctg aaa ctg gat
ctg acc cgt ggt aaa ccg agc tct gaa cag ctg gat 144Leu Lys Leu Asp
Leu Thr Arg Gly Lys Pro Ser Ser Glu Gln Leu Asp 35
40 45 ttt gcg gac gaa
ctg ctg gcc ctg ccg ggc aag ggt gat ttc aaa gcg 192Phe Ala Asp Glu
Leu Leu Ala Leu Pro Gly Lys Gly Asp Phe Lys Ala 50
55 60 gcc gat ggc acc
gac gtt cgt aac tat ggc ggt ctg gat ggt att gtc 240Ala Asp Gly Thr
Asp Val Arg Asn Tyr Gly Gly Leu Asp Gly Ile Val 65
70 75 80 gac atc cgc cag
att tgg gcg gat ctg ctg ggc gtg ccg gtt gaa caa 288Asp Ile Arg Gln
Ile Trp Ala Asp Leu Leu Gly Val Pro Val Glu Gln
85 90 95 gtg ctg gca ggt
gat gct agt tcc ctg aat atc atg ttt gac gtg att 336Val Leu Ala Gly
Asp Ala Ser Ser Leu Asn Ile Met Phe Asp Val Ile 100
105 110 agc tgg tct tat
atc ttc ggc aac aat gat tca gtt cag ccg tgg tcg 384Ser Trp Ser Tyr
Ile Phe Gly Asn Asn Asp Ser Val Gln Pro Trp Ser 115
120 125 aaa gaa gaa acg
gtg aaa tgg att tgc ccg gtt ccg ggc tac gac cgt 432Lys Glu Glu Thr
Val Lys Trp Ile Cys Pro Val Pro Gly Tyr Asp Arg 130
135 140 cat ttt tct att
acc gaa cgc ttt ggt ttc gaa atg atc agt gtt ccg 480His Phe Ser Ile
Thr Glu Arg Phe Gly Phe Glu Met Ile Ser Val Pro 145
150 155 160 atg aac gaa gat
ggc ccg gat atg gac gca gtt gaa gaa ctg gtc aaa 528Met Asn Glu Asp
Gly Pro Asp Met Asp Ala Val Glu Glu Leu Val Lys
165 170 175 gac ccg caa gtc
aaa ggc atg tgg gtg gtt ccg gtg ttt agt aat ccg 576Asp Pro Gln Val
Lys Gly Met Trp Val Val Pro Val Phe Ser Asn Pro 180
185 190 acc ggc ttc acg
gtg tcc gaa gat gtt gcc aaa cgt ctg tca acc atg 624Thr Gly Phe Thr
Val Ser Glu Asp Val Ala Lys Arg Leu Ser Thr Met 195
200 205 gaa acg gca gct
ccg gat ttt cgc gtc gtg tgg gac aat gcg tac gcc 672Glu Thr Ala Ala
Pro Asp Phe Arg Val Val Trp Asp Asn Ala Tyr Ala 210
215 220 gtg cac acc ctg
acg gat gaa ttc ccg gaa gtc att gac atc gtg ggt 720Val His Thr Leu
Thr Asp Glu Phe Pro Glu Val Ile Asp Ile Val Gly 225
230 235 240 ctg ggt gaa gcg
gcc ggt aac ccg aat cgt ttt tgg gcg ttc acc agt 768Leu Gly Glu Ala
Ala Gly Asn Pro Asn Arg Phe Trp Ala Phe Thr Ser
245 250 255 acg tcc aaa att
acc ctg gca ggc gct ggt gtc agc ttt ttc atg acg 816Thr Ser Lys Ile
Thr Leu Ala Gly Ala Gly Val Ser Phe Phe Met Thr 260
265 270 agc gcg gaa aac
cgt aaa tgg tat agc ggc cat gct ggt atc cgc ggc 864Ser Ala Glu Asn
Arg Lys Trp Tyr Ser Gly His Ala Gly Ile Arg Gly 275
280 285 att ggt ccg aac
aaa gtt aat cag ctg gcg cac gcc cgc tac ttt ggc 912Ile Gly Pro Asn
Lys Val Asn Gln Leu Ala His Ala Arg Tyr Phe Gly 290
295 300 gat gca gaa ggt
gtc cgt gct gtg atg cgc aaa cat gca gct tcc ctg 960Asp Ala Glu Gly
Val Arg Ala Val Met Arg Lys His Ala Ala Ser Leu 305
310 315 320 gcg ccg aaa ttc
aat aaa gtg ctg gaa atc ctg gat agt cgt ctg gcg 1008Ala Pro Lys Phe
Asn Lys Val Leu Glu Ile Leu Asp Ser Arg Leu Ala
325 330 335 gaa tat ggt gtt
gca cag tgg acc gtc ccg gcc ggc ggt tac ttt att 1056Glu Tyr Gly Val
Ala Gln Trp Thr Val Pro Ala Gly Gly Tyr Phe Ile 340
345 350 tcg ctg gac gtt
gtc ccg ggt acg gca agc cgc gtt gcg gaa ctg gcc 1104Ser Leu Asp Val
Val Pro Gly Thr Ala Ser Arg Val Ala Glu Leu Ala 355
360 365 aaa gaa gca ggc
atc gct ctg acc ggc gcg ggt tca tcg tat ccg ctg 1152Lys Glu Ala Gly
Ile Ala Leu Thr Gly Ala Gly Ser Ser Tyr Pro Leu 370
375 380 cgt caa gat ccg
gaa aac aaa aat ctg cgt ctg gca ccg agc ctg ccg 1200Arg Gln Asp Pro
Glu Asn Lys Asn Leu Arg Leu Ala Pro Ser Leu Pro 385
390 395 400 ccg gtg gaa gaa
ctg gaa gtt gcg atg gat ggt gtg gct acc tgc gtg 1248Pro Val Glu Glu
Leu Glu Val Ala Met Asp Gly Val Ala Thr Cys Val
405 410 415 ctg ctg gct gcc
gcc gaa cat tat gct tct ctc gag tga 1287Leu Leu Ala Ala
Ala Glu His Tyr Ala Ser Leu Glu 420
425
67428PRTCorynebacterium glutamicum 67Met Ser Ser Val Ser Leu Gln Asp Phe
Asp Ala Glu Arg Ile Gly Leu 1 5 10
15 Phe His Glu Asp Ile Lys Arg Lys Phe Asp Glu Leu Lys Ser
Lys Asn 20 25 30
Leu Lys Leu Asp Leu Thr Arg Gly Lys Pro Ser Ser Glu Gln Leu Asp
35 40 45 Phe Ala Asp Glu
Leu Leu Ala Leu Pro Gly Lys Gly Asp Phe Lys Ala 50
55 60 Ala Asp Gly Thr Asp Val Arg Asn
Tyr Gly Gly Leu Asp Gly Ile Val 65 70
75 80 Asp Ile Arg Gln Ile Trp Ala Asp Leu Leu Gly Val
Pro Val Glu Gln 85 90
95 Val Leu Ala Gly Asp Ala Ser Ser Leu Asn Ile Met Phe Asp Val Ile
100 105 110 Ser Trp Ser
Tyr Ile Phe Gly Asn Asn Asp Ser Val Gln Pro Trp Ser 115
120 125 Lys Glu Glu Thr Val Lys Trp Ile
Cys Pro Val Pro Gly Tyr Asp Arg 130 135
140 His Phe Ser Ile Thr Glu Arg Phe Gly Phe Glu Met Ile
Ser Val Pro 145 150 155
160 Met Asn Glu Asp Gly Pro Asp Met Asp Ala Val Glu Glu Leu Val Lys
165 170 175 Asp Pro Gln Val
Lys Gly Met Trp Val Val Pro Val Phe Ser Asn Pro 180
185 190 Thr Gly Phe Thr Val Ser Glu Asp Val
Ala Lys Arg Leu Ser Thr Met 195 200
205 Glu Thr Ala Ala Pro Asp Phe Arg Val Val Trp Asp Asn Ala
Tyr Ala 210 215 220
Val His Thr Leu Thr Asp Glu Phe Pro Glu Val Ile Asp Ile Val Gly 225
230 235 240 Leu Gly Glu Ala Ala
Gly Asn Pro Asn Arg Phe Trp Ala Phe Thr Ser 245
250 255 Thr Ser Lys Ile Thr Leu Ala Gly Ala Gly
Val Ser Phe Phe Met Thr 260 265
270 Ser Ala Glu Asn Arg Lys Trp Tyr Ser Gly His Ala Gly Ile Arg
Gly 275 280 285 Ile
Gly Pro Asn Lys Val Asn Gln Leu Ala His Ala Arg Tyr Phe Gly 290
295 300 Asp Ala Glu Gly Val Arg
Ala Val Met Arg Lys His Ala Ala Ser Leu 305 310
315 320 Ala Pro Lys Phe Asn Lys Val Leu Glu Ile Leu
Asp Ser Arg Leu Ala 325 330
335 Glu Tyr Gly Val Ala Gln Trp Thr Val Pro Ala Gly Gly Tyr Phe Ile
340 345 350 Ser Leu
Asp Val Val Pro Gly Thr Ala Ser Arg Val Ala Glu Leu Ala 355
360 365 Lys Glu Ala Gly Ile Ala Leu
Thr Gly Ala Gly Ser Ser Tyr Pro Leu 370 375
380 Arg Gln Asp Pro Glu Asn Lys Asn Leu Arg Leu Ala
Pro Ser Leu Pro 385 390 395
400 Pro Val Glu Glu Leu Glu Val Ala Met Asp Gly Val Ala Thr Cys Val
405 410 415 Leu Leu Ala
Ala Ala Glu His Tyr Ala Ser Leu Glu 420 425
681200DNAThermus thermophilusCDS(1)..(1200) 68atg aaa ccg ctg
tcc tgg tct gaa gcg ttc ggc aaa ggt gct ggt cgt 48Met Lys Pro Leu
Ser Trp Ser Glu Ala Phe Gly Lys Gly Ala Gly Arg 1
5 10 15 atc caa gcc tct
acc att cgt gaa ctg ctg aaa ctg acg cag cgc ccg 96Ile Gln Ala Ser
Thr Ile Arg Glu Leu Leu Lys Leu Thr Gln Arg Pro 20
25 30 ggt att ctg agc
ttt gca ggc ggt ctg ccg gct ccg gaa ctg ttc ccg 144Gly Ile Leu Ser
Phe Ala Gly Gly Leu Pro Ala Pro Glu Leu Phe Pro 35
40 45 aaa gaa gaa gcg
gcc gaa gca gct gcg cgt atc ctg cgt gaa aaa ggt 192Lys Glu Glu Ala
Ala Glu Ala Ala Ala Arg Ile Leu Arg Glu Lys Gly 50
55 60 gaa gtt gca ctg
caa tat agc ccg acc gaa ggt tac gct ccg ctg cgt 240Glu Val Ala Leu
Gln Tyr Ser Pro Thr Glu Gly Tyr Ala Pro Leu Arg 65
70 75 80 gca ttt gtc gct
gaa tgg att ggt gtt cgt ccg gaa gaa gtc ctg atc 288Ala Phe Val Ala
Glu Trp Ile Gly Val Arg Pro Glu Glu Val Leu Ile
85 90 95 acc acg ggc tct
cag caa gcg ctg gat ctg gtg ggt aaa gtt ttc ctg 336Thr Thr Gly Ser
Gln Gln Ala Leu Asp Leu Val Gly Lys Val Phe Leu 100
105 110 gac gaa ggc agt
ccg gtt ctg ctg gaa gcc ccg tcc tat atg ggt gcg 384Asp Glu Gly Ser
Pro Val Leu Leu Glu Ala Pro Ser Tyr Met Gly Ala 115
120 125 att cag gcc ttt
cgc ctg caa ggt ccg cgt ttc ctg acc gtc ccg gca 432Ile Gln Ala Phe
Arg Leu Gln Gly Pro Arg Phe Leu Thr Val Pro Ala 130
135 140 ggt gaa gaa ggc
ccg gat ctg gac gct ctg gaa gaa gtg ctg aaa cgt 480Gly Glu Glu Gly
Pro Asp Leu Asp Ala Leu Glu Glu Val Leu Lys Arg 145
150 155 160 gaa cgc ccg cgt
ttt ctg tac ctg atc ccg agc ttc cag aac ccg acc 528Glu Arg Pro Arg
Phe Leu Tyr Leu Ile Pro Ser Phe Gln Asn Pro Thr
165 170 175 ggc ggt ctg acg
ccg ctg ccg gca cgt aaa cgt ctg ctg caa atg gtg 576Gly Gly Leu Thr
Pro Leu Pro Ala Arg Lys Arg Leu Leu Gln Met Val 180
185 190 atg gaa cgt ggt
ctg gtg gtt gtc gaa gat gac gcg tat cgc gaa ctg 624Met Glu Arg Gly
Leu Val Val Val Glu Asp Asp Ala Tyr Arg Glu Leu 195
200 205 tac ttt ggc gaa
gcc cgt ctg ccg tca ctg ttc gaa ctg gca cgc gaa 672Tyr Phe Gly Glu
Ala Arg Leu Pro Ser Leu Phe Glu Leu Ala Arg Glu 210
215 220 gct ggt tat ccg
ggc gtg att tac ctg ggt agc ttt tct aaa gtt ctg 720Ala Gly Tyr Pro
Gly Val Ile Tyr Leu Gly Ser Phe Ser Lys Val Leu 225
230 235 240 tcg ccg ggt ctg
cgt gtg gca ttc gca gtt gct cat ccg gaa gcg ctg 768Ser Pro Gly Leu
Arg Val Ala Phe Ala Val Ala His Pro Glu Ala Leu
245 250 255 caa aaa ctg gtt
cag gcg aaa caa ggt gcc gat ctg cat acc ccg atg 816Gln Lys Leu Val
Gln Ala Lys Gln Gly Ala Asp Leu His Thr Pro Met 260
265 270 ctg aac caa atg
ctg gtg cac gaa ctg ctg aaa gaa ggc ttt tct gaa 864Leu Asn Gln Met
Leu Val His Glu Leu Leu Lys Glu Gly Phe Ser Glu 275
280 285 cgt ctg gaa cgt
gtc cgt cgc gtg tat cgc gaa aaa gcg cag gcc atg 912Arg Leu Glu Arg
Val Arg Arg Val Tyr Arg Glu Lys Ala Gln Ala Met 290
295 300 ctg cac gca ctg
gac cgt gaa gtc ccg aaa gaa gtg cgc tac acg cgt 960Leu His Ala Leu
Asp Arg Glu Val Pro Lys Glu Val Arg Tyr Thr Arg 305
310 315 320 ccg aaa ggc ggt
atg ttt gtg tgg atg gaa ctg ccg aaa ggt ctg agt 1008Pro Lys Gly Gly
Met Phe Val Trp Met Glu Leu Pro Lys Gly Leu Ser
325 330 335 gcc gaa ggc ctg
ttt cgt cgc gcg ctg gaa gaa aat gtt gcc ttc gtc 1056Ala Glu Gly Leu
Phe Arg Arg Ala Leu Glu Glu Asn Val Ala Phe Val 340
345 350 ccg ggc ggt ccg
ttt ttc gca aac ggc ggt ggc gaa aat acc ctg cgc 1104Pro Gly Gly Pro
Phe Phe Ala Asn Gly Gly Gly Glu Asn Thr Leu Arg 355
360 365 ctg tcc tat gca
acg ctg gat cgt gaa ggc atc gcc gaa ggt gtc cgc 1152Leu Ser Tyr Ala
Thr Leu Asp Arg Glu Gly Ile Ala Glu Gly Val Arg 370
375 380 cgc ctg ggt cgt
gct ctg aaa ggt ctg ctg gcc ctg gtt ctc gag tga 1200Arg Leu Gly Arg
Ala Leu Lys Gly Leu Leu Ala Leu Val Leu Glu 385
390 395 69399PRTThermus
thermophilus 69Met Lys Pro Leu Ser Trp Ser Glu Ala Phe Gly Lys Gly Ala
Gly Arg 1 5 10 15
Ile Gln Ala Ser Thr Ile Arg Glu Leu Leu Lys Leu Thr Gln Arg Pro
20 25 30 Gly Ile Leu Ser Phe
Ala Gly Gly Leu Pro Ala Pro Glu Leu Phe Pro 35
40 45 Lys Glu Glu Ala Ala Glu Ala Ala Ala
Arg Ile Leu Arg Glu Lys Gly 50 55
60 Glu Val Ala Leu Gln Tyr Ser Pro Thr Glu Gly Tyr Ala
Pro Leu Arg 65 70 75
80 Ala Phe Val Ala Glu Trp Ile Gly Val Arg Pro Glu Glu Val Leu Ile
85 90 95 Thr Thr Gly Ser
Gln Gln Ala Leu Asp Leu Val Gly Lys Val Phe Leu 100
105 110 Asp Glu Gly Ser Pro Val Leu Leu Glu
Ala Pro Ser Tyr Met Gly Ala 115 120
125 Ile Gln Ala Phe Arg Leu Gln Gly Pro Arg Phe Leu Thr Val
Pro Ala 130 135 140
Gly Glu Glu Gly Pro Asp Leu Asp Ala Leu Glu Glu Val Leu Lys Arg 145
150 155 160 Glu Arg Pro Arg Phe
Leu Tyr Leu Ile Pro Ser Phe Gln Asn Pro Thr 165
170 175 Gly Gly Leu Thr Pro Leu Pro Ala Arg Lys
Arg Leu Leu Gln Met Val 180 185
190 Met Glu Arg Gly Leu Val Val Val Glu Asp Asp Ala Tyr Arg Glu
Leu 195 200 205 Tyr
Phe Gly Glu Ala Arg Leu Pro Ser Leu Phe Glu Leu Ala Arg Glu 210
215 220 Ala Gly Tyr Pro Gly Val
Ile Tyr Leu Gly Ser Phe Ser Lys Val Leu 225 230
235 240 Ser Pro Gly Leu Arg Val Ala Phe Ala Val Ala
His Pro Glu Ala Leu 245 250
255 Gln Lys Leu Val Gln Ala Lys Gln Gly Ala Asp Leu His Thr Pro Met
260 265 270 Leu Asn
Gln Met Leu Val His Glu Leu Leu Lys Glu Gly Phe Ser Glu 275
280 285 Arg Leu Glu Arg Val Arg Arg
Val Tyr Arg Glu Lys Ala Gln Ala Met 290 295
300 Leu His Ala Leu Asp Arg Glu Val Pro Lys Glu Val
Arg Tyr Thr Arg 305 310 315
320 Pro Lys Gly Gly Met Phe Val Trp Met Glu Leu Pro Lys Gly Leu Ser
325 330 335 Ala Glu Gly
Leu Phe Arg Arg Ala Leu Glu Glu Asn Val Ala Phe Val 340
345 350 Pro Gly Gly Pro Phe Phe Ala Asn
Gly Gly Gly Glu Asn Thr Leu Arg 355 360
365 Leu Ser Tyr Ala Thr Leu Asp Arg Glu Gly Ile Ala Glu
Gly Val Arg 370 375 380
Arg Leu Gly Arg Ala Leu Lys Gly Leu Leu Ala Leu Val Leu Glu 385
390 395 701248DNAThermotoga
maritimaCDS(1)..(1248) 70atg gtc gtc aat ctg gaa ggt aaa atc tct aaa atc
ggt caa aat atg 48Met Val Val Asn Leu Glu Gly Lys Ile Ser Lys Ile
Gly Gln Asn Met 1 5 10
15 aaa tcg agc att atc cgt gaa atc ctg aaa ttc gct
gcg gat aaa gac 96Lys Ser Ser Ile Ile Arg Glu Ile Leu Lys Phe Ala
Ala Asp Lys Asp 20 25
30 gcg att agc ttt ggc ggt ggc gtg ccg gat ccg gaa
acc ttc ccg cgt 144Ala Ile Ser Phe Gly Gly Gly Val Pro Asp Pro Glu
Thr Phe Pro Arg 35 40
45 aaa gaa ctg gca gaa atc gct aaa gaa atc atc gaa
aaa gaa tac cat 192Lys Glu Leu Ala Glu Ile Ala Lys Glu Ile Ile Glu
Lys Glu Tyr His 50 55 60
tac acc ctg caa tac tct acc acg gaa ggc gat ccg
gtt ctg aaa cag 240Tyr Thr Leu Gln Tyr Ser Thr Thr Glu Gly Asp Pro
Val Leu Lys Gln 65 70 75
80 caa att ctg aaa ctg ctg gaa cgt atg tac ggt att
acc ggc ctg gat 288Gln Ile Leu Lys Leu Leu Glu Arg Met Tyr Gly Ile
Thr Gly Leu Asp 85 90
95 gaa gac aac ctg atc ttt acg gtc ggc tca cag caa
gcc ctg gat ctg 336Glu Asp Asn Leu Ile Phe Thr Val Gly Ser Gln Gln
Ala Leu Asp Leu 100 105
110 att ggt aaa ctg ttc ctg gat gac gaa tcg tat tgc
gtt ctg gat gac 384Ile Gly Lys Leu Phe Leu Asp Asp Glu Ser Tyr Cys
Val Leu Asp Asp 115 120
125 ccg gca tac ctg ggt gca atc aac gca ttt cgc cag
tat ctg gcc aat 432Pro Ala Tyr Leu Gly Ala Ile Asn Ala Phe Arg Gln
Tyr Leu Ala Asn 130 135 140
ttc gtg gtt gtc ccg ctg gaa gat gac ggc atg gat
ctg aac gtg ctg 480Phe Val Val Val Pro Leu Glu Asp Asp Gly Met Asp
Leu Asn Val Leu 145 150 155
160 gaa cgt aaa ctg tca gaa ttt gac aaa aac ggt aaa
atc aaa caa gtt 528Glu Arg Lys Leu Ser Glu Phe Asp Lys Asn Gly Lys
Ile Lys Gln Val 165 170
175 aaa ttc atc tac gtg gtt agc aac ttc cat aat ccg
gca ggt gtg acc 576Lys Phe Ile Tyr Val Val Ser Asn Phe His Asn Pro
Ala Gly Val Thr 180 185
190 acg tct ctg gaa aaa cgc aaa gcg ctg gtt gaa att
gcc gaa aaa tac 624Thr Ser Leu Glu Lys Arg Lys Ala Leu Val Glu Ile
Ala Glu Lys Tyr 195 200
205 gac ctg ttt atc gtc gaa gat gac ccg tat ggc gct
ctg cgc tac gaa 672Asp Leu Phe Ile Val Glu Asp Asp Pro Tyr Gly Ala
Leu Arg Tyr Glu 210 215 220
ggt gaa acc gtg gac ccg att ttt aaa atc ggt ggc
ccg gaa cgt gtc 720Gly Glu Thr Val Asp Pro Ile Phe Lys Ile Gly Gly
Pro Glu Arg Val 225 230 235
240 gtg ctg ctg aac acg ttc agt aaa gtt ctg gca ccg
ggt ctg cgc att 768Val Leu Leu Asn Thr Phe Ser Lys Val Leu Ala Pro
Gly Leu Arg Ile 245 250
255 ggc atg gtc gct ggt tcc aaa gaa ttc atc cgt aaa
atc gtt cag gca 816Gly Met Val Ala Gly Ser Lys Glu Phe Ile Arg Lys
Ile Val Gln Ala 260 265
270 aaa caa agt gct gat ctg tgc tcc ccg gca att acc
cac cgt ctg gca 864Lys Gln Ser Ala Asp Leu Cys Ser Pro Ala Ile Thr
His Arg Leu Ala 275 280
285 gca cgc tat ctg gaa cgt tac gac ctg ctg gaa cag
ctg aaa ccg acc 912Ala Arg Tyr Leu Glu Arg Tyr Asp Leu Leu Glu Gln
Leu Lys Pro Thr 290 295 300
atc gaa ctg tat cgt cgc aaa cgc acg gtg atg ctg
aat gca ctg gaa 960Ile Glu Leu Tyr Arg Arg Lys Arg Thr Val Met Leu
Asn Ala Leu Glu 305 310 315
320 gaa tac ttt tca gat att ccg ggc gtt aaa tgg gtc
aaa tcg gaa ggt 1008Glu Tyr Phe Ser Asp Ile Pro Gly Val Lys Trp Val
Lys Ser Glu Gly 325 330
335 ggc ctg ttc atc tgg ctg acc ctg ccg gaa ggt ttt
gat acg tgg gaa 1056Gly Leu Phe Ile Trp Leu Thr Leu Pro Glu Gly Phe
Asp Thr Trp Glu 340 345
350 atg ttc gaa tat gcc aaa cgc aaa aaa gtg ttt tac
gtt ccg ggt cgt 1104Met Phe Glu Tyr Ala Lys Arg Lys Lys Val Phe Tyr
Val Pro Gly Arg 355 360
365 gtc ttc aaa gtg tat gat gaa ccg agc ccg tct atg
cgt ctg tcc ttt 1152Val Phe Lys Val Tyr Asp Glu Pro Ser Pro Ser Met
Arg Leu Ser Phe 370 375 380
tgt ctg ccg ccg gac gaa aaa atc gtg gaa ggc atc
aaa cgt ctg cgt 1200Cys Leu Pro Pro Asp Glu Lys Ile Val Glu Gly Ile
Lys Arg Leu Arg 385 390 395
400 gaa gtt gtg ctg gaa tac ggt aaa gaa aaa cat ctg
ctg ctc gag tga 1248Glu Val Val Leu Glu Tyr Gly Lys Glu Lys His Leu
Leu Leu Glu 405 410
415 71415PRTThermotoga maritima 71Met Val Val Asn
Leu Glu Gly Lys Ile Ser Lys Ile Gly Gln Asn Met 1 5
10 15 Lys Ser Ser Ile Ile Arg Glu Ile Leu
Lys Phe Ala Ala Asp Lys Asp 20 25
30 Ala Ile Ser Phe Gly Gly Gly Val Pro Asp Pro Glu Thr Phe
Pro Arg 35 40 45
Lys Glu Leu Ala Glu Ile Ala Lys Glu Ile Ile Glu Lys Glu Tyr His 50
55 60 Tyr Thr Leu Gln Tyr
Ser Thr Thr Glu Gly Asp Pro Val Leu Lys Gln 65 70
75 80 Gln Ile Leu Lys Leu Leu Glu Arg Met Tyr
Gly Ile Thr Gly Leu Asp 85 90
95 Glu Asp Asn Leu Ile Phe Thr Val Gly Ser Gln Gln Ala Leu Asp
Leu 100 105 110 Ile
Gly Lys Leu Phe Leu Asp Asp Glu Ser Tyr Cys Val Leu Asp Asp 115
120 125 Pro Ala Tyr Leu Gly Ala
Ile Asn Ala Phe Arg Gln Tyr Leu Ala Asn 130 135
140 Phe Val Val Val Pro Leu Glu Asp Asp Gly Met
Asp Leu Asn Val Leu 145 150 155
160 Glu Arg Lys Leu Ser Glu Phe Asp Lys Asn Gly Lys Ile Lys Gln Val
165 170 175 Lys Phe
Ile Tyr Val Val Ser Asn Phe His Asn Pro Ala Gly Val Thr 180
185 190 Thr Ser Leu Glu Lys Arg Lys
Ala Leu Val Glu Ile Ala Glu Lys Tyr 195 200
205 Asp Leu Phe Ile Val Glu Asp Asp Pro Tyr Gly Ala
Leu Arg Tyr Glu 210 215 220
Gly Glu Thr Val Asp Pro Ile Phe Lys Ile Gly Gly Pro Glu Arg Val 225
230 235 240 Val Leu Leu
Asn Thr Phe Ser Lys Val Leu Ala Pro Gly Leu Arg Ile 245
250 255 Gly Met Val Ala Gly Ser Lys Glu
Phe Ile Arg Lys Ile Val Gln Ala 260 265
270 Lys Gln Ser Ala Asp Leu Cys Ser Pro Ala Ile Thr His
Arg Leu Ala 275 280 285
Ala Arg Tyr Leu Glu Arg Tyr Asp Leu Leu Glu Gln Leu Lys Pro Thr 290
295 300 Ile Glu Leu Tyr
Arg Arg Lys Arg Thr Val Met Leu Asn Ala Leu Glu 305 310
315 320 Glu Tyr Phe Ser Asp Ile Pro Gly Val
Lys Trp Val Lys Ser Glu Gly 325 330
335 Gly Leu Phe Ile Trp Leu Thr Leu Pro Glu Gly Phe Asp Thr
Trp Glu 340 345 350
Met Phe Glu Tyr Ala Lys Arg Lys Lys Val Phe Tyr Val Pro Gly Arg
355 360 365 Val Phe Lys Val
Tyr Asp Glu Pro Ser Pro Ser Met Arg Leu Ser Phe 370
375 380 Cys Leu Pro Pro Asp Glu Lys Ile
Val Glu Gly Ile Lys Arg Leu Arg 385 390
395 400 Glu Val Val Leu Glu Tyr Gly Lys Glu Lys His Leu
Leu Leu Glu 405 410 415
721218DNAPyrococcus horikoshiiCDS(1)..(1218) 72atg ctg ggc gat gtg gaa
cgc ttc ttc tcg aaa aaa gct ctg gaa atg 48Met Leu Gly Asp Val Glu
Arg Phe Phe Ser Lys Lys Ala Leu Glu Met 1 5
10 15 cgt gcg tct gaa gtc cgt
gaa ctg ctg aaa ctg gtg gaa acc agt gat 96Arg Ala Ser Glu Val Arg
Glu Leu Leu Lys Leu Val Glu Thr Ser Asp 20
25 30 att atc tcc ctg gcg ggc
ggt ctg ccg aac ccg aaa acg ttc ccg aaa 144Ile Ile Ser Leu Ala Gly
Gly Leu Pro Asn Pro Lys Thr Phe Pro Lys 35
40 45 gaa att atc cgt gat atc
ctg gtt gaa atc atg gaa aaa tat gca gac 192Glu Ile Ile Arg Asp Ile
Leu Val Glu Ile Met Glu Lys Tyr Ala Asp 50
55 60 aaa gct ctg caa tac ggc
acc acg aaa ggt ttt acc ccg ctg cgt gaa 240Lys Ala Leu Gln Tyr Gly
Thr Thr Lys Gly Phe Thr Pro Leu Arg Glu 65 70
75 80 acg ctg atg aaa tgg ctg
ggc aaa cgc tat ggt att tcc cag gat aat 288Thr Leu Met Lys Trp Leu
Gly Lys Arg Tyr Gly Ile Ser Gln Asp Asn 85
90 95 gac att atg atc acc agc
ggt tct cag caa gcc ctg gat ctg att ggc 336Asp Ile Met Ile Thr Ser
Gly Ser Gln Gln Ala Leu Asp Leu Ile Gly 100
105 110 cgc gtg ttc ctg aac ccg
ggt gac atc gtg gtt gtc gaa gca ccg acc 384Arg Val Phe Leu Asn Pro
Gly Asp Ile Val Val Val Glu Ala Pro Thr 115
120 125 tac ctg gcg gcc ctg caa
gct ttt aat ttc tat gaa ccg cag tac att 432Tyr Leu Ala Ala Leu Gln
Ala Phe Asn Phe Tyr Glu Pro Gln Tyr Ile 130
135 140 caa atc ccg ctg gat gac
gaa ggc atg aaa gtt gaa atc ctg gaa gaa 480Gln Ile Pro Leu Asp Asp
Glu Gly Met Lys Val Glu Ile Leu Glu Glu 145 150
155 160 aaa ctg aaa gaa ctg aaa
agc cag ggt aaa aaa gtc aaa gtg gtt tat 528Lys Leu Lys Glu Leu Lys
Ser Gln Gly Lys Lys Val Lys Val Val Tyr 165
170 175 acc gtg ccg acg ttc caa
aac ccg gcg ggt gtg acc atg aat gaa gat 576Thr Val Pro Thr Phe Gln
Asn Pro Ala Gly Val Thr Met Asn Glu Asp 180
185 190 cgt cgc aaa tat ctg ctg
gaa ctg gcc tca gaa tac gac ttt atc gtc 624Arg Arg Lys Tyr Leu Leu
Glu Leu Ala Ser Glu Tyr Asp Phe Ile Val 195
200 205 gtg gaa gat gac ccg tat
ggc gaa ctg cgt tac tcg ggt aac ccg gaa 672Val Glu Asp Asp Pro Tyr
Gly Glu Leu Arg Tyr Ser Gly Asn Pro Glu 210
215 220 aag aaa att aaa gcc ctg
gat aat gaa ggc cgc gtt atc tac ctg ggt 720Lys Lys Ile Lys Ala Leu
Asp Asn Glu Gly Arg Val Ile Tyr Leu Gly 225 230
235 240 acc ttt agc aaa att ctg
gca ccg ggc ttc cgt atc ggt tgg atg gtc 768Thr Phe Ser Lys Ile Leu
Ala Pro Gly Phe Arg Ile Gly Trp Met Val 245
250 255 ggc gat ccg ggt att atc
cgc aaa atg gaa att gca aaa cag tct acc 816Gly Asp Pro Gly Ile Ile
Arg Lys Met Glu Ile Ala Lys Gln Ser Thr 260
265 270 gac ctg tgc acg aac gtt
ttt ggc caa gtt gtc gct tgg cgt tat gtc 864Asp Leu Cys Thr Asn Val
Phe Gly Gln Val Val Ala Trp Arg Tyr Val 275
280 285 gat ggc ggt tac ctg gaa
aaa cat att ccg gaa atc cgc aaa ttt tat 912Asp Gly Gly Tyr Leu Glu
Lys His Ile Pro Glu Ile Arg Lys Phe Tyr 290
295 300 aaa ccg cgt cgc gat gca
atg ctg gaa gct ctg gaa gaa ttc atg ccg 960Lys Pro Arg Arg Asp Ala
Met Leu Glu Ala Leu Glu Glu Phe Met Pro 305 310
315 320 gaa ggt gtc aaa tgg acc
aaa ccg gaa ggc ggt atg ttt att tgg gtg 1008Glu Gly Val Lys Trp Thr
Lys Pro Glu Gly Gly Met Phe Ile Trp Val 325
330 335 acg ctg ccg gat ggc atc
gac agc aag aaa atg ctg gaa cgt gcg atc 1056Thr Leu Pro Asp Gly Ile
Asp Ser Lys Lys Met Leu Glu Arg Ala Ile 340
345 350 aaa aaa ggc gtg gcc tat
gtt ccg ggt gaa gcg ttt tac gcc cac cgt 1104Lys Lys Gly Val Ala Tyr
Val Pro Gly Glu Ala Phe Tyr Ala His Arg 355
360 365 gat gtg aaa aac acc atg
cgc ctg aat ttc acg tat gtt gac gaa gac 1152Asp Val Lys Asn Thr Met
Arg Leu Asn Phe Thr Tyr Val Asp Glu Asp 370
375 380 aaa atc atg gaa ggt atc
aaa cgc ctg gca gaa acg atc aaa gaa gaa 1200Lys Ile Met Glu Gly Ile
Lys Arg Leu Ala Glu Thr Ile Lys Glu Glu 385 390
395 400 ctg aaa gcg ctc gag tga
1218Leu Lys Ala Leu Glu
405
73405PRTPyrococcus
horikoshii 73Met Leu Gly Asp Val Glu Arg Phe Phe Ser Lys Lys Ala Leu Glu
Met 1 5 10 15 Arg
Ala Ser Glu Val Arg Glu Leu Leu Lys Leu Val Glu Thr Ser Asp
20 25 30 Ile Ile Ser Leu Ala
Gly Gly Leu Pro Asn Pro Lys Thr Phe Pro Lys 35
40 45 Glu Ile Ile Arg Asp Ile Leu Val Glu
Ile Met Glu Lys Tyr Ala Asp 50 55
60 Lys Ala Leu Gln Tyr Gly Thr Thr Lys Gly Phe Thr Pro
Leu Arg Glu 65 70 75
80 Thr Leu Met Lys Trp Leu Gly Lys Arg Tyr Gly Ile Ser Gln Asp Asn
85 90 95 Asp Ile Met Ile
Thr Ser Gly Ser Gln Gln Ala Leu Asp Leu Ile Gly 100
105 110 Arg Val Phe Leu Asn Pro Gly Asp Ile
Val Val Val Glu Ala Pro Thr 115 120
125 Tyr Leu Ala Ala Leu Gln Ala Phe Asn Phe Tyr Glu Pro Gln
Tyr Ile 130 135 140
Gln Ile Pro Leu Asp Asp Glu Gly Met Lys Val Glu Ile Leu Glu Glu 145
150 155 160 Lys Leu Lys Glu Leu
Lys Ser Gln Gly Lys Lys Val Lys Val Val Tyr 165
170 175 Thr Val Pro Thr Phe Gln Asn Pro Ala Gly
Val Thr Met Asn Glu Asp 180 185
190 Arg Arg Lys Tyr Leu Leu Glu Leu Ala Ser Glu Tyr Asp Phe Ile
Val 195 200 205 Val
Glu Asp Asp Pro Tyr Gly Glu Leu Arg Tyr Ser Gly Asn Pro Glu 210
215 220 Lys Lys Ile Lys Ala Leu
Asp Asn Glu Gly Arg Val Ile Tyr Leu Gly 225 230
235 240 Thr Phe Ser Lys Ile Leu Ala Pro Gly Phe Arg
Ile Gly Trp Met Val 245 250
255 Gly Asp Pro Gly Ile Ile Arg Lys Met Glu Ile Ala Lys Gln Ser Thr
260 265 270 Asp Leu
Cys Thr Asn Val Phe Gly Gln Val Val Ala Trp Arg Tyr Val 275
280 285 Asp Gly Gly Tyr Leu Glu Lys
His Ile Pro Glu Ile Arg Lys Phe Tyr 290 295
300 Lys Pro Arg Arg Asp Ala Met Leu Glu Ala Leu Glu
Glu Phe Met Pro 305 310 315
320 Glu Gly Val Lys Trp Thr Lys Pro Glu Gly Gly Met Phe Ile Trp Val
325 330 335 Thr Leu Pro
Asp Gly Ile Asp Ser Lys Lys Met Leu Glu Arg Ala Ile 340
345 350 Lys Lys Gly Val Ala Tyr Val Pro
Gly Glu Ala Phe Tyr Ala His Arg 355 360
365 Asp Val Lys Asn Thr Met Arg Leu Asn Phe Thr Tyr Val
Asp Glu Asp 370 375 380
Lys Ile Met Glu Gly Ile Lys Arg Leu Ala Glu Thr Ile Lys Glu Glu 385
390 395 400 Leu Lys Ala Leu
Glu 405 741173DNAPhormidium lapideumCDS(1)..(1173) 74atg
aaa ctg gct gcc cgt gtt gaa agt gtg tcc ccg agt atg acc ctg 48Met
Lys Leu Ala Ala Arg Val Glu Ser Val Ser Pro Ser Met Thr Leu 1
5 10 15 att
att gat gcg aaa gca aaa gcg atg aaa gcg gaa ggc att gat gtg 96Ile
Ile Asp Ala Lys Ala Lys Ala Met Lys Ala Glu Gly Ile Asp Val
20 25 30 tgc
agt ttt tcc gcc ggt gaa ccg gac ttc aac acg ccg aaa cat atc 144Cys
Ser Phe Ser Ala Gly Glu Pro Asp Phe Asn Thr Pro Lys His Ile
35 40 45 gtt
gaa gcg gcc aaa gca gct ctg gaa cag ggt aaa acc cgt tat ggt 192Val
Glu Ala Ala Lys Ala Ala Leu Glu Gln Gly Lys Thr Arg Tyr Gly
50 55 60 ccg
gcg gcc ggt gaa ccg cgt ctg cgt gaa gcg att gcc cag aaa ctg 240Pro
Ala Ala Gly Glu Pro Arg Leu Arg Glu Ala Ile Ala Gln Lys Leu 65
70 75 80 caa
cgt gat aac ggc ctg tgt tac ggt gcg gac aac atc ctg gtt acc 288Gln
Arg Asp Asn Gly Leu Cys Tyr Gly Ala Asp Asn Ile Leu Val Thr
85 90 95 aat
ggc ggt aaa cag agt att ttt aat ctg atg ctg gcg atg atc gaa 336Asn
Gly Gly Lys Gln Ser Ile Phe Asn Leu Met Leu Ala Met Ile Glu
100 105 110 ccg
ggt gat gaa gtg att atc ccg gcc ccg ttc tgg gtc tcc tat ccg 384Pro
Gly Asp Glu Val Ile Ile Pro Ala Pro Phe Trp Val Ser Tyr Pro
115 120 125 gaa
atg gtg aaa ctg gcc gaa ggc acg ccg gtt att ctg ccg acc acg 432Glu
Met Val Lys Leu Ala Glu Gly Thr Pro Val Ile Leu Pro Thr Thr
130 135 140 gtc
gaa acc cag ttt aaa gtg agc ccg gaa cag att cgc caa gcg atc 480Val
Glu Thr Gln Phe Lys Val Ser Pro Glu Gln Ile Arg Gln Ala Ile 145
150 155 160 acc
ccg aaa acg aaa ctg ctg gtt ttc aac acc ccg tct aat ccg acg 528Thr
Pro Lys Thr Lys Leu Leu Val Phe Asn Thr Pro Ser Asn Pro Thr
165 170 175 ggt
atg gtt tac acc ccg gat gaa gtc cgt gca att gct cag gtc gca 576Gly
Met Val Tyr Thr Pro Asp Glu Val Arg Ala Ile Ala Gln Val Ala
180 185 190 gtg
gaa gca ggt ctg tgg gtg ctg agt gat gaa atc tac gaa aaa atc 624Val
Glu Ala Gly Leu Trp Val Leu Ser Asp Glu Ile Tyr Glu Lys Ile
195 200 205 ctg
tac gat gac gca caa cat ctg agt atc ggt gca gct tcc ccg gaa 672Leu
Tyr Asp Asp Ala Gln His Leu Ser Ile Gly Ala Ala Ser Pro Glu
210 215 220 gcg
tat gaa cgc agc gtg gtt tgc tct ggc ttt gcg aaa acc tac gcc 720Ala
Tyr Glu Arg Ser Val Val Cys Ser Gly Phe Ala Lys Thr Tyr Ala 225
230 235 240 atg
acg ggt tgg cgt gtt ggt ttc ctg gca ggt ccg gtt ccg ctg gtc 768Met
Thr Gly Trp Arg Val Gly Phe Leu Ala Gly Pro Val Pro Leu Val
245 250 255 aaa
gca gcc acc aaa att cag ggt cac tca acg tcg aac gtc tgc acc 816Lys
Ala Ala Thr Lys Ile Gln Gly His Ser Thr Ser Asn Val Cys Thr
260 265 270 ttt
gca caa tat ggc gct atc gca gct tac gaa aat tct cag gat tgt 864Phe
Ala Gln Tyr Gly Ala Ile Ala Ala Tyr Glu Asn Ser Gln Asp Cys
275 280 285 gtg
caa gaa atg ctg gcg gcc ttt gcg gaa cgt cgc cgt tat atg ctg 912Val
Gln Glu Met Leu Ala Ala Phe Ala Glu Arg Arg Arg Tyr Met Leu
290 295 300 gat
gca ctg aat gct atg ccg ggt ctg gaa tgt ccg aaa ccg gac ggc 960Asp
Ala Leu Asn Ala Met Pro Gly Leu Glu Cys Pro Lys Pro Asp Gly 305
310 315 320 gcg
ttt tac atg ttc ccg tca att gcc aaa acc ggt cgc agc tct ctg 1008Ala
Phe Tyr Met Phe Pro Ser Ile Ala Lys Thr Gly Arg Ser Ser Leu
325 330 335 gat
ttt tgc tcg gaa ctg ctg gac cag cac caa gtg gca acg gtt ccg 1056Asp
Phe Cys Ser Glu Leu Leu Asp Gln His Gln Val Ala Thr Val Pro
340 345 350 ggt
gca gct ttc ggc gct gat gac tgt atc cgt ctg agc tat gca acc 1104Gly
Ala Ala Phe Gly Ala Asp Asp Cys Ile Arg Leu Ser Tyr Ala Thr
355 360 365 gac
ctg gac acg atc aaa cgc ggt atg gaa cgc ctg gaa aaa ttt ctg 1152Asp
Leu Asp Thr Ile Lys Arg Gly Met Glu Arg Leu Glu Lys Phe Leu
370 375 380 cac
ggc att ctg ctc gag tga 1173His
Gly Ile Leu Leu Glu 385
390
75390PRTPhormidium lapideum 75Met Lys Leu Ala Ala Arg Val Glu Ser Val Ser
Pro Ser Met Thr Leu 1 5 10
15 Ile Ile Asp Ala Lys Ala Lys Ala Met Lys Ala Glu Gly Ile Asp Val
20 25 30 Cys Ser
Phe Ser Ala Gly Glu Pro Asp Phe Asn Thr Pro Lys His Ile 35
40 45 Val Glu Ala Ala Lys Ala Ala
Leu Glu Gln Gly Lys Thr Arg Tyr Gly 50 55
60 Pro Ala Ala Gly Glu Pro Arg Leu Arg Glu Ala Ile
Ala Gln Lys Leu 65 70 75
80 Gln Arg Asp Asn Gly Leu Cys Tyr Gly Ala Asp Asn Ile Leu Val Thr
85 90 95 Asn Gly Gly
Lys Gln Ser Ile Phe Asn Leu Met Leu Ala Met Ile Glu 100
105 110 Pro Gly Asp Glu Val Ile Ile Pro
Ala Pro Phe Trp Val Ser Tyr Pro 115 120
125 Glu Met Val Lys Leu Ala Glu Gly Thr Pro Val Ile Leu
Pro Thr Thr 130 135 140
Val Glu Thr Gln Phe Lys Val Ser Pro Glu Gln Ile Arg Gln Ala Ile 145
150 155 160 Thr Pro Lys Thr
Lys Leu Leu Val Phe Asn Thr Pro Ser Asn Pro Thr 165
170 175 Gly Met Val Tyr Thr Pro Asp Glu Val
Arg Ala Ile Ala Gln Val Ala 180 185
190 Val Glu Ala Gly Leu Trp Val Leu Ser Asp Glu Ile Tyr Glu
Lys Ile 195 200 205
Leu Tyr Asp Asp Ala Gln His Leu Ser Ile Gly Ala Ala Ser Pro Glu 210
215 220 Ala Tyr Glu Arg Ser
Val Val Cys Ser Gly Phe Ala Lys Thr Tyr Ala 225 230
235 240 Met Thr Gly Trp Arg Val Gly Phe Leu Ala
Gly Pro Val Pro Leu Val 245 250
255 Lys Ala Ala Thr Lys Ile Gln Gly His Ser Thr Ser Asn Val Cys
Thr 260 265 270 Phe
Ala Gln Tyr Gly Ala Ile Ala Ala Tyr Glu Asn Ser Gln Asp Cys 275
280 285 Val Gln Glu Met Leu Ala
Ala Phe Ala Glu Arg Arg Arg Tyr Met Leu 290 295
300 Asp Ala Leu Asn Ala Met Pro Gly Leu Glu Cys
Pro Lys Pro Asp Gly 305 310 315
320 Ala Phe Tyr Met Phe Pro Ser Ile Ala Lys Thr Gly Arg Ser Ser Leu
325 330 335 Asp Phe
Cys Ser Glu Leu Leu Asp Gln His Gln Val Ala Thr Val Pro 340
345 350 Gly Ala Ala Phe Gly Ala Asp
Asp Cys Ile Arg Leu Ser Tyr Ala Thr 355 360
365 Asp Leu Asp Thr Ile Lys Arg Gly Met Glu Arg Leu
Glu Lys Phe Leu 370 375 380
His Gly Ile Leu Leu Glu 385 390 761164DNAThermus
thermophilusCDS(1)..(1164) 76atg cgt ggt ctg tcg cgt cgt gtc caa gca atg
aaa ccg tca gca acc 48Met Arg Gly Leu Ser Arg Arg Val Gln Ala Met
Lys Pro Ser Ala Thr 1 5 10
15 gtc gcc gtt aat gcc aaa gcc ctg gaa ctg cgt
cgt cag ggt gtc gat 96Val Ala Val Asn Ala Lys Ala Leu Glu Leu Arg
Arg Gln Gly Val Asp 20 25
30 ctg gtg gca ctg acc gct ggc gaa ccg gat ttt
gac acg ccg gaa cat 144Leu Val Ala Leu Thr Ala Gly Glu Pro Asp Phe
Asp Thr Pro Glu His 35 40
45 gtt aaa gaa gcg gca cgt cgc gca ctg gca caa
ggt aaa acc aaa tat 192Val Lys Glu Ala Ala Arg Arg Ala Leu Ala Gln
Gly Lys Thr Lys Tyr 50 55
60 gca ccg ccg gcg ggt att ccg gaa ctg cgt gaa
gca ctg gct gaa aaa 240Ala Pro Pro Ala Gly Ile Pro Glu Leu Arg Glu
Ala Leu Ala Glu Lys 65 70 75
80 ttc cgt cgc gaa aac ggt ctg agc gtg acg ccg
gaa gaa acc atc gtt 288Phe Arg Arg Glu Asn Gly Leu Ser Val Thr Pro
Glu Glu Thr Ile Val 85 90
95 acg gtc ggc ggt aaa cag gcg ctg ttt aac ctg
ttt caa gcc att ctg 336Thr Val Gly Gly Lys Gln Ala Leu Phe Asn Leu
Phe Gln Ala Ile Leu 100 105
110 gat ccg ggc gac gaa gtg atc gtt ctg tca ccg
tat tgg gtg tcg tac 384Asp Pro Gly Asp Glu Val Ile Val Leu Ser Pro
Tyr Trp Val Ser Tyr 115 120
125 ccg gaa atg gtt cgt ttt gcg ggc ggt gtg gtt
gtc gaa gtg gaa acc 432Pro Glu Met Val Arg Phe Ala Gly Gly Val Val
Val Glu Val Glu Thr 130 135
140 ctg ccg gaa gaa ggt ttc gtc ccg gat ccg gaa
cgt gtg cgt cgc gca 480Leu Pro Glu Glu Gly Phe Val Pro Asp Pro Glu
Arg Val Arg Arg Ala 145 150 155
160 att acc ccg cgc acg aaa gct ctg gtg gtt aac
tct ccg aac aat ccg 528Ile Thr Pro Arg Thr Lys Ala Leu Val Val Asn
Ser Pro Asn Asn Pro 165 170
175 acc ggc gca gtt tat ccg aaa gaa gtc ctg gaa
gca ctg gca cgt ctg 576Thr Gly Ala Val Tyr Pro Lys Glu Val Leu Glu
Ala Leu Ala Arg Leu 180 185
190 gca gtt gaa cat gat ttt tac ctg gtc agc gac
gaa atc tat gaa cat 624Ala Val Glu His Asp Phe Tyr Leu Val Ser Asp
Glu Ile Tyr Glu His 195 200
205 ctg ctg tac gaa ggt gaa cac ttc tcc ccg ggt
cgt gtc gca ccg gaa 672Leu Leu Tyr Glu Gly Glu His Phe Ser Pro Gly
Arg Val Ala Pro Glu 210 215
220 cac acc ctg acg gtg aac ggt gca gct aaa gca
ttt gct atg acc ggc 720His Thr Leu Thr Val Asn Gly Ala Ala Lys Ala
Phe Ala Met Thr Gly 225 230 235
240 tgg cgc att ggt tat gca tgc ggc ccg aaa gaa
gtg atc aaa gcg atg 768Trp Arg Ile Gly Tyr Ala Cys Gly Pro Lys Glu
Val Ile Lys Ala Met 245 250
255 gcc tca gtt agc tct cag agt acc acg tcc ccg
gat acg att gca caa 816Ala Ser Val Ser Ser Gln Ser Thr Thr Ser Pro
Asp Thr Ile Ala Gln 260 265
270 tgg gct acc ctg gaa gcg ctg acg aat cag gaa
gcg tcg cgt gcc ttt 864Trp Ala Thr Leu Glu Ala Leu Thr Asn Gln Glu
Ala Ser Arg Ala Phe 275 280
285 gtt gaa atg gca cgc gaa gct tat cgt cgc cgt
cgc gac ctg ctg ctg 912Val Glu Met Ala Arg Glu Ala Tyr Arg Arg Arg
Arg Asp Leu Leu Leu 290 295
300 gaa ggt ctg acc gca ctg ggc ctg aaa gct gtt
cgt ccg agc ggt gcg 960Glu Gly Leu Thr Ala Leu Gly Leu Lys Ala Val
Arg Pro Ser Gly Ala 305 310 315
320 ttc tac gtc ctg atg gat acg tct ccg atc gca
ccg gac gaa gtt cgt 1008Phe Tyr Val Leu Met Asp Thr Ser Pro Ile Ala
Pro Asp Glu Val Arg 325 330
335 gcg gcc gaa cgt ctg ctg gaa gcc ggt gtg gca
gtc gtg ccg ggc acc 1056Ala Ala Glu Arg Leu Leu Glu Ala Gly Val Ala
Val Val Pro Gly Thr 340 345
350 gat ttt gca gct ttc ggc cac gtg cgc ctg tca
tac gcc acc tcc gaa 1104Asp Phe Ala Ala Phe Gly His Val Arg Leu Ser
Tyr Ala Thr Ser Glu 355 360
365 gaa aat ctg cgt aaa gcc ctg gaa cgt ttt gct
cgt gtc ctg ggt cgt 1152Glu Asn Leu Arg Lys Ala Leu Glu Arg Phe Ala
Arg Val Leu Gly Arg 370 375
380 gcg ctc gag tga
1164Ala Leu Glu
385
77387PRTThermus thermophilus 77Met Arg Gly
Leu Ser Arg Arg Val Gln Ala Met Lys Pro Ser Ala Thr 1 5
10 15 Val Ala Val Asn Ala Lys Ala Leu
Glu Leu Arg Arg Gln Gly Val Asp 20 25
30 Leu Val Ala Leu Thr Ala Gly Glu Pro Asp Phe Asp Thr
Pro Glu His 35 40 45
Val Lys Glu Ala Ala Arg Arg Ala Leu Ala Gln Gly Lys Thr Lys Tyr 50
55 60 Ala Pro Pro Ala
Gly Ile Pro Glu Leu Arg Glu Ala Leu Ala Glu Lys 65 70
75 80 Phe Arg Arg Glu Asn Gly Leu Ser Val
Thr Pro Glu Glu Thr Ile Val 85 90
95 Thr Val Gly Gly Lys Gln Ala Leu Phe Asn Leu Phe Gln Ala
Ile Leu 100 105 110
Asp Pro Gly Asp Glu Val Ile Val Leu Ser Pro Tyr Trp Val Ser Tyr
115 120 125 Pro Glu Met Val
Arg Phe Ala Gly Gly Val Val Val Glu Val Glu Thr 130
135 140 Leu Pro Glu Glu Gly Phe Val Pro
Asp Pro Glu Arg Val Arg Arg Ala 145 150
155 160 Ile Thr Pro Arg Thr Lys Ala Leu Val Val Asn Ser
Pro Asn Asn Pro 165 170
175 Thr Gly Ala Val Tyr Pro Lys Glu Val Leu Glu Ala Leu Ala Arg Leu
180 185 190 Ala Val Glu
His Asp Phe Tyr Leu Val Ser Asp Glu Ile Tyr Glu His 195
200 205 Leu Leu Tyr Glu Gly Glu His Phe
Ser Pro Gly Arg Val Ala Pro Glu 210 215
220 His Thr Leu Thr Val Asn Gly Ala Ala Lys Ala Phe Ala
Met Thr Gly 225 230 235
240 Trp Arg Ile Gly Tyr Ala Cys Gly Pro Lys Glu Val Ile Lys Ala Met
245 250 255 Ala Ser Val Ser
Ser Gln Ser Thr Thr Ser Pro Asp Thr Ile Ala Gln 260
265 270 Trp Ala Thr Leu Glu Ala Leu Thr Asn
Gln Glu Ala Ser Arg Ala Phe 275 280
285 Val Glu Met Ala Arg Glu Ala Tyr Arg Arg Arg Arg Asp Leu
Leu Leu 290 295 300
Glu Gly Leu Thr Ala Leu Gly Leu Lys Ala Val Arg Pro Ser Gly Ala 305
310 315 320 Phe Tyr Val Leu Met
Asp Thr Ser Pro Ile Ala Pro Asp Glu Val Arg 325
330 335 Ala Ala Glu Arg Leu Leu Glu Ala Gly Val
Ala Val Val Pro Gly Thr 340 345
350 Asp Phe Ala Ala Phe Gly His Val Arg Leu Ser Tyr Ala Thr Ser
Glu 355 360 365 Glu
Asn Leu Arg Lys Ala Leu Glu Arg Phe Ala Arg Val Leu Gly Arg 370
375 380 Ala Leu Glu 385
781176DNAPyrococcus horikoshiiCDS(1)..(1176) 78atg gcc ctg tcg gac cgt
ctg gaa ctg gtg tcg gcg agt gaa att cgt 48Met Ala Leu Ser Asp Arg
Leu Glu Leu Val Ser Ala Ser Glu Ile Arg 1 5
10 15 aaa ctg ttt gat att gcc
gct ggt atg aaa gat gtt att agt ctg ggc 96Lys Leu Phe Asp Ile Ala
Ala Gly Met Lys Asp Val Ile Ser Leu Gly 20
25 30 att ggt gaa ccg gat ttc
gac acc ccg cag cat atc aaa gaa tat gct 144Ile Gly Glu Pro Asp Phe
Asp Thr Pro Gln His Ile Lys Glu Tyr Ala 35
40 45 aaa gaa gcg ctg gat aaa
ggt ctg acg cac tac ggc ccg aac att ggt 192Lys Glu Ala Leu Asp Lys
Gly Leu Thr His Tyr Gly Pro Asn Ile Gly 50
55 60 ctg ctg gaa ctg cgc gaa
gcc atc gca gaa aaa ctg aaa aaa cag aac 240Leu Leu Glu Leu Arg Glu
Ala Ile Ala Glu Lys Leu Lys Lys Gln Asn 65 70
75 80 ggc att gaa gct gat ccg
aaa acc gaa atc atg gtt ctg ctg ggc gcc 288Gly Ile Glu Ala Asp Pro
Lys Thr Glu Ile Met Val Leu Leu Gly Ala 85
90 95 aat caa gca ttt ctg atg
ggt ctg agt gcc ttc ctg aaa gac ggc gaa 336Asn Gln Ala Phe Leu Met
Gly Leu Ser Ala Phe Leu Lys Asp Gly Glu 100
105 110 gaa gtg ctg att ccg acc
ccg gct ttc gtc tcc tat gct ccg gcg gtg 384Glu Val Leu Ile Pro Thr
Pro Ala Phe Val Ser Tyr Ala Pro Ala Val 115
120 125 atc ctg gcg ggc ggt aaa
ccg gtt gaa gtc ccg acg tat gaa gaa gat 432Ile Leu Ala Gly Gly Lys
Pro Val Glu Val Pro Thr Tyr Glu Glu Asp 130
135 140 gaa ttt cgc ctg aat gtt
gac gaa ctg aaa aaa tac gtc acc gat aaa 480Glu Phe Arg Leu Asn Val
Asp Glu Leu Lys Lys Tyr Val Thr Asp Lys 145 150
155 160 acg cgt gcc ctg att atc
aac tca ccg tgc aat ccg acc ggt gcc gtt 528Thr Arg Ala Leu Ile Ile
Asn Ser Pro Cys Asn Pro Thr Gly Ala Val 165
170 175 ctg acg aaa aaa gat ctg
gaa gaa att gca gac ttt gtg gtt gaa cat 576Leu Thr Lys Lys Asp Leu
Glu Glu Ile Ala Asp Phe Val Val Glu His 180
185 190 gat ctg att gtg atc tcg
gac gaa gtt tat gaa cat ttc att tac gat 624Asp Leu Ile Val Ile Ser
Asp Glu Val Tyr Glu His Phe Ile Tyr Asp 195
200 205 gac gct cgc cac tac agc
atc gcg tct ctg gat ggc atg ttt gaa cgt 672Asp Ala Arg His Tyr Ser
Ile Ala Ser Leu Asp Gly Met Phe Glu Arg 210
215 220 acc atc acg gtg aac ggc
ttt agc aaa acc ttc gca atg acg ggt tgg 720Thr Ile Thr Val Asn Gly
Phe Ser Lys Thr Phe Ala Met Thr Gly Trp 225 230
235 240 cgt ctg ggt ttt gtg gca
gca ccg tct tgg att atc gaa cgt atg gtt 768Arg Leu Gly Phe Val Ala
Ala Pro Ser Trp Ile Ile Glu Arg Met Val 245
250 255 aaa ttc cag atg tac aac
gcg acc tgt ccg gtc acg ttc att caa tac 816Lys Phe Gln Met Tyr Asn
Ala Thr Cys Pro Val Thr Phe Ile Gln Tyr 260
265 270 gca gct gcg aaa gcc ctg
aaa gat gaa cgc tct tgg aaa gca gtt gaa 864Ala Ala Ala Lys Ala Leu
Lys Asp Glu Arg Ser Trp Lys Ala Val Glu 275
280 285 gaa atg cgt aaa gaa tat
gac cgt cgc cgt aaa ctg gtg tgg aaa cgt 912Glu Met Arg Lys Glu Tyr
Asp Arg Arg Arg Lys Leu Val Trp Lys Arg 290
295 300 ctg aac gaa atg ggt ctg
ccg acc gtt aaa ccg aaa ggc gcg ttt tac 960Leu Asn Glu Met Gly Leu
Pro Thr Val Lys Pro Lys Gly Ala Phe Tyr 305 310
315 320 att ttc ccg cgc atc cgt
gat acc ggc ctg acg agt aaa aaa ttc tcc 1008Ile Phe Pro Arg Ile Arg
Asp Thr Gly Leu Thr Ser Lys Lys Phe Ser 325
330 335 gaa ctg atg ctg aaa gaa
gct cgc gtc gca gtc gtg ccg ggt tca gca 1056Glu Leu Met Leu Lys Glu
Ala Arg Val Ala Val Val Pro Gly Ser Ala 340
345 350 ttt ggt aaa gca ggc gaa
ggt tat gtg cgt att tcg tat gcc acc gca 1104Phe Gly Lys Ala Gly Glu
Gly Tyr Val Arg Ile Ser Tyr Ala Thr Ala 355
360 365 tac gaa aaa ctg gaa gaa
gcg atg gac cgt atg gaa cgt gtg ctg aaa 1152Tyr Glu Lys Leu Glu Glu
Ala Met Asp Arg Met Glu Arg Val Leu Lys 370
375 380 gaa cgc aaa ctg gtg ctc
gag tga 1176Glu Arg Lys Leu Val Leu
Glu 385 390
79391PRTPyrococcus
horikoshii 79Met Ala Leu Ser Asp Arg Leu Glu Leu Val Ser Ala Ser Glu Ile
Arg 1 5 10 15 Lys
Leu Phe Asp Ile Ala Ala Gly Met Lys Asp Val Ile Ser Leu Gly
20 25 30 Ile Gly Glu Pro Asp
Phe Asp Thr Pro Gln His Ile Lys Glu Tyr Ala 35
40 45 Lys Glu Ala Leu Asp Lys Gly Leu Thr
His Tyr Gly Pro Asn Ile Gly 50 55
60 Leu Leu Glu Leu Arg Glu Ala Ile Ala Glu Lys Leu Lys
Lys Gln Asn 65 70 75
80 Gly Ile Glu Ala Asp Pro Lys Thr Glu Ile Met Val Leu Leu Gly Ala
85 90 95 Asn Gln Ala Phe
Leu Met Gly Leu Ser Ala Phe Leu Lys Asp Gly Glu 100
105 110 Glu Val Leu Ile Pro Thr Pro Ala Phe
Val Ser Tyr Ala Pro Ala Val 115 120
125 Ile Leu Ala Gly Gly Lys Pro Val Glu Val Pro Thr Tyr Glu
Glu Asp 130 135 140
Glu Phe Arg Leu Asn Val Asp Glu Leu Lys Lys Tyr Val Thr Asp Lys 145
150 155 160 Thr Arg Ala Leu Ile
Ile Asn Ser Pro Cys Asn Pro Thr Gly Ala Val 165
170 175 Leu Thr Lys Lys Asp Leu Glu Glu Ile Ala
Asp Phe Val Val Glu His 180 185
190 Asp Leu Ile Val Ile Ser Asp Glu Val Tyr Glu His Phe Ile Tyr
Asp 195 200 205 Asp
Ala Arg His Tyr Ser Ile Ala Ser Leu Asp Gly Met Phe Glu Arg 210
215 220 Thr Ile Thr Val Asn Gly
Phe Ser Lys Thr Phe Ala Met Thr Gly Trp 225 230
235 240 Arg Leu Gly Phe Val Ala Ala Pro Ser Trp Ile
Ile Glu Arg Met Val 245 250
255 Lys Phe Gln Met Tyr Asn Ala Thr Cys Pro Val Thr Phe Ile Gln Tyr
260 265 270 Ala Ala
Ala Lys Ala Leu Lys Asp Glu Arg Ser Trp Lys Ala Val Glu 275
280 285 Glu Met Arg Lys Glu Tyr Asp
Arg Arg Arg Lys Leu Val Trp Lys Arg 290 295
300 Leu Asn Glu Met Gly Leu Pro Thr Val Lys Pro Lys
Gly Ala Phe Tyr 305 310 315
320 Ile Phe Pro Arg Ile Arg Asp Thr Gly Leu Thr Ser Lys Lys Phe Ser
325 330 335 Glu Leu Met
Leu Lys Glu Ala Arg Val Ala Val Val Pro Gly Ser Ala 340
345 350 Phe Gly Lys Ala Gly Glu Gly Tyr
Val Arg Ile Ser Tyr Ala Thr Ala 355 360
365 Tyr Glu Lys Leu Glu Glu Ala Met Asp Arg Met Glu Arg
Val Leu Lys 370 375 380
Glu Arg Lys Leu Val Leu Glu 385 390
801119DNAMethanococcus jannaschiiCDS(1)..(1119) 80atg ctg tct aaa cgc ctg
ctg aat ttt gaa tct ttt gaa gtt atg gac 48Met Leu Ser Lys Arg Leu
Leu Asn Phe Glu Ser Phe Glu Val Met Asp 1 5
10 15 atc ctg gca ctg gca cag
aaa ctg gaa tcg gaa ggt aaa aaa gtg att 96Ile Leu Ala Leu Ala Gln
Lys Leu Glu Ser Glu Gly Lys Lys Val Ile 20
25 30 cat ctg gaa atc ggt gaa
ccg gat ttt aac acc ccg aaa ccg att gtt 144His Leu Glu Ile Gly Glu
Pro Asp Phe Asn Thr Pro Lys Pro Ile Val 35
40 45 gac gaa ggt atc aaa agc
ctg aaa gaa ggc aaa acc cac tat acg gat 192Asp Glu Gly Ile Lys Ser
Leu Lys Glu Gly Lys Thr His Tyr Thr Asp 50
55 60 tct cgt ggc att ctg gaa
ctg cgc gaa aaa atc agt gaa ctg tac aaa 240Ser Arg Gly Ile Leu Glu
Leu Arg Glu Lys Ile Ser Glu Leu Tyr Lys 65 70
75 80 gac aaa tac aaa gca gat
atc atc ccg gac aat att atc att acg ggc 288Asp Lys Tyr Lys Ala Asp
Ile Ile Pro Asp Asn Ile Ile Ile Thr Gly 85
90 95 ggt agc tct ctg ggt ctg
ttt ttc gct ctg agt tcc atc att gat gac 336Gly Ser Ser Leu Gly Leu
Phe Phe Ala Leu Ser Ser Ile Ile Asp Asp 100
105 110 ggc gat gaa gtg ctg att
cag aac ccg tgc tat ccg tgt tac aaa aat 384Gly Asp Glu Val Leu Ile
Gln Asn Pro Cys Tyr Pro Cys Tyr Lys Asn 115
120 125 ttt atc cgt ttc ctg ggt
gca aaa ccg gtc ttt tgc gat ttc acc gtg 432Phe Ile Arg Phe Leu Gly
Ala Lys Pro Val Phe Cys Asp Phe Thr Val 130
135 140 gaa agt ctg gaa gaa gca
ctg tcc gac aaa acg aaa gct atc att atc 480Glu Ser Leu Glu Glu Ala
Leu Ser Asp Lys Thr Lys Ala Ile Ile Ile 145 150
155 160 aac tca ccg tcg aat ccg
ctg ggc gaa gtc att gat cgc gaa atc tat 528Asn Ser Pro Ser Asn Pro
Leu Gly Glu Val Ile Asp Arg Glu Ile Tyr 165
170 175 gaa ttt gcg tac gaa aac
att ccg tac att atc tcc gac gaa atc tat 576Glu Phe Ala Tyr Glu Asn
Ile Pro Tyr Ile Ile Ser Asp Glu Ile Tyr 180
185 190 aat ggt ctg gtg tac gaa
ggc aaa tgt tat tct gcc att gaa ttc gat 624Asn Gly Leu Val Tyr Glu
Gly Lys Cys Tyr Ser Ala Ile Glu Phe Asp 195
200 205 gaa aac ctg gaa aaa acc
att ctg atc aat ggt ttt agc aaa ctg tac 672Glu Asn Leu Glu Lys Thr
Ile Leu Ile Asn Gly Phe Ser Lys Leu Tyr 210
215 220 gcg atg acg ggt tgg cgt
att ggc tac gtt atc agt aac gat gaa atc 720Ala Met Thr Gly Trp Arg
Ile Gly Tyr Val Ile Ser Asn Asp Glu Ile 225 230
235 240 atc gaa gcc att ctg aaa
ctg caa caa aac ctg ttt att agc gca ccg 768Ile Glu Ala Ile Leu Lys
Leu Gln Gln Asn Leu Phe Ile Ser Ala Pro 245
250 255 acc atc tct caa tat gcg
gcc ctg aaa gct ttc gaa aaa gaa acg gaa 816Thr Ile Ser Gln Tyr Ala
Ala Leu Lys Ala Phe Glu Lys Glu Thr Glu 260
265 270 cgc gaa att aac agc atg
atc aaa gaa ttc gat cgt cgc cgt cgc ctg 864Arg Glu Ile Asn Ser Met
Ile Lys Glu Phe Asp Arg Arg Arg Arg Leu 275
280 285 gtg ctg aaa tac gtt aaa
gac ttt ggt tgg gaa gtt aac aat ccg att 912Val Leu Lys Tyr Val Lys
Asp Phe Gly Trp Glu Val Asn Asn Pro Ile 290
295 300 ggc gcc tat tac gtc ttc
ccg aac atc ggt gaa gat ggc cgt gaa ttc 960Gly Ala Tyr Tyr Val Phe
Pro Asn Ile Gly Glu Asp Gly Arg Glu Phe 305 310
315 320 gcg tac aaa ctg ctg aaa
gaa aaa ttc gtc gcc ctg acc ccg ggc att 1008Ala Tyr Lys Leu Leu Lys
Glu Lys Phe Val Ala Leu Thr Pro Gly Ile 325
330 335 ggt ttt ggc tca aaa ggc
aaa aat tac att cgc atc tcg tat gcc aac 1056Gly Phe Gly Ser Lys Gly
Lys Asn Tyr Ile Arg Ile Ser Tyr Ala Asn 340
345 350 agt tac gaa aac atc aaa
gaa ggt ctg gaa cgc atc aaa gaa ttc ctg 1104Ser Tyr Glu Asn Ile Lys
Glu Gly Leu Glu Arg Ile Lys Glu Phe Leu 355
360 365 aac aaa ctc gag tga
1119Asn Lys Leu Glu
370
81372PRTMethanococcus
jannaschii 81Met Leu Ser Lys Arg Leu Leu Asn Phe Glu Ser Phe Glu Val Met
Asp 1 5 10 15 Ile
Leu Ala Leu Ala Gln Lys Leu Glu Ser Glu Gly Lys Lys Val Ile
20 25 30 His Leu Glu Ile Gly
Glu Pro Asp Phe Asn Thr Pro Lys Pro Ile Val 35
40 45 Asp Glu Gly Ile Lys Ser Leu Lys Glu
Gly Lys Thr His Tyr Thr Asp 50 55
60 Ser Arg Gly Ile Leu Glu Leu Arg Glu Lys Ile Ser Glu
Leu Tyr Lys 65 70 75
80 Asp Lys Tyr Lys Ala Asp Ile Ile Pro Asp Asn Ile Ile Ile Thr Gly
85 90 95 Gly Ser Ser Leu
Gly Leu Phe Phe Ala Leu Ser Ser Ile Ile Asp Asp 100
105 110 Gly Asp Glu Val Leu Ile Gln Asn Pro
Cys Tyr Pro Cys Tyr Lys Asn 115 120
125 Phe Ile Arg Phe Leu Gly Ala Lys Pro Val Phe Cys Asp Phe
Thr Val 130 135 140
Glu Ser Leu Glu Glu Ala Leu Ser Asp Lys Thr Lys Ala Ile Ile Ile 145
150 155 160 Asn Ser Pro Ser Asn
Pro Leu Gly Glu Val Ile Asp Arg Glu Ile Tyr 165
170 175 Glu Phe Ala Tyr Glu Asn Ile Pro Tyr Ile
Ile Ser Asp Glu Ile Tyr 180 185
190 Asn Gly Leu Val Tyr Glu Gly Lys Cys Tyr Ser Ala Ile Glu Phe
Asp 195 200 205 Glu
Asn Leu Glu Lys Thr Ile Leu Ile Asn Gly Phe Ser Lys Leu Tyr 210
215 220 Ala Met Thr Gly Trp Arg
Ile Gly Tyr Val Ile Ser Asn Asp Glu Ile 225 230
235 240 Ile Glu Ala Ile Leu Lys Leu Gln Gln Asn Leu
Phe Ile Ser Ala Pro 245 250
255 Thr Ile Ser Gln Tyr Ala Ala Leu Lys Ala Phe Glu Lys Glu Thr Glu
260 265 270 Arg Glu
Ile Asn Ser Met Ile Lys Glu Phe Asp Arg Arg Arg Arg Leu 275
280 285 Val Leu Lys Tyr Val Lys Asp
Phe Gly Trp Glu Val Asn Asn Pro Ile 290 295
300 Gly Ala Tyr Tyr Val Phe Pro Asn Ile Gly Glu Asp
Gly Arg Glu Phe 305 310 315
320 Ala Tyr Lys Leu Leu Lys Glu Lys Phe Val Ala Leu Thr Pro Gly Ile
325 330 335 Gly Phe Gly
Ser Lys Gly Lys Asn Tyr Ile Arg Ile Ser Tyr Ala Asn 340
345 350 Ser Tyr Glu Asn Ile Lys Glu Gly
Leu Glu Arg Ile Lys Glu Phe Leu 355 360
365 Asn Lys Leu Glu 370 821200DNAThermotoga
maritimaCDS(1)..(1200) 82atg gat gtc ttt agc gac cgt gtc ctg ctg acc gaa
gaa tca ccg atc 48Met Asp Val Phe Ser Asp Arg Val Leu Leu Thr Glu
Glu Ser Pro Ile 1 5 10
15 cgc aaa ctg gtt ccg ttt gct gaa atg gcg aaa aaa
cgc ggc gtc cgt 96Arg Lys Leu Val Pro Phe Ala Glu Met Ala Lys Lys
Arg Gly Val Arg 20 25
30 att cat cac ctg aac atc ggt cag ccg gat ctg aaa
acc ccg gaa gtg 144Ile His His Leu Asn Ile Gly Gln Pro Asp Leu Lys
Thr Pro Glu Val 35 40
45 ttt ttc gaa cgc atc tat gaa aat aaa ccg gaa gtg
gtt tat tac agc 192Phe Phe Glu Arg Ile Tyr Glu Asn Lys Pro Glu Val
Val Tyr Tyr Ser 50 55 60
cat agc gcg ggc att tgg gaa ctg cgt gaa gcg ttt
gcc agc tat tac 240His Ser Ala Gly Ile Trp Glu Leu Arg Glu Ala Phe
Ala Ser Tyr Tyr 65 70 75
80 aaa cgt cgc caa cgc gtt gat gtc aaa ccg gaa aac
gtg ctg gtt acc 288Lys Arg Arg Gln Arg Val Asp Val Lys Pro Glu Asn
Val Leu Val Thr 85 90
95 aat ggc ggt tct gaa gcc att ctg ttt agt ttc gca
gtc atc gct aac 336Asn Gly Gly Ser Glu Ala Ile Leu Phe Ser Phe Ala
Val Ile Ala Asn 100 105
110 ccg ggt gac gaa att ctg gtg ctg gaa ccg ttt tat
gcg aac tac aat 384Pro Gly Asp Glu Ile Leu Val Leu Glu Pro Phe Tyr
Ala Asn Tyr Asn 115 120
125 gca ttc gct aaa att gcc ggc gtg aaa ctg atc ccg
gtt acg cgt cgc 432Ala Phe Ala Lys Ile Ala Gly Val Lys Leu Ile Pro
Val Thr Arg Arg 130 135 140
atg gaa gaa ggt ttt gcg atc ccg cag aac ctg gaa
tcg ttc atc aat 480Met Glu Glu Gly Phe Ala Ile Pro Gln Asn Leu Glu
Ser Phe Ile Asn 145 150 155
160 gaa cgt acc aaa ggc att gtt ctg agc aac ccg tgc
aat ccg acg ggc 528Glu Arg Thr Lys Gly Ile Val Leu Ser Asn Pro Cys
Asn Pro Thr Gly 165 170
175 gtc gtg tat ggt aaa gat gaa atg cgt tac ctg gtt
gaa att gcc gaa 576Val Val Tyr Gly Lys Asp Glu Met Arg Tyr Leu Val
Glu Ile Ala Glu 180 185
190 cgc cac ggc ctg ttt ctg atc gtc gac gaa gtg tac
agt gaa att gtg 624Arg His Gly Leu Phe Leu Ile Val Asp Glu Val Tyr
Ser Glu Ile Val 195 200
205 ttt cgc ggt gaa ttc gcg tca gcc ctg tcg atc gaa
agc gat aaa gtt 672Phe Arg Gly Glu Phe Ala Ser Ala Leu Ser Ile Glu
Ser Asp Lys Val 210 215 220
gtc gtg att gac agt gtt tcc aaa aaa ttc tct gcg
tgc ggc gcc cgt 720Val Val Ile Asp Ser Val Ser Lys Lys Phe Ser Ala
Cys Gly Ala Arg 225 230 235
240 gtc ggt tgt ctg atc acc cgc aac gaa gaa ctg att
agt cat gca atg 768Val Gly Cys Leu Ile Thr Arg Asn Glu Glu Leu Ile
Ser His Ala Met 245 250
255 aaa ctg gct cag ggt cgt ctg gca ccg ccg ctg ctg
gaa caa atc ggc 816Lys Leu Ala Gln Gly Arg Leu Ala Pro Pro Leu Leu
Glu Gln Ile Gly 260 265
270 tcc gtg ggt ctg ctg aat ctg gat gac tca ttt ttc
gat ttt gtt cgt 864Ser Val Gly Leu Leu Asn Leu Asp Asp Ser Phe Phe
Asp Phe Val Arg 275 280
285 gaa acc tat cgt gaa cgc gtt gaa acg gtc ctg aaa
aaa ctg gaa gaa 912Glu Thr Tyr Arg Glu Arg Val Glu Thr Val Leu Lys
Lys Leu Glu Glu 290 295 300
cac ggc ctg aaa cgc ttt acc aaa ccg tcc ggt gca
ttc tac att acg 960His Gly Leu Lys Arg Phe Thr Lys Pro Ser Gly Ala
Phe Tyr Ile Thr 305 310 315
320 gct gaa ctg ccg gtg gaa gac gcg gaa gaa ttt gcc
cgc tgg atg ctg 1008Ala Glu Leu Pro Val Glu Asp Ala Glu Glu Phe Ala
Arg Trp Met Leu 325 330
335 acc gat ttc aat atg gac ggc gaa acc acg atg gtt
gca ccg ctg cgt 1056Thr Asp Phe Asn Met Asp Gly Glu Thr Thr Met Val
Ala Pro Leu Arg 340 345
350 ggt ttt tat ctg acg ccg ggc ctg ggt aaa aaa gaa
att cgc atc gct 1104Gly Phe Tyr Leu Thr Pro Gly Leu Gly Lys Lys Glu
Ile Arg Ile Ala 355 360
365 tgt gtg ctg gaa aaa gat ctg ctg tct cgt gcg att
gat gtt ctg atg 1152Cys Val Leu Glu Lys Asp Leu Leu Ser Arg Ala Ile
Asp Val Leu Met 370 375 380
gaa ggt ctg aaa atg ttc tgt agc agc cgt atc tcc
tgt ctc gag tga 1200Glu Gly Leu Lys Met Phe Cys Ser Ser Arg Ile Ser
Cys Leu Glu 385 390 395
83399PRTThermotoga maritima 83Met Asp Val Phe
Ser Asp Arg Val Leu Leu Thr Glu Glu Ser Pro Ile 1 5
10 15 Arg Lys Leu Val Pro Phe Ala Glu Met
Ala Lys Lys Arg Gly Val Arg 20 25
30 Ile His His Leu Asn Ile Gly Gln Pro Asp Leu Lys Thr Pro
Glu Val 35 40 45
Phe Phe Glu Arg Ile Tyr Glu Asn Lys Pro Glu Val Val Tyr Tyr Ser 50
55 60 His Ser Ala Gly Ile
Trp Glu Leu Arg Glu Ala Phe Ala Ser Tyr Tyr 65 70
75 80 Lys Arg Arg Gln Arg Val Asp Val Lys Pro
Glu Asn Val Leu Val Thr 85 90
95 Asn Gly Gly Ser Glu Ala Ile Leu Phe Ser Phe Ala Val Ile Ala
Asn 100 105 110 Pro
Gly Asp Glu Ile Leu Val Leu Glu Pro Phe Tyr Ala Asn Tyr Asn 115
120 125 Ala Phe Ala Lys Ile Ala
Gly Val Lys Leu Ile Pro Val Thr Arg Arg 130 135
140 Met Glu Glu Gly Phe Ala Ile Pro Gln Asn Leu
Glu Ser Phe Ile Asn 145 150 155
160 Glu Arg Thr Lys Gly Ile Val Leu Ser Asn Pro Cys Asn Pro Thr Gly
165 170 175 Val Val
Tyr Gly Lys Asp Glu Met Arg Tyr Leu Val Glu Ile Ala Glu 180
185 190 Arg His Gly Leu Phe Leu Ile
Val Asp Glu Val Tyr Ser Glu Ile Val 195 200
205 Phe Arg Gly Glu Phe Ala Ser Ala Leu Ser Ile Glu
Ser Asp Lys Val 210 215 220
Val Val Ile Asp Ser Val Ser Lys Lys Phe Ser Ala Cys Gly Ala Arg 225
230 235 240 Val Gly Cys
Leu Ile Thr Arg Asn Glu Glu Leu Ile Ser His Ala Met 245
250 255 Lys Leu Ala Gln Gly Arg Leu Ala
Pro Pro Leu Leu Glu Gln Ile Gly 260 265
270 Ser Val Gly Leu Leu Asn Leu Asp Asp Ser Phe Phe Asp
Phe Val Arg 275 280 285
Glu Thr Tyr Arg Glu Arg Val Glu Thr Val Leu Lys Lys Leu Glu Glu 290
295 300 His Gly Leu Lys
Arg Phe Thr Lys Pro Ser Gly Ala Phe Tyr Ile Thr 305 310
315 320 Ala Glu Leu Pro Val Glu Asp Ala Glu
Glu Phe Ala Arg Trp Met Leu 325 330
335 Thr Asp Phe Asn Met Asp Gly Glu Thr Thr Met Val Ala Pro
Leu Arg 340 345 350
Gly Phe Tyr Leu Thr Pro Gly Leu Gly Lys Lys Glu Ile Arg Ile Ala
355 360 365 Cys Val Leu Glu
Lys Asp Leu Leu Ser Arg Ala Ile Asp Val Leu Met 370
375 380 Glu Gly Leu Lys Met Phe Cys Ser
Ser Arg Ile Ser Cys Leu Glu 385 390 395
841263DNASaccharomyces cerevisiaeCDS(1)..(1263) 84atg tca
gcc acc ctg ttc aat aat atc gaa ctg ctg ccg ccg gac gcc 48Met Ser
Ala Thr Leu Phe Asn Asn Ile Glu Leu Leu Pro Pro Asp Ala 1
5 10 15 ctg ttt
ggt atc aaa caa cgc tat ggt caa gat caa cgc gcc acc aaa 96Leu Phe
Gly Ile Lys Gln Arg Tyr Gly Gln Asp Gln Arg Ala Thr Lys
20 25 30 gtt gac
ctg ggc att ggt gca tat cgt gat gac aat ggc aaa ccg tgg 144Val Asp
Leu Gly Ile Gly Ala Tyr Arg Asp Asp Asn Gly Lys Pro Trp
35 40 45 gtc ctg
ccg agt gtg aaa gcg gcc gaa aaa ctg atc cat aac gat agc 192Val Leu
Pro Ser Val Lys Ala Ala Glu Lys Leu Ile His Asn Asp Ser 50
55 60 tct tac
aat cac gaa tac ctg ggc atc acc ggt ctg ccg agc ctg acg 240Ser Tyr
Asn His Glu Tyr Leu Gly Ile Thr Gly Leu Pro Ser Leu Thr 65
70 75 80 tct aac
gca gct aaa att atc ttt ggt acc cag agc gac gcg ctg caa 288Ser Asn
Ala Ala Lys Ile Ile Phe Gly Thr Gln Ser Asp Ala Leu Gln
85 90 95 gaa gat
cgc gtc att tct gtg cag tca ctg tcg ggc acg ggt gca ctg 336Glu Asp
Arg Val Ile Ser Val Gln Ser Leu Ser Gly Thr Gly Ala Leu
100 105 110 cat atc
agt gct aaa ttt ttc tcc aaa ttt ttc ccg gac aaa ctg gtt 384His Ile
Ser Ala Lys Phe Phe Ser Lys Phe Phe Pro Asp Lys Leu Val
115 120 125 tac ctg
tca aaa ccg acc tgg gca aac cac atg gct att ttc gaa aat 432Tyr Leu
Ser Lys Pro Thr Trp Ala Asn His Met Ala Ile Phe Glu Asn 130
135 140 cag ggc
ctg aaa acc gct acg tat ccg tac tgg gcc aat gaa acg aaa 480Gln Gly
Leu Lys Thr Ala Thr Tyr Pro Tyr Trp Ala Asn Glu Thr Lys 145
150 155 160 tcg ctg
gat ctg aac ggc ttt ctg aat gcg att caa aaa gcc ccg gaa 528Ser Leu
Asp Leu Asn Gly Phe Leu Asn Ala Ile Gln Lys Ala Pro Glu
165 170 175 ggt tca
atc ttc gtg ctg cat tcg tgc gcg cac aat ccg acc ggt ctg 576Gly Ser
Ile Phe Val Leu His Ser Cys Ala His Asn Pro Thr Gly Leu
180 185 190 gac ccg
acg agt gaa cag tgg gtt caa att gtc gat gcg atc gcc tcc 624Asp Pro
Thr Ser Glu Gln Trp Val Gln Ile Val Asp Ala Ile Ala Ser
195 200 205 aaa aac
cat atc gcg ctg ttt gat acc gcc tat cag ggc ttc gca acg 672Lys Asn
His Ile Ala Leu Phe Asp Thr Ala Tyr Gln Gly Phe Ala Thr 210
215 220 ggt gat
ctg gac aaa gat gca tac gct gtt cgt ctg ggc gtc gaa aaa 720Gly Asp
Leu Asp Lys Asp Ala Tyr Ala Val Arg Leu Gly Val Glu Lys 225
230 235 240 ctg agt
acc gtt tcc ccg gtg ttt gtt tgc caa tca ttc gcg aaa aac 768Leu Ser
Thr Val Ser Pro Val Phe Val Cys Gln Ser Phe Ala Lys Asn
245 250 255 gcc ggc
atg tat ggt gaa cgc gtc ggt tgt ttt cat ctg gct ctg acc 816Ala Gly
Met Tyr Gly Glu Arg Val Gly Cys Phe His Leu Ala Leu Thr
260 265 270 aaa cag
gcg caa aat aaa acc att aaa ccg gca gtg acg tct cag ctg 864Lys Gln
Ala Gln Asn Lys Thr Ile Lys Pro Ala Val Thr Ser Gln Leu
275 280 285 gcg aaa
att atc cgt tca gaa gtg tcg aac ccg ccg gca tac ggc gct 912Ala Lys
Ile Ile Arg Ser Glu Val Ser Asn Pro Pro Ala Tyr Gly Ala 290
295 300 aaa atc
gtt gcc aaa ctg ctg gaa acc ccg gaa ctg acg gaa cag tgg 960Lys Ile
Val Ala Lys Leu Leu Glu Thr Pro Glu Leu Thr Glu Gln Trp 305
310 315 320 cac aaa
gat atg gtg acc atg agt tcc cgc att acg aaa atg cgt cat 1008His Lys
Asp Met Val Thr Met Ser Ser Arg Ile Thr Lys Met Arg His
325 330 335 gcg ctg
cgc gac cac ctg gtc aaa ctg ggc acc ccg ggt aac tgg gat 1056Ala Leu
Arg Asp His Leu Val Lys Leu Gly Thr Pro Gly Asn Trp Asp
340 345 350 cat atc
gtg aat cag tgt ggc atg ttt agc ttc acc ggt ctg acg ccg 1104His Ile
Val Asn Gln Cys Gly Met Phe Ser Phe Thr Gly Leu Thr Pro
355 360 365 caa atg
gtt aaa cgt ctg gaa gaa acc cac gcc gtg tat ctg gtt gca 1152Gln Met
Val Lys Arg Leu Glu Glu Thr His Ala Val Tyr Leu Val Ala 370
375 380 agc ggt
cgc gcg tct att gcc ggc ctg aac cag ggt aat gtc gaa tat 1200Ser Gly
Arg Ala Ser Ile Ala Gly Leu Asn Gln Gly Asn Val Glu Tyr 385
390 395 400 gtc gca
aaa gcc att gac gaa gtg gtc cgt ttc tac gca acc gaa gca 1248Val Ala
Lys Ala Ile Asp Glu Val Val Arg Phe Tyr Ala Thr Glu Ala
405 410 415 aaa ctg
ctc gag tga 1263Lys Leu
Leu Glu
420
85420PRTSaccharomyces cerevisiae 85Met Ser Ala Thr Leu Phe Asn Asn Ile
Glu Leu Leu Pro Pro Asp Ala 1 5 10
15 Leu Phe Gly Ile Lys Gln Arg Tyr Gly Gln Asp Gln Arg Ala
Thr Lys 20 25 30
Val Asp Leu Gly Ile Gly Ala Tyr Arg Asp Asp Asn Gly Lys Pro Trp
35 40 45 Val Leu Pro Ser
Val Lys Ala Ala Glu Lys Leu Ile His Asn Asp Ser 50
55 60 Ser Tyr Asn His Glu Tyr Leu Gly
Ile Thr Gly Leu Pro Ser Leu Thr 65 70
75 80 Ser Asn Ala Ala Lys Ile Ile Phe Gly Thr Gln Ser
Asp Ala Leu Gln 85 90
95 Glu Asp Arg Val Ile Ser Val Gln Ser Leu Ser Gly Thr Gly Ala Leu
100 105 110 His Ile Ser
Ala Lys Phe Phe Ser Lys Phe Phe Pro Asp Lys Leu Val 115
120 125 Tyr Leu Ser Lys Pro Thr Trp Ala
Asn His Met Ala Ile Phe Glu Asn 130 135
140 Gln Gly Leu Lys Thr Ala Thr Tyr Pro Tyr Trp Ala Asn
Glu Thr Lys 145 150 155
160 Ser Leu Asp Leu Asn Gly Phe Leu Asn Ala Ile Gln Lys Ala Pro Glu
165 170 175 Gly Ser Ile Phe
Val Leu His Ser Cys Ala His Asn Pro Thr Gly Leu 180
185 190 Asp Pro Thr Ser Glu Gln Trp Val Gln
Ile Val Asp Ala Ile Ala Ser 195 200
205 Lys Asn His Ile Ala Leu Phe Asp Thr Ala Tyr Gln Gly Phe
Ala Thr 210 215 220
Gly Asp Leu Asp Lys Asp Ala Tyr Ala Val Arg Leu Gly Val Glu Lys 225
230 235 240 Leu Ser Thr Val Ser
Pro Val Phe Val Cys Gln Ser Phe Ala Lys Asn 245
250 255 Ala Gly Met Tyr Gly Glu Arg Val Gly Cys
Phe His Leu Ala Leu Thr 260 265
270 Lys Gln Ala Gln Asn Lys Thr Ile Lys Pro Ala Val Thr Ser Gln
Leu 275 280 285 Ala
Lys Ile Ile Arg Ser Glu Val Ser Asn Pro Pro Ala Tyr Gly Ala 290
295 300 Lys Ile Val Ala Lys Leu
Leu Glu Thr Pro Glu Leu Thr Glu Gln Trp 305 310
315 320 His Lys Asp Met Val Thr Met Ser Ser Arg Ile
Thr Lys Met Arg His 325 330
335 Ala Leu Arg Asp His Leu Val Lys Leu Gly Thr Pro Gly Asn Trp Asp
340 345 350 His Ile
Val Asn Gln Cys Gly Met Phe Ser Phe Thr Gly Leu Thr Pro 355
360 365 Gln Met Val Lys Arg Leu Glu
Glu Thr His Ala Val Tyr Leu Val Ala 370 375
380 Ser Gly Arg Ala Ser Ile Ala Gly Leu Asn Gln Gly
Asn Val Glu Tyr 385 390 395
400 Val Ala Lys Ala Ile Asp Glu Val Val Arg Phe Tyr Ala Thr Glu Ala
405 410 415 Lys Leu Leu
Glu 420 861200DNAEubacterium rectaleCDS(1)..(1200) 86atg gtc
gtc aat gaa tca atg tat caa ctg ggc tcg gtc cgc tcg gca 48Met Val
Val Asn Glu Ser Met Tyr Gln Leu Gly Ser Val Arg Ser Ala 1
5 10 15 atc cgt
gaa ctg ttc gaa tat ggc aaa aaa cgt gct gcg att gtt ggc 96Ile Arg
Glu Leu Phe Glu Tyr Gly Lys Lys Arg Ala Ala Ile Val Gly
20 25 30 aaa gaa
aac gtc tat gat ttt agc att ggt aat ccg tct atc ccg gcc 144Lys Glu
Asn Val Tyr Asp Phe Ser Ile Gly Asn Pro Ser Ile Pro Ala
35 40 45 ccg cag
att gtt aac gac acc atc aaa gaa ctg gtg acg gat tat gac 192Pro Gln
Ile Val Asn Asp Thr Ile Lys Glu Leu Val Thr Asp Tyr Asp 50
55 60 tct gtt
gct ctg cat ggc tac acc agt gcg caa ggt gat gtg gaa acg 240Ser Val
Ala Leu His Gly Tyr Thr Ser Ala Gln Gly Asp Val Glu Thr 65
70 75 80 cgt gcg
gcc att gct gaa ttt ctg aac aat acc cat ggc acg cac ttc 288Arg Ala
Ala Ile Ala Glu Phe Leu Asn Asn Thr His Gly Thr His Phe
85 90 95 aac gcc
gac aat ctg tac atg acc atg ggt gca gct gcg agc ctg tct 336Asn Ala
Asp Asn Leu Tyr Met Thr Met Gly Ala Ala Ala Ser Leu Ser
100 105 110 atc tgc
ttt cgt gcc ctg acc agc gat gcg tat gat gaa ttc att acg 384Ile Cys
Phe Arg Ala Leu Thr Ser Asp Ala Tyr Asp Glu Phe Ile Thr
115 120 125 atc gcg
ccg tat ttt ccg gaa tac aaa gtg ttc gtt aat gcc gca ggc 432Ile Ala
Pro Tyr Phe Pro Glu Tyr Lys Val Phe Val Asn Ala Ala Gly 130
135 140 gca cgc
ctg gtc gaa gtg ccg gca gat acc gaa cat ttt cag att gat 480Ala Arg
Leu Val Glu Val Pro Ala Asp Thr Glu His Phe Gln Ile Asp 145
150 155 160 ttc gac
gct ctg gaa gaa cgt atc aac gcg cac acg cgc ggc gtt att 528Phe Asp
Ala Leu Glu Glu Arg Ile Asn Ala His Thr Arg Gly Val Ile
165 170 175 atc aat
agt ccg aac aat ccg tcc ggt acc gtc tat tca gaa gaa acg 576Ile Asn
Ser Pro Asn Asn Pro Ser Gly Thr Val Tyr Ser Glu Glu Thr
180 185 190 atc aaa
aaa ctg tcg gat ctg ctg gaa aag aaa agc aaa gaa att ggc 624Ile Lys
Lys Leu Ser Asp Leu Leu Glu Lys Lys Ser Lys Glu Ile Gly
195 200 205 cgt ccg
atc ttt att atc gcg gat gaa ccg tat cgc gaa att gtt tac 672Arg Pro
Ile Phe Ile Ile Ala Asp Glu Pro Tyr Arg Glu Ile Val Tyr 210
215 220 gac ggt
atc aaa gtg ccg ttc gtt acc aaa tat tac gat aac acg ctg 720Asp Gly
Ile Lys Val Pro Phe Val Thr Lys Tyr Tyr Asp Asn Thr Leu 225
230 235 240 gtg tgc
tat agt tac tcc aaa tca ctg tcg ctg ccg ggc gaa cgt atc 768Val Cys
Tyr Ser Tyr Ser Lys Ser Leu Ser Leu Pro Gly Glu Arg Ile
245 250 255 ggt tac
gtt ctg gtc ccg gat gaa gtt tat gac aaa gca gaa ctg tac 816Gly Tyr
Val Leu Val Pro Asp Glu Val Tyr Asp Lys Ala Glu Leu Tyr
260 265 270 gct gcg
gtc tgc ggt gct ggt cgt gca ctg ggt tat gtg tgt gcg ccg 864Ala Ala
Val Cys Gly Ala Gly Arg Ala Leu Gly Tyr Val Cys Ala Pro
275 280 285 agt ctg
ttc cag aaa atg atc gtt aaa tgt caa ggc gcc acc ggt gat 912Ser Leu
Phe Gln Lys Met Ile Val Lys Cys Gln Gly Ala Thr Gly Asp 290
295 300 atc aac
gca tat aaa gaa aat cgt gac ctg ctg tac gaa ggc ctg acc 960Ile Asn
Ala Tyr Lys Glu Asn Arg Asp Leu Leu Tyr Glu Gly Leu Thr 305
310 315 320 cgc att
ggt tat cac tgc ttc aaa ccg gat ggc gcc ttt tac atg ttc 1008Arg Ile
Gly Tyr His Cys Phe Lys Pro Asp Gly Ala Phe Tyr Met Phe
325 330 335 gtg aaa
gca ctg gaa gat gac tcc aat gct ttt tgt gaa aaa gcg aaa 1056Val Lys
Ala Leu Glu Asp Asp Ser Asn Ala Phe Cys Glu Lys Ala Lys
340 345 350 gaa gaa
gat gtc ctg att gtg gcc gca gac ggt ttc ggt tgc ccg ggt 1104Glu Glu
Asp Val Leu Ile Val Ala Ala Asp Gly Phe Gly Cys Pro Gly
355 360 365 tgg gtc
cgt atc tct tat tgt gtg gat cgt gaa atg att aaa cac agc 1152Trp Val
Arg Ile Ser Tyr Cys Val Asp Arg Glu Met Ile Lys His Ser 370
375 380 atg ccg
gcc ttt gaa aaa atc tat aaa aaa tac aat aaa ctc gag tga 1200Met Pro
Ala Phe Glu Lys Ile Tyr Lys Lys Tyr Asn Lys Leu Glu 385
390 395
87399PRTEubacterium rectale 87Met Val Val Asn Glu Ser Met Tyr Gln Leu Gly
Ser Val Arg Ser Ala 1 5 10
15 Ile Arg Glu Leu Phe Glu Tyr Gly Lys Lys Arg Ala Ala Ile Val Gly
20 25 30 Lys Glu
Asn Val Tyr Asp Phe Ser Ile Gly Asn Pro Ser Ile Pro Ala 35
40 45 Pro Gln Ile Val Asn Asp Thr
Ile Lys Glu Leu Val Thr Asp Tyr Asp 50 55
60 Ser Val Ala Leu His Gly Tyr Thr Ser Ala Gln Gly
Asp Val Glu Thr 65 70 75
80 Arg Ala Ala Ile Ala Glu Phe Leu Asn Asn Thr His Gly Thr His Phe
85 90 95 Asn Ala Asp
Asn Leu Tyr Met Thr Met Gly Ala Ala Ala Ser Leu Ser 100
105 110 Ile Cys Phe Arg Ala Leu Thr Ser
Asp Ala Tyr Asp Glu Phe Ile Thr 115 120
125 Ile Ala Pro Tyr Phe Pro Glu Tyr Lys Val Phe Val Asn
Ala Ala Gly 130 135 140
Ala Arg Leu Val Glu Val Pro Ala Asp Thr Glu His Phe Gln Ile Asp 145
150 155 160 Phe Asp Ala Leu
Glu Glu Arg Ile Asn Ala His Thr Arg Gly Val Ile 165
170 175 Ile Asn Ser Pro Asn Asn Pro Ser Gly
Thr Val Tyr Ser Glu Glu Thr 180 185
190 Ile Lys Lys Leu Ser Asp Leu Leu Glu Lys Lys Ser Lys Glu
Ile Gly 195 200 205
Arg Pro Ile Phe Ile Ile Ala Asp Glu Pro Tyr Arg Glu Ile Val Tyr 210
215 220 Asp Gly Ile Lys Val
Pro Phe Val Thr Lys Tyr Tyr Asp Asn Thr Leu 225 230
235 240 Val Cys Tyr Ser Tyr Ser Lys Ser Leu Ser
Leu Pro Gly Glu Arg Ile 245 250
255 Gly Tyr Val Leu Val Pro Asp Glu Val Tyr Asp Lys Ala Glu Leu
Tyr 260 265 270 Ala
Ala Val Cys Gly Ala Gly Arg Ala Leu Gly Tyr Val Cys Ala Pro 275
280 285 Ser Leu Phe Gln Lys Met
Ile Val Lys Cys Gln Gly Ala Thr Gly Asp 290 295
300 Ile Asn Ala Tyr Lys Glu Asn Arg Asp Leu Leu
Tyr Glu Gly Leu Thr 305 310 315
320 Arg Ile Gly Tyr His Cys Phe Lys Pro Asp Gly Ala Phe Tyr Met Phe
325 330 335 Val Lys
Ala Leu Glu Asp Asp Ser Asn Ala Phe Cys Glu Lys Ala Lys 340
345 350 Glu Glu Asp Val Leu Ile Val
Ala Ala Asp Gly Phe Gly Cys Pro Gly 355 360
365 Trp Val Arg Ile Ser Tyr Cys Val Asp Arg Glu Met
Ile Lys His Ser 370 375 380
Met Pro Ala Phe Glu Lys Ile Tyr Lys Lys Tyr Asn Lys Leu Glu 385
390 395 881314DNABacillus
pumilusCDS(1)..(1314) 88atg tcg ggc ttt acg gca ctg tcg gaa gcg gaa ctg
aat gac ctg tat 48Met Ser Gly Phe Thr Ala Leu Ser Glu Ala Glu Leu
Asn Asp Leu Tyr 1 5 10
15 gcg gca ctg caa gat gaa tat gaa acc tac aaa tcc
aaa aac ctg cat 96Ala Ala Leu Gln Asp Glu Tyr Glu Thr Tyr Lys Ser
Lys Asn Leu His 20 25
30 ctg gac atg agc cgt ggc aaa ccg tct ccg aaa cag
ctg gat ctg agt 144Leu Asp Met Ser Arg Gly Lys Pro Ser Pro Lys Gln
Leu Asp Leu Ser 35 40
45 atg ggt atg ctg gat gtg gtt acc tcc aaa gac gca
atg acc gct gaa 192Met Gly Met Leu Asp Val Val Thr Ser Lys Asp Ala
Met Thr Ala Glu 50 55 60
gat ggc acg gac gtg cgc aac tat ggc ggt ctg acg
ggt ctg agc gaa 240Asp Gly Thr Asp Val Arg Asn Tyr Gly Gly Leu Thr
Gly Leu Ser Glu 65 70 75
80 acc aaa acg ttt ttc gcg gat gtt ctg aat ctg aaa
ccg gaa caa att 288Thr Lys Thr Phe Phe Ala Asp Val Leu Asn Leu Lys
Pro Glu Gln Ile 85 90
95 atc att ggc ggt aac agc tct ctg aat atg atg cat
gac acc atc gca 336Ile Ile Gly Gly Asn Ser Ser Leu Asn Met Met His
Asp Thr Ile Ala 100 105
110 cgt gct atg acg cac ggc gtt tat gat tct aaa acc
ccg tgg ggt aaa 384Arg Ala Met Thr His Gly Val Tyr Asp Ser Lys Thr
Pro Trp Gly Lys 115 120
125 ctg ccg aaa gtc aaa ttt ctg gca ccg tca ccg ggt
tac gat cgt cac 432Leu Pro Lys Val Lys Phe Leu Ala Pro Ser Pro Gly
Tyr Asp Arg His 130 135 140
ttt tcg att tgc gaa ctg ttc aac atc gaa atg att
acc gtc gat atg 480Phe Ser Ile Cys Glu Leu Phe Asn Ile Glu Met Ile
Thr Val Asp Met 145 150 155
160 aaa gcc gac ggt ccg gat atg gac cag gtg gaa aaa
ctg gtt gcg gaa 528Lys Ala Asp Gly Pro Asp Met Asp Gln Val Glu Lys
Leu Val Ala Glu 165 170
175 gac gaa gcc atc aaa ggc att tgg tgt gtt ccg aaa
tat agc aat ccg 576Asp Glu Ala Ile Lys Gly Ile Trp Cys Val Pro Lys
Tyr Ser Asn Pro 180 185
190 gat ggt att acc tac tct gat gaa gtc gtg gac cgt
ctg gcg agc atg 624Asp Gly Ile Thr Tyr Ser Asp Glu Val Val Asp Arg
Leu Ala Ser Met 195 200
205 aaa acg aaa gcc gat gac ttt cgc atc ttc tgg gat
gac gcg tat gcc 672Lys Thr Lys Ala Asp Asp Phe Arg Ile Phe Trp Asp
Asp Ala Tyr Ala 210 215 220
gtg cat cac ctg acc gat acg ccg gac acc ctg aaa
gat att ttt cag 720Val His His Leu Thr Asp Thr Pro Asp Thr Leu Lys
Asp Ile Phe Gln 225 230 235
240 gca gtc gaa gaa gct ggc cat ccg cac cgt gtg ttt
atg ttc gca tca 768Ala Val Glu Glu Ala Gly His Pro His Arg Val Phe
Met Phe Ala Ser 245 250
255 acc tcg aaa atc acg ttt ccg ggc agc ggt att gcg
ctg atg gcc agt 816Thr Ser Lys Ile Thr Phe Pro Gly Ser Gly Ile Ala
Leu Met Ala Ser 260 265
270 tcc ctg gat aac gtt agt ttc acc cag aaa caa ctg
tcc atc cag acg 864Ser Leu Asp Asn Val Ser Phe Thr Gln Lys Gln Leu
Ser Ile Gln Thr 275 280
285 att ggc ccg gat aaa atc aac caa ctg cgt cat ctg
cgc ttt ttc aaa 912Ile Gly Pro Asp Lys Ile Asn Gln Leu Arg His Leu
Arg Phe Phe Lys 290 295 300
aat ccg gaa ggt ctg aaa gaa cac atg cgc aaa cac
gcg gcc atc att 960Asn Pro Glu Gly Leu Lys Glu His Met Arg Lys His
Ala Ala Ile Ile 305 310 315
320 aaa ccg aaa ttt gat ctg gtg ctg tca atc ctg gac
gaa acc ctg ggc 1008Lys Pro Lys Phe Asp Leu Val Leu Ser Ile Leu Asp
Glu Thr Leu Gly 325 330
335 ggt aaa gat att gcc gaa tgg cat aaa ccg aac ggc
ggt tac ttc att 1056Gly Lys Asp Ile Ala Glu Trp His Lys Pro Asn Gly
Gly Tyr Phe Ile 340 345
350 tcg ctg aat acc ctg gat cac tgc gca aaa gct gtt
gtc cag aaa gca 1104Ser Leu Asn Thr Leu Asp His Cys Ala Lys Ala Val
Val Gln Lys Ala 355 360
365 aaa gaa gct ggt gtt acc atg acg ggt gca ggt gca
acc tat ccg tac 1152Lys Glu Ala Gly Val Thr Met Thr Gly Ala Gly Ala
Thr Tyr Pro Tyr 370 375 380
ggt aaa gat ccg ctg gac cgt aat atc cgc att gca
ccg acc ttt ccg 1200Gly Lys Asp Pro Leu Asp Arg Asn Ile Arg Ile Ala
Pro Thr Phe Pro 385 390 395
400 agt ctg gaa gaa ctg gaa caa gct atc gat att ttc
acg ctg tgt gtt 1248Ser Leu Glu Glu Leu Glu Gln Ala Ile Asp Ile Phe
Thr Leu Cys Val 405 410
415 caa ctg gtg tct atc gaa aaa ctg ctg tcc aaa aaa
tct caa tct gct 1296Gln Leu Val Ser Ile Glu Lys Leu Leu Ser Lys Lys
Ser Gln Ser Ala 420 425
430 ccg acc gtg ctc gag tga
1314Pro Thr Val Leu Glu
435
89437PRTBacillus pumilus 89Met Ser Gly Phe Thr
Ala Leu Ser Glu Ala Glu Leu Asn Asp Leu Tyr 1 5
10 15 Ala Ala Leu Gln Asp Glu Tyr Glu Thr Tyr
Lys Ser Lys Asn Leu His 20 25
30 Leu Asp Met Ser Arg Gly Lys Pro Ser Pro Lys Gln Leu Asp Leu
Ser 35 40 45 Met
Gly Met Leu Asp Val Val Thr Ser Lys Asp Ala Met Thr Ala Glu 50
55 60 Asp Gly Thr Asp Val Arg
Asn Tyr Gly Gly Leu Thr Gly Leu Ser Glu 65 70
75 80 Thr Lys Thr Phe Phe Ala Asp Val Leu Asn Leu
Lys Pro Glu Gln Ile 85 90
95 Ile Ile Gly Gly Asn Ser Ser Leu Asn Met Met His Asp Thr Ile Ala
100 105 110 Arg Ala
Met Thr His Gly Val Tyr Asp Ser Lys Thr Pro Trp Gly Lys 115
120 125 Leu Pro Lys Val Lys Phe Leu
Ala Pro Ser Pro Gly Tyr Asp Arg His 130 135
140 Phe Ser Ile Cys Glu Leu Phe Asn Ile Glu Met Ile
Thr Val Asp Met 145 150 155
160 Lys Ala Asp Gly Pro Asp Met Asp Gln Val Glu Lys Leu Val Ala Glu
165 170 175 Asp Glu Ala
Ile Lys Gly Ile Trp Cys Val Pro Lys Tyr Ser Asn Pro 180
185 190 Asp Gly Ile Thr Tyr Ser Asp Glu
Val Val Asp Arg Leu Ala Ser Met 195 200
205 Lys Thr Lys Ala Asp Asp Phe Arg Ile Phe Trp Asp Asp
Ala Tyr Ala 210 215 220
Val His His Leu Thr Asp Thr Pro Asp Thr Leu Lys Asp Ile Phe Gln 225
230 235 240 Ala Val Glu Glu
Ala Gly His Pro His Arg Val Phe Met Phe Ala Ser 245
250 255 Thr Ser Lys Ile Thr Phe Pro Gly Ser
Gly Ile Ala Leu Met Ala Ser 260 265
270 Ser Leu Asp Asn Val Ser Phe Thr Gln Lys Gln Leu Ser Ile
Gln Thr 275 280 285
Ile Gly Pro Asp Lys Ile Asn Gln Leu Arg His Leu Arg Phe Phe Lys 290
295 300 Asn Pro Glu Gly Leu
Lys Glu His Met Arg Lys His Ala Ala Ile Ile 305 310
315 320 Lys Pro Lys Phe Asp Leu Val Leu Ser Ile
Leu Asp Glu Thr Leu Gly 325 330
335 Gly Lys Asp Ile Ala Glu Trp His Lys Pro Asn Gly Gly Tyr Phe
Ile 340 345 350 Ser
Leu Asn Thr Leu Asp His Cys Ala Lys Ala Val Val Gln Lys Ala 355
360 365 Lys Glu Ala Gly Val Thr
Met Thr Gly Ala Gly Ala Thr Tyr Pro Tyr 370 375
380 Gly Lys Asp Pro Leu Asp Arg Asn Ile Arg Ile
Ala Pro Thr Phe Pro 385 390 395
400 Ser Leu Glu Glu Leu Glu Gln Ala Ile Asp Ile Phe Thr Leu Cys Val
405 410 415 Gln Leu
Val Ser Ile Glu Lys Leu Leu Ser Lys Lys Ser Gln Ser Ala 420
425 430 Pro Thr Val Leu Glu
435 90 1296DNABacillus cellulosilyticusCDS(1)..(1296) 90atg
agc gat tac acc gtg ctg tca acg caa gaa ctg caa caa gtc cac 48Met
Ser Asp Tyr Thr Val Leu Ser Thr Gln Glu Leu Gln Gln Val His 1
5 10 15 atg
gac ctg ctg gaa aaa ttc aac aaa ctg aaa gac gaa aat ctg gcg 96Met
Asp Leu Leu Glu Lys Phe Asn Lys Leu Lys Asp Glu Asn Leu Ala
20 25 30 ctg
gat atg agt cgt ggt aaa ccg tcc ccg gat caa ctg gac ctg tca 144Leu
Asp Met Ser Arg Gly Lys Pro Ser Pro Asp Gln Leu Asp Leu Ser
35 40 45 aac
ggc atg ctg gat att atc tcg gcg gac acc ccg ctg aaa gcc gaa 192Asn
Gly Met Leu Asp Ile Ile Ser Ala Asp Thr Pro Leu Lys Ala Glu
50 55 60 gat
ggt acg gac gtg cgc aat tat ggc ggt ctg gat ggc ctg ccg gaa 240Asp
Gly Thr Asp Val Arg Asn Tyr Gly Gly Leu Asp Gly Leu Pro Glu 65
70 75 80 gcg
aaa gcc ttt ttc agc aac att ctg aat gtt agc tct aac gaa att 288Ala
Lys Ala Phe Phe Ser Asn Ile Leu Asn Val Ser Ser Asn Glu Ile
85 90 95 atc
att ggc ggt aac agt tcc ctg aat ctg atg cat gat acc gtt gca 336Ile
Ile Gly Gly Asn Ser Ser Leu Asn Leu Met His Asp Thr Val Ala
100 105 110 cgt
gct atg ctg ttt ggt gtc aat gac ggc gaa acg gcg tgg gcc aaa 384Arg
Ala Met Leu Phe Gly Val Asn Asp Gly Glu Thr Ala Trp Ala Lys
115 120 125 ctg
ccg aaa gtc aaa ttc ctg tgc ccg agc ccg ggt tac gat cgt cac 432Leu
Pro Lys Val Lys Phe Leu Cys Pro Ser Pro Gly Tyr Asp Arg His
130 135 140 ttc
tct att tgt gaa ctg ttc aac atc gaa atg att cgc gtt gat atg 480Phe
Ser Ile Cys Glu Leu Phe Asn Ile Glu Met Ile Arg Val Asp Met 145
150 155 160 ctg
gaa gac ggc ccg aac atg gat cag atc gaa aaa ctg gtg caa gaa 528Leu
Glu Asp Gly Pro Asn Met Asp Gln Ile Glu Lys Leu Val Gln Glu
165 170 175 gac
gaa agt atc aaa ggt att tgg tgc gtt ccg aaa tat agt aac ccg 576Asp
Glu Ser Ile Lys Gly Ile Trp Cys Val Pro Lys Tyr Ser Asn Pro
180 185 190 gat
ggc att acc tac tcc gat gaa gtg gtt gac cgt ttt gca agc atg 624Asp
Gly Ile Thr Tyr Ser Asp Glu Val Val Asp Arg Phe Ala Ser Met
195 200 205 aaa
acg aaa gct aaa gac ttt cgc att ttc tgg gat gac gcg tat acc 672Lys
Thr Lys Ala Lys Asp Phe Arg Ile Phe Trp Asp Asp Ala Tyr Thr
210 215 220 gtg
cat cac ctg acg gat aaa ccg gac gaa ctg aaa aat atc ctg acc 720Val
His His Leu Thr Asp Lys Pro Asp Glu Leu Lys Asn Ile Leu Thr 225
230 235 240 gca
tgt aaa cag gct ggc aac gaa gat cgc gtt ctg atc ttt tca tcg 768Ala
Cys Lys Gln Ala Gly Asn Glu Asp Arg Val Leu Ile Phe Ser Ser
245 250 255 acc
agc aaa att acg ttc gcg ggc ggt ggc atc ggt gtc ctg gcc agc 816Thr
Ser Lys Ile Thr Phe Ala Gly Gly Gly Ile Gly Val Leu Ala Ser
260 265 270 tct
gaa aac aac atc cag tac ttc aaa aaa ctg ctg gca atg caa acc 864Ser
Glu Asn Asn Ile Gln Tyr Phe Lys Lys Leu Leu Ala Met Gln Thr
275 280 285 atc
ggc ccg gat aaa ctg aat cag atc cgt cat att cgc ttt ttc aaa 912Ile
Gly Pro Asp Lys Leu Asn Gln Ile Arg His Ile Arg Phe Phe Lys
290 295 300 aac
gtg gaa aat ctg tca acc cac atg aaa aaa cac gcc tcg atc att 960Asn
Val Glu Asn Leu Ser Thr His Met Lys Lys His Ala Ser Ile Ile 305
310 315 320 aaa
ccg aaa ttt gat atg gtc ctg aac aaa ctg gaa agc gaa atc ggt 1008Lys
Pro Lys Phe Asp Met Val Leu Asn Lys Leu Glu Ser Glu Ile Gly
325 330 335 ggc
aaa aac att ggt tct tgg gtg gaa ccg aat ggt ggc tat ttt att 1056Gly
Lys Asn Ile Gly Ser Trp Val Glu Pro Asn Gly Gly Tyr Phe Ile
340 345 350 agt
ttc aac acc ctg gat ggt tgc gcc aaa acg gtc gtg tcc atg gcg 1104Ser
Phe Asn Thr Leu Asp Gly Cys Ala Lys Thr Val Val Ser Met Ala
355 360 365 aaa
gaa gcc ggc gtt aaa ctg acc ggt gca ggc gct acg ttt ccg tac 1152Lys
Glu Ala Gly Val Lys Leu Thr Gly Ala Gly Ala Thr Phe Pro Tyr
370 375 380 ggt
cac gat ccg cgt gac cgc aat atc cgt att gca ccg acc ttc ccg 1200Gly
His Asp Pro Arg Asp Arg Asn Ile Arg Ile Ala Pro Thr Phe Pro 385
390 395 400 tct
ctg atc gaa ctg gaa cgc gct atg gat gtc ttc tgc ctg tgt gtg 1248Ser
Leu Ile Glu Leu Glu Arg Ala Met Asp Val Phe Cys Leu Cys Val
405 410 415 caa
ctg gcg tcg gtg gaa aaa ctg ctg aaa gaa caa ctg ctc gag tga 1296Gln
Leu Ala Ser Val Glu Lys Leu Leu Lys Glu Gln Leu Leu Glu
420 425 430
91431PRTBacillus cellulosilyticus 91Met Ser Asp Tyr Thr Val Leu Ser Thr
Gln Glu Leu Gln Gln Val His 1 5 10
15 Met Asp Leu Leu Glu Lys Phe Asn Lys Leu Lys Asp Glu Asn
Leu Ala 20 25 30
Leu Asp Met Ser Arg Gly Lys Pro Ser Pro Asp Gln Leu Asp Leu Ser
35 40 45 Asn Gly Met Leu
Asp Ile Ile Ser Ala Asp Thr Pro Leu Lys Ala Glu 50
55 60 Asp Gly Thr Asp Val Arg Asn Tyr
Gly Gly Leu Asp Gly Leu Pro Glu 65 70
75 80 Ala Lys Ala Phe Phe Ser Asn Ile Leu Asn Val Ser
Ser Asn Glu Ile 85 90
95 Ile Ile Gly Gly Asn Ser Ser Leu Asn Leu Met His Asp Thr Val Ala
100 105 110 Arg Ala Met
Leu Phe Gly Val Asn Asp Gly Glu Thr Ala Trp Ala Lys 115
120 125 Leu Pro Lys Val Lys Phe Leu Cys
Pro Ser Pro Gly Tyr Asp Arg His 130 135
140 Phe Ser Ile Cys Glu Leu Phe Asn Ile Glu Met Ile Arg
Val Asp Met 145 150 155
160 Leu Glu Asp Gly Pro Asn Met Asp Gln Ile Glu Lys Leu Val Gln Glu
165 170 175 Asp Glu Ser Ile
Lys Gly Ile Trp Cys Val Pro Lys Tyr Ser Asn Pro 180
185 190 Asp Gly Ile Thr Tyr Ser Asp Glu Val
Val Asp Arg Phe Ala Ser Met 195 200
205 Lys Thr Lys Ala Lys Asp Phe Arg Ile Phe Trp Asp Asp Ala
Tyr Thr 210 215 220
Val His His Leu Thr Asp Lys Pro Asp Glu Leu Lys Asn Ile Leu Thr 225
230 235 240 Ala Cys Lys Gln Ala
Gly Asn Glu Asp Arg Val Leu Ile Phe Ser Ser 245
250 255 Thr Ser Lys Ile Thr Phe Ala Gly Gly Gly
Ile Gly Val Leu Ala Ser 260 265
270 Ser Glu Asn Asn Ile Gln Tyr Phe Lys Lys Leu Leu Ala Met Gln
Thr 275 280 285 Ile
Gly Pro Asp Lys Leu Asn Gln Ile Arg His Ile Arg Phe Phe Lys 290
295 300 Asn Val Glu Asn Leu Ser
Thr His Met Lys Lys His Ala Ser Ile Ile 305 310
315 320 Lys Pro Lys Phe Asp Met Val Leu Asn Lys Leu
Glu Ser Glu Ile Gly 325 330
335 Gly Lys Asn Ile Gly Ser Trp Val Glu Pro Asn Gly Gly Tyr Phe Ile
340 345 350 Ser Phe
Asn Thr Leu Asp Gly Cys Ala Lys Thr Val Val Ser Met Ala 355
360 365 Lys Glu Ala Gly Val Lys Leu
Thr Gly Ala Gly Ala Thr Phe Pro Tyr 370 375
380 Gly His Asp Pro Arg Asp Arg Asn Ile Arg Ile Ala
Pro Thr Phe Pro 385 390 395
400 Ser Leu Ile Glu Leu Glu Arg Ala Met Asp Val Phe Cys Leu Cys Val
405 410 415 Gln Leu Ala
Ser Val Glu Lys Leu Leu Lys Glu Gln Leu Leu Glu 420
425 430 921185DNABacillus sp.CDS(1)..(1185)
92atg aaa gaa ctg ctg gca aac cgt gtg aaa acc ctg acc ccg tct acg
48Met Lys Glu Leu Leu Ala Asn Arg Val Lys Thr Leu Thr Pro Ser Thr
1 5 10 15
acc ctg gcg att acc gca aaa gcg aaa gaa atg aaa gcg cag ggt att
96Thr Leu Ala Ile Thr Ala Lys Ala Lys Glu Met Lys Ala Gln Gly Ile
20 25 30
gat gtg atc ggt ctg ggt gca ggt gaa ccg gac ttt aac acc ccg cag
144Asp Val Ile Gly Leu Gly Ala Gly Glu Pro Asp Phe Asn Thr Pro Gln
35 40 45
aat att atg gat gcg gcc atc gac tcg atg cag caa ggc tat acc aaa
192Asn Ile Met Asp Ala Ala Ile Asp Ser Met Gln Gln Gly Tyr Thr Lys
50 55 60
tac acg ccg agc ggc ggt ctg ccg gca ctg aaa cag gct atc atc gaa
240Tyr Thr Pro Ser Gly Gly Leu Pro Ala Leu Lys Gln Ala Ile Ile Glu
65 70 75 80
aaa ttc aaa cgt gat aac caa ctg gaa tat aaa ccg aat gaa att atc
288Lys Phe Lys Arg Asp Asn Gln Leu Glu Tyr Lys Pro Asn Glu Ile Ile
85 90 95
gtc ggt gtg ggc gcg aaa cat gtg ctg tac acc ctg ttc cag gtt att
336Val Gly Val Gly Ala Lys His Val Leu Tyr Thr Leu Phe Gln Val Ile
100 105 110
ctg aac gaa ggt gac gaa gtt att atc ccg atc ccg tat tgg gtt tct
384Leu Asn Glu Gly Asp Glu Val Ile Ile Pro Ile Pro Tyr Trp Val Ser
115 120 125
tac ccg gaa cag gtc aaa ctg gcc ggc ggt gtt ccg gtc tat att gaa
432Tyr Pro Glu Gln Val Lys Leu Ala Gly Gly Val Pro Val Tyr Ile Glu
130 135 140
gca acc agt gaa cag aac tac aaa att acg gct gaa caa ctg aaa aat
480Ala Thr Ser Glu Gln Asn Tyr Lys Ile Thr Ala Glu Gln Leu Lys Asn
145 150 155 160
gcg atc acc gat aaa acg aaa gcc gtc att atc aac agc ccg tct aat
528Ala Ile Thr Asp Lys Thr Lys Ala Val Ile Ile Asn Ser Pro Ser Asn
165 170 175
ccg acc ggc atg gtg tat acg cgt gaa gaa ctg gaa gat att gca aaa
576Pro Thr Gly Met Val Tyr Thr Arg Glu Glu Leu Glu Asp Ile Ala Lys
180 185 190
atc gct ctg gaa aac aat att ctg atc gtg tcc gac gaa att tat gaa
624Ile Ala Leu Glu Asn Asn Ile Leu Ile Val Ser Asp Glu Ile Tyr Glu
195 200 205
aaa ctg ctg tac aac ggt gcc gaa cat ttc agt att gca cag atc tcc
672Lys Leu Leu Tyr Asn Gly Ala Glu His Phe Ser Ile Ala Gln Ile Ser
210 215 220
gaa gaa gtt aaa gca caa acc att gtc atc aat ggc gtg agt aaa tcc
720Glu Glu Val Lys Ala Gln Thr Ile Val Ile Asn Gly Val Ser Lys Ser
225 230 235 240
cac tca atg acg ggc tgg cgc att ggt tat gca gct ggc aac gcg gat
768His Ser Met Thr Gly Trp Arg Ile Gly Tyr Ala Ala Gly Asn Ala Asp
245 250 255
att atc aat gcc atg acc gac ctg gca tcg cac agc acg tct aac ccg
816Ile Ile Asn Ala Met Thr Asp Leu Ala Ser His Ser Thr Ser Asn Pro
260 265 270
acc acg gct agc cag tat gcg gcc atc gaa gcg tac aat ggt ccg caa
864Thr Thr Ala Ser Gln Tyr Ala Ala Ile Glu Ala Tyr Asn Gly Pro Gln
275 280 285
gat agt gtg gaa gaa atg cgt aaa gcg ttt gaa tcc cgc ctg gaa acc
912Asp Ser Val Glu Glu Met Arg Lys Ala Phe Glu Ser Arg Leu Glu Thr
290 295 300
att tat ccg aaa ctg tca gct atc ccg ggt ttt aaa gtg gtt aaa ccg
960Ile Tyr Pro Lys Leu Ser Ala Ile Pro Gly Phe Lys Val Val Lys Pro
305 310 315 320
cag ggc gcc ttc tac ctg ctg ccg gat gtt tct gaa gca gct caa aaa
1008Gln Gly Ala Phe Tyr Leu Leu Pro Asp Val Ser Glu Ala Ala Gln Lys
325 330 335
acc ggc ttt gca agc gtc gac gaa ttc gcg tct gcc ctg ctg acg gaa
1056Thr Gly Phe Ala Ser Val Asp Glu Phe Ala Ser Ala Leu Leu Thr Glu
340 345 350
gcg aat gtg gcc gtt att ccg ggt agc ggt ttc ggt gca ccg tca acc
1104Ala Asn Val Ala Val Ile Pro Gly Ser Gly Phe Gly Ala Pro Ser Thr
355 360 365
att cgc atc tcg tac gca acc tct ctg aac ctg att gaa gaa gcg att
1152Ile Arg Ile Ser Tyr Ala Thr Ser Leu Asn Leu Ile Glu Glu Ala Ile
370 375 380
gaa cgc att gac cgt ttt gtg aaa ctc gag tga
1185Glu Arg Ile Asp Arg Phe Val Lys Leu Glu
385 390
93394PRTBacillus sp. 93Met Lys Glu Leu Leu Ala Asn Arg Val Lys Thr
Leu Thr Pro Ser Thr 1 5 10
15 Thr Leu Ala Ile Thr Ala Lys Ala Lys Glu Met Lys Ala Gln Gly Ile
20 25 30 Asp Val
Ile Gly Leu Gly Ala Gly Glu Pro Asp Phe Asn Thr Pro Gln 35
40 45 Asn Ile Met Asp Ala Ala Ile
Asp Ser Met Gln Gln Gly Tyr Thr Lys 50 55
60 Tyr Thr Pro Ser Gly Gly Leu Pro Ala Leu Lys Gln
Ala Ile Ile Glu 65 70 75
80 Lys Phe Lys Arg Asp Asn Gln Leu Glu Tyr Lys Pro Asn Glu Ile Ile
85 90 95 Val Gly Val
Gly Ala Lys His Val Leu Tyr Thr Leu Phe Gln Val Ile 100
105 110 Leu Asn Glu Gly Asp Glu Val Ile
Ile Pro Ile Pro Tyr Trp Val Ser 115 120
125 Tyr Pro Glu Gln Val Lys Leu Ala Gly Gly Val Pro Val
Tyr Ile Glu 130 135 140
Ala Thr Ser Glu Gln Asn Tyr Lys Ile Thr Ala Glu Gln Leu Lys Asn 145
150 155 160 Ala Ile Thr Asp
Lys Thr Lys Ala Val Ile Ile Asn Ser Pro Ser Asn 165
170 175 Pro Thr Gly Met Val Tyr Thr Arg Glu
Glu Leu Glu Asp Ile Ala Lys 180 185
190 Ile Ala Leu Glu Asn Asn Ile Leu Ile Val Ser Asp Glu Ile
Tyr Glu 195 200 205
Lys Leu Leu Tyr Asn Gly Ala Glu His Phe Ser Ile Ala Gln Ile Ser 210
215 220 Glu Glu Val Lys Ala
Gln Thr Ile Val Ile Asn Gly Val Ser Lys Ser 225 230
235 240 His Ser Met Thr Gly Trp Arg Ile Gly Tyr
Ala Ala Gly Asn Ala Asp 245 250
255 Ile Ile Asn Ala Met Thr Asp Leu Ala Ser His Ser Thr Ser Asn
Pro 260 265 270 Thr
Thr Ala Ser Gln Tyr Ala Ala Ile Glu Ala Tyr Asn Gly Pro Gln 275
280 285 Asp Ser Val Glu Glu Met
Arg Lys Ala Phe Glu Ser Arg Leu Glu Thr 290 295
300 Ile Tyr Pro Lys Leu Ser Ala Ile Pro Gly Phe
Lys Val Val Lys Pro 305 310 315
320 Gln Gly Ala Phe Tyr Leu Leu Pro Asp Val Ser Glu Ala Ala Gln Lys
325 330 335 Thr Gly
Phe Ala Ser Val Asp Glu Phe Ala Ser Ala Leu Leu Thr Glu 340
345 350 Ala Asn Val Ala Val Ile Pro
Gly Ser Gly Phe Gly Ala Pro Ser Thr 355 360
365 Ile Arg Ile Ser Tyr Ala Thr Ser Leu Asn Leu Ile
Glu Glu Ala Ile 370 375 380
Glu Arg Ile Asp Arg Phe Val Lys Leu Glu 385 390
941239DNASinorhizobium melilotiCDS(1)..(1239) 94atg acc atc
aat gcc acg gtt aaa gaa gcg ggt ttc cag ccg gcg tcg 48Met Thr Ile
Asn Ala Thr Val Lys Glu Ala Gly Phe Gln Pro Ala Ser 1
5 10 15 cgt att agt
agt atc ggt gtg tct gaa atc ctg aaa atc ggt gcc cgc 96Arg Ile Ser
Ser Ile Gly Val Ser Glu Ile Leu Lys Ile Gly Ala Arg
20 25 30 gcg gcc gca
atg aaa cgt gaa ggc aaa ccg gtt att atc ctg ggc gca 144Ala Ala Ala
Met Lys Arg Glu Gly Lys Pro Val Ile Ile Leu Gly Ala 35
40 45 ggt gaa ccg
gat ttt gac acc ccg gaa cat gtc aaa caa gct gcg agc 192Gly Glu Pro
Asp Phe Asp Thr Pro Glu His Val Lys Gln Ala Ala Ser 50
55 60 gat gcc att
cac cgc ggt gaa acc aaa tat acg gca ctg gac ggc acg 240Asp Ala Ile
His Arg Gly Glu Thr Lys Tyr Thr Ala Leu Asp Gly Thr 65
70 75 80 ccg gaa ctg
aaa aaa gct atc cgc gaa aaa ttt caa cgt gaa aac ggt 288Pro Glu Leu
Lys Lys Ala Ile Arg Glu Lys Phe Gln Arg Glu Asn Gly
85 90 95 ctg gcg tac
gaa ctg gat gaa att acc gtg gct acg ggc gcg aaa cag 336Leu Ala Tyr
Glu Leu Asp Glu Ile Thr Val Ala Thr Gly Ala Lys Gln
100 105 110 atc ctg ttc
aat gcc atg atg gca tct ctg gat ccg ggt gac gaa gtt 384Ile Leu Phe
Asn Ala Met Met Ala Ser Leu Asp Pro Gly Asp Glu Val 115
120 125 att atc ccg
acc ccg tat tgg acg tca tac tcg gat att gtc cat atc 432Ile Ile Pro
Thr Pro Tyr Trp Thr Ser Tyr Ser Asp Ile Val His Ile 130
135 140 tgc gaa ggt
aaa ccg gtg ctg att gct tgt gac gcg agc tct ggc ttt 480Cys Glu Gly
Lys Pro Val Leu Ile Ala Cys Asp Ala Ser Ser Gly Phe 145
150 155 160 cgt ctg acc
gcc gaa aaa ctg gaa gca gca atc acc ccg cgt acg cgt 528Arg Leu Thr
Ala Glu Lys Leu Glu Ala Ala Ile Thr Pro Arg Thr Arg
165 170 175 tgg gtc ctg
ctg aac agc ccg tct aat ccg agc ggt gct gcg tat tct 576Trp Val Leu
Leu Asn Ser Pro Ser Asn Pro Ser Gly Ala Ala Tyr Ser
180 185 190 gcc gca gat
tac cgc ccg ctg ctg gaa gtt ctg ctg cgt cat ccg cac 624Ala Ala Asp
Tyr Arg Pro Leu Leu Glu Val Leu Leu Arg His Pro His 195
200 205 gtc tgg ctg
ctg gtt gat gac atg tat gaa cac atc gtg tac gat ggc 672Val Trp Leu
Leu Val Asp Asp Met Tyr Glu His Ile Val Tyr Asp Gly 210
215 220 ttt cgc ttc
gtt acc ccg gcg cag ctg gaa ccg ggt ctg aaa aac cgt 720Phe Arg Phe
Val Thr Pro Ala Gln Leu Glu Pro Gly Leu Lys Asn Arg 225
230 235 240 acc ctg acg
gtg aat ggc gtt agc aaa gct tat gcg atg acg ggt tgg 768Thr Leu Thr
Val Asn Gly Val Ser Lys Ala Tyr Ala Met Thr Gly Trp
245 250 255 cgt att ggt
tac gcg ggc ggt ccg cgt gaa ctg atc aaa gcc atg gca 816Arg Ile Gly
Tyr Ala Gly Gly Pro Arg Glu Leu Ile Lys Ala Met Ala
260 265 270 gtg gtt cag
agt caa gcc acc tcc tgc ccg agt tcc att tca cag gct 864Val Val Gln
Ser Gln Ala Thr Ser Cys Pro Ser Ser Ile Ser Gln Ala 275
280 285 gcg tcg gtg
gca gca ctg aac ggt ccg caa gat ttt ctg aaa gaa cgc 912Ala Ser Val
Ala Ala Leu Asn Gly Pro Gln Asp Phe Leu Lys Glu Arg 290
295 300 acc gaa agc
ttc cag cgt cgc cgt gac ctg gtc gtg aac ggt ctg aat 960Thr Glu Ser
Phe Gln Arg Arg Arg Asp Leu Val Val Asn Gly Leu Asn 305
310 315 320 gcg att gat
ggc ctg gac tgc cgt gtt ccg gaa ggt gct ttt tat acc 1008Ala Ile Asp
Gly Leu Asp Cys Arg Val Pro Glu Gly Ala Phe Tyr Thr
325 330 335 ttc tca ggc
tgt gcg ggt gtt ctg ggc aaa gtc acg ccg tcg ggc aaa 1056Phe Ser Gly
Cys Ala Gly Val Leu Gly Lys Val Thr Pro Ser Gly Lys
340 345 350 cgc atc aaa
acc gat acg gac ttt tgt gcc tat ctg ctg gaa gat gcc 1104Arg Ile Lys
Thr Asp Thr Asp Phe Cys Ala Tyr Leu Leu Glu Asp Ala 355
360 365 cat gtg gca
gtt gtc ccg ggt agt gca ttc ggc ctg tcc ccg ttt ttc 1152His Val Ala
Val Val Pro Gly Ser Ala Phe Gly Leu Ser Pro Phe Phe 370
375 380 cgt att agt
tac gcg acc tcc gaa gcc gaa ctg aaa gaa gcc ctg gaa 1200Arg Ile Ser
Tyr Ala Thr Ser Glu Ala Glu Leu Lys Glu Ala Leu Glu 385
390 395 400 cgc att gct
gcc gcc tgt gac cgt ctg tcg ctc gag tga 1239Arg Ile Ala
Ala Ala Cys Asp Arg Leu Ser Leu Glu
405 410
95412PRTSinorhizobium meliloti 95Met Thr Ile Asn Ala Thr Val Lys Glu Ala
Gly Phe Gln Pro Ala Ser 1 5 10
15 Arg Ile Ser Ser Ile Gly Val Ser Glu Ile Leu Lys Ile Gly Ala
Arg 20 25 30 Ala
Ala Ala Met Lys Arg Glu Gly Lys Pro Val Ile Ile Leu Gly Ala 35
40 45 Gly Glu Pro Asp Phe Asp
Thr Pro Glu His Val Lys Gln Ala Ala Ser 50 55
60 Asp Ala Ile His Arg Gly Glu Thr Lys Tyr Thr
Ala Leu Asp Gly Thr 65 70 75
80 Pro Glu Leu Lys Lys Ala Ile Arg Glu Lys Phe Gln Arg Glu Asn Gly
85 90 95 Leu Ala
Tyr Glu Leu Asp Glu Ile Thr Val Ala Thr Gly Ala Lys Gln 100
105 110 Ile Leu Phe Asn Ala Met Met
Ala Ser Leu Asp Pro Gly Asp Glu Val 115 120
125 Ile Ile Pro Thr Pro Tyr Trp Thr Ser Tyr Ser Asp
Ile Val His Ile 130 135 140
Cys Glu Gly Lys Pro Val Leu Ile Ala Cys Asp Ala Ser Ser Gly Phe 145
150 155 160 Arg Leu Thr
Ala Glu Lys Leu Glu Ala Ala Ile Thr Pro Arg Thr Arg 165
170 175 Trp Val Leu Leu Asn Ser Pro Ser
Asn Pro Ser Gly Ala Ala Tyr Ser 180 185
190 Ala Ala Asp Tyr Arg Pro Leu Leu Glu Val Leu Leu Arg
His Pro His 195 200 205
Val Trp Leu Leu Val Asp Asp Met Tyr Glu His Ile Val Tyr Asp Gly 210
215 220 Phe Arg Phe Val
Thr Pro Ala Gln Leu Glu Pro Gly Leu Lys Asn Arg 225 230
235 240 Thr Leu Thr Val Asn Gly Val Ser Lys
Ala Tyr Ala Met Thr Gly Trp 245 250
255 Arg Ile Gly Tyr Ala Gly Gly Pro Arg Glu Leu Ile Lys Ala
Met Ala 260 265 270
Val Val Gln Ser Gln Ala Thr Ser Cys Pro Ser Ser Ile Ser Gln Ala
275 280 285 Ala Ser Val Ala
Ala Leu Asn Gly Pro Gln Asp Phe Leu Lys Glu Arg 290
295 300 Thr Glu Ser Phe Gln Arg Arg Arg
Asp Leu Val Val Asn Gly Leu Asn 305 310
315 320 Ala Ile Asp Gly Leu Asp Cys Arg Val Pro Glu Gly
Ala Phe Tyr Thr 325 330
335 Phe Ser Gly Cys Ala Gly Val Leu Gly Lys Val Thr Pro Ser Gly Lys
340 345 350 Arg Ile Lys
Thr Asp Thr Asp Phe Cys Ala Tyr Leu Leu Glu Asp Ala 355
360 365 His Val Ala Val Val Pro Gly Ser
Ala Phe Gly Leu Ser Pro Phe Phe 370 375
380 Arg Ile Ser Tyr Ala Thr Ser Glu Ala Glu Leu Lys Glu
Ala Leu Glu 385 390 395
400 Arg Ile Ala Ala Ala Cys Asp Arg Leu Ser Leu Glu 405
410 96999DNAMethanothermobacter
thermautotrophicusCDS(1)..(999) 96atg cgt ctg tgg cgt gct ctg tat cgc ccg
ccg acc att acc tac ccg 48Met Arg Leu Trp Arg Ala Leu Tyr Arg Pro
Pro Thr Ile Thr Tyr Pro 1 5 10
15 tca aaa agc ccg gaa gtc att att atg tct
tgt gaa gcg tcc ggc aaa 96Ser Lys Ser Pro Glu Val Ile Ile Met Ser
Cys Glu Ala Ser Gly Lys 20 25
30 att tgg ctg aac ggt gaa atg gtt gaa tgg
gaa gaa gca acc gtt cat 144Ile Trp Leu Asn Gly Glu Met Val Glu Trp
Glu Glu Ala Thr Val His 35 40
45 gtc ctg tca cat gtg gtt cac tat ggc agc
tct gtg ttt gaa ggt att 192Val Leu Ser His Val Val His Tyr Gly Ser
Ser Val Phe Glu Gly Ile 50 55
60 cgt tgc tac cgc aat tcg aaa ggt agc gcg
atc ttt cgt ctg cgc gaa 240Arg Cys Tyr Arg Asn Ser Lys Gly Ser Ala
Ile Phe Arg Leu Arg Glu 65 70
75 80 cac gtt aaa cgt ctg ttc gat tcc gcc aaa
att tat cgc atg gac atc 288His Val Lys Arg Leu Phe Asp Ser Ala Lys
Ile Tyr Arg Met Asp Ile 85 90
95 ccg tac acc cag gaa caa att tgc gat gcc
atc gtt gaa acg gtc cgt 336Pro Tyr Thr Gln Glu Gln Ile Cys Asp Ala
Ile Val Glu Thr Val Arg 100 105
110 gaa aac ggt ctg gaa gaa tgt tat atc cgt
ccg gtc gtg ttc cgc ggc 384Glu Asn Gly Leu Glu Glu Cys Tyr Ile Arg
Pro Val Val Phe Arg Gly 115 120
125 tac ggt gaa atg ggc gtg cat ccg gtt aat
tgt ccg gtg gac gtt gca 432Tyr Gly Glu Met Gly Val His Pro Val Asn
Cys Pro Val Asp Val Ala 130 135
140 gtc gca gca tgg gaa tgg ggt gca tat ctg
ggt gca gaa gca ctg gaa 480Val Ala Ala Trp Glu Trp Gly Ala Tyr Leu
Gly Ala Glu Ala Leu Glu 145 150
155 160 gtg ggc gtt gat gca ggt gtt tct acc tgg
cgt cgc atg gct ccg aac 528Val Gly Val Asp Ala Gly Val Ser Thr Trp
Arg Arg Met Ala Pro Asn 165 170
175 acg atg ccg aat atg gca aaa gct ggc ggt
aac tat ctg aat tca cag 576Thr Met Pro Asn Met Ala Lys Ala Gly Gly
Asn Tyr Leu Asn Ser Gln 180 185
190 ctg gca aaa atg gaa gct gtg cgc cat ggc
tac gat gaa gcg att atg 624Leu Ala Lys Met Glu Ala Val Arg His Gly
Tyr Asp Glu Ala Ile Met 195 200
205 ctg gac tat cac ggt tac atc tct gaa ggc
agt ggt gaa aac att ttt 672Leu Asp Tyr His Gly Tyr Ile Ser Glu Gly
Ser Gly Glu Asn Ile Phe 210 215
220 ctg gtc tcg gaa ggc gaa atc tat acc ccg
cct gtg agt tcc tca ctg 720Leu Val Ser Glu Gly Glu Ile Tyr Thr Pro
Pro Val Ser Ser Ser Leu 225 230
235 240 ctg cgt ggt att acg cgc gat agc gtg att
aaa atc gca cgt acc gaa 768Leu Arg Gly Ile Thr Arg Asp Ser Val Ile
Lys Ile Ala Arg Thr Glu 245 250
255 ggc gtc acg gtg cac gaa gaa ccg att acc
cgc gaa atg ctg tac atc 816Gly Val Thr Val His Glu Glu Pro Ile Thr
Arg Glu Met Leu Tyr Ile 260 265
270 gcg gat gaa gcc ttt ttc acc ggc acg gca
gct gaa att acc ccg atc 864Ala Asp Glu Ala Phe Phe Thr Gly Thr Ala
Ala Glu Ile Thr Pro Ile 275 280
285 cgt agc gtt gac ggc att gaa atc ggt gct
ggt cgt cgc ggt ccg gtc 912Arg Ser Val Asp Gly Ile Glu Ile Gly Ala
Gly Arg Arg Gly Pro Val 290 295
300 acg aaa ctg ctg caa gat gaa ttt ttc cgc
atc atc cgt gcc gaa acc 960Thr Lys Leu Leu Gln Asp Glu Phe Phe Arg
Ile Ile Arg Ala Glu Thr 305 310
315 320 gaa gat agc ttt ggc tgg ctg acc tac att
ctc gag tga 999Glu Asp Ser Phe Gly Trp Leu Thr Tyr Ile
Leu Glu 325 330
97332PRTMethanothermobacter
thermautotrophicus 97Met Arg Leu Trp Arg Ala Leu Tyr Arg Pro Pro Thr Ile
Thr Tyr Pro 1 5 10 15
Ser Lys Ser Pro Glu Val Ile Ile Met Ser Cys Glu Ala Ser Gly Lys
20 25 30 Ile Trp Leu Asn
Gly Glu Met Val Glu Trp Glu Glu Ala Thr Val His 35
40 45 Val Leu Ser His Val Val His Tyr Gly
Ser Ser Val Phe Glu Gly Ile 50 55
60 Arg Cys Tyr Arg Asn Ser Lys Gly Ser Ala Ile Phe Arg
Leu Arg Glu 65 70 75
80 His Val Lys Arg Leu Phe Asp Ser Ala Lys Ile Tyr Arg Met Asp Ile
85 90 95 Pro Tyr Thr Gln
Glu Gln Ile Cys Asp Ala Ile Val Glu Thr Val Arg 100
105 110 Glu Asn Gly Leu Glu Glu Cys Tyr Ile
Arg Pro Val Val Phe Arg Gly 115 120
125 Tyr Gly Glu Met Gly Val His Pro Val Asn Cys Pro Val Asp
Val Ala 130 135 140
Val Ala Ala Trp Glu Trp Gly Ala Tyr Leu Gly Ala Glu Ala Leu Glu 145
150 155 160 Val Gly Val Asp Ala
Gly Val Ser Thr Trp Arg Arg Met Ala Pro Asn 165
170 175 Thr Met Pro Asn Met Ala Lys Ala Gly Gly
Asn Tyr Leu Asn Ser Gln 180 185
190 Leu Ala Lys Met Glu Ala Val Arg His Gly Tyr Asp Glu Ala Ile
Met 195 200 205 Leu
Asp Tyr His Gly Tyr Ile Ser Glu Gly Ser Gly Glu Asn Ile Phe 210
215 220 Leu Val Ser Glu Gly Glu
Ile Tyr Thr Pro Pro Val Ser Ser Ser Leu 225 230
235 240 Leu Arg Gly Ile Thr Arg Asp Ser Val Ile Lys
Ile Ala Arg Thr Glu 245 250
255 Gly Val Thr Val His Glu Glu Pro Ile Thr Arg Glu Met Leu Tyr Ile
260 265 270 Ala Asp
Glu Ala Phe Phe Thr Gly Thr Ala Ala Glu Ile Thr Pro Ile 275
280 285 Arg Ser Val Asp Gly Ile Glu
Ile Gly Ala Gly Arg Arg Gly Pro Val 290 295
300 Thr Lys Leu Leu Gln Asp Glu Phe Phe Arg Ile Ile
Arg Ala Glu Thr 305 310 315
320 Glu Asp Ser Phe Gly Trp Leu Thr Tyr Ile Leu Glu 325
330 981605DNALactobacillus
acidophilusCDS(1)..(1605) 98atg gac aac tcc gaa gaa aaa aaa ctg gaa gcc
ctg ggt gcc ttt gaa 48Met Asp Asn Ser Glu Glu Lys Lys Leu Glu Ala
Leu Gly Ala Phe Glu 1 5 10
15 atc tca cgt aaa atg ctg gcg ctg gcg cag aaa
aat gaa aaa agc aac 96Ile Ser Arg Lys Met Leu Ala Leu Ala Gln Lys
Asn Glu Lys Ser Asn 20 25
30 att ttt ctg aat gcg ggc cgt ggt aac ccg aat
tgg atc cag acc ctg 144Ile Phe Leu Asn Ala Gly Arg Gly Asn Pro Asn
Trp Ile Gln Thr Leu 35 40
45 gca cgt ctg gca ttt gtg cgt ctg gtt caa ttc
ggt gtt acg gaa tct 192Ala Arg Leu Ala Phe Val Arg Leu Val Gln Phe
Gly Val Thr Glu Ser 50 55
60 aaa ctg acc att aac aat ggt atc atg gcc ggc
tat att aac acg gat 240Lys Leu Thr Ile Asn Asn Gly Ile Met Ala Gly
Tyr Ile Asn Thr Asp 65 70 75
80 ggc atc cgt gaa cgc ctg ttt gca ttc ctg gat
ccg gac aaa aac gat 288Gly Ile Arg Glu Arg Leu Phe Ala Phe Leu Asp
Pro Asp Lys Asn Asp 85 90
95 gaa gac aaa ttc ctg atc gat gcc gtg aac tac
tgc cat acc gaa ctg 336Glu Asp Lys Phe Leu Ile Asp Ala Val Asn Tyr
Cys His Thr Glu Leu 100 105
110 ggt ctg aat cgt gac aaa gtg gtt gca gaa tgg
gtt aac ggc gca gtc 384Gly Leu Asn Arg Asp Lys Val Val Ala Glu Trp
Val Asn Gly Ala Val 115 120
125 gct aac aat tat ccg gtc ccg gat cgc tgt ctg
gtg aac acg gaa aaa 432Ala Asn Asn Tyr Pro Val Pro Asp Arg Cys Leu
Val Asn Thr Glu Lys 130 135
140 atc atc aac tat ttt ctg caa gaa ctg tca tac
aaa gat gca aat ctg 480Ile Ile Asn Tyr Phe Leu Gln Glu Leu Ser Tyr
Lys Asp Ala Asn Leu 145 150 155
160 gct gaa caa acc gac ctg ttt ccg acg gaa ggc
ggt acc gcg gcc att 528Ala Glu Gln Thr Asp Leu Phe Pro Thr Glu Gly
Gly Thr Ala Ala Ile 165 170
175 gtt tac gcg ttc cat tcg ctg gcc gaa aac cac
ctg ctg aaa aaa ggt 576Val Tyr Ala Phe His Ser Leu Ala Glu Asn His
Leu Leu Lys Lys Gly 180 185
190 gat aaa atc gcc atc aac gaa ccg atc ttc acc
ccg tac ctg cgt atc 624Asp Lys Ile Ala Ile Asn Glu Pro Ile Phe Thr
Pro Tyr Leu Arg Ile 195 200
205 ccg gaa ctg aaa gat tat gaa ctg gtt gaa gtc
gac ctg cac agc tat 672Pro Glu Leu Lys Asp Tyr Glu Leu Val Glu Val
Asp Leu His Ser Tyr 210 215
220 gag aaa aac gat tgg gaa att gaa ccg aat gaa
atc gaa aaa ctg aaa 720Glu Lys Asn Asp Trp Glu Ile Glu Pro Asn Glu
Ile Glu Lys Leu Lys 225 230 235
240 gac ccg agc att aaa gcg ctg atc gtc gtg aac
ccg acg aat ccg acc 768Asp Pro Ser Ile Lys Ala Leu Ile Val Val Asn
Pro Thr Asn Pro Thr 245 250
255 tct aaa gaa ttt gat acc aac gcg ctg aat gcc
att aaa cag gct gtc 816Ser Lys Glu Phe Asp Thr Asn Ala Leu Asn Ala
Ile Lys Gln Ala Val 260 265
270 gag aaa aac ccg aaa ctg atg att atc agc gac
gaa gtg tat ggt gcc 864Glu Lys Asn Pro Lys Leu Met Ile Ile Ser Asp
Glu Val Tyr Gly Ala 275 280
285 ttt gtt ccg aac ttc aaa agc atc tat tct gtt
gtc ccg tac aat acg 912Phe Val Pro Asn Phe Lys Ser Ile Tyr Ser Val
Val Pro Tyr Asn Thr 290 295
300 atg ctg gtt tat agt tac tcc aaa ctg ttt ggt
tgc acc ggc tgg cgc 960Met Leu Val Tyr Ser Tyr Ser Lys Leu Phe Gly
Cys Thr Gly Trp Arg 305 310 315
320 ctg ggc gtt att gct ctg aac gag aaa aac gtc
ttc gat gac aat atc 1008Leu Gly Val Ile Ala Leu Asn Glu Lys Asn Val
Phe Asp Asp Asn Ile 325 330
335 gcg cat ctg gat aaa gtg gaa ctg cgt cag ctg
cac aaa cgc tac agc 1056Ala His Leu Asp Lys Val Glu Leu Arg Gln Leu
His Lys Arg Tyr Ser 340 345
350 tct gtg gtt ctg gat ccg gac aaa atg aaa ttt
att gat cgt ctg tgt 1104Ser Val Val Leu Asp Pro Asp Lys Met Lys Phe
Ile Asp Arg Leu Cys 355 360
365 gcg gac tca cgc tcg atc ggt ctg tat cat acg
gcc ggc ctg tca acc 1152Ala Asp Ser Arg Ser Ile Gly Leu Tyr His Thr
Ala Gly Leu Ser Thr 370 375
380 ccg cag caa att atg gaa gca ctg ttc tcg atg
acc cac ctg ctg acc 1200Pro Gln Gln Ile Met Glu Ala Leu Phe Ser Met
Thr His Leu Leu Thr 385 390 395
400 agt acg aac ggc ggt tcc gat gac ccg tac att
gat atc gca cgt aaa 1248Ser Thr Asn Gly Gly Ser Asp Asp Pro Tyr Ile
Asp Ile Ala Arg Lys 405 410
415 ctg gtg tct gaa cgc tat gat cag ctg cat gac
gca atg caa gct ccg 1296Leu Val Ser Glu Arg Tyr Asp Gln Leu His Asp
Ala Met Gln Ala Pro 420 425
430 aaa gat gaa acc gac acg aat acc cac tat tac
tcc ctg att gat atc 1344Lys Asp Glu Thr Asp Thr Asn Thr His Tyr Tyr
Ser Leu Ile Asp Ile 435 440
445 tat cgt ctg gcg gaa aaa atc tac ggc aaa gaa
ttt cgc gat tat ctg 1392Tyr Arg Leu Ala Glu Lys Ile Tyr Gly Lys Glu
Phe Arg Asp Tyr Leu 450 455
460 acg aac aat ttt gaa cag gtg gac ttc ctg ctg
aaa ctg gct gag aaa 1440Thr Asn Asn Phe Glu Gln Val Asp Phe Leu Leu
Lys Leu Ala Glu Lys 465 470 475
480 aac ggt gtc gtg ctg gtc gat ggc gtg ggt ttc
ggc gcg aaa ccg ggc 1488Asn Gly Val Val Leu Val Asp Gly Val Gly Phe
Gly Ala Lys Pro Gly 485 490
495 gaa ctg cgc gtt agt caa gca aat ctg ccg acc
gaa gat tat gct ctg 1536Glu Leu Arg Val Ser Gln Ala Asn Leu Pro Thr
Glu Asp Tyr Ala Leu 500 505
510 att ggc aaa caa gtc ctg gaa ctg ctg aaa gaa
tac tat gaa gaa ttt 1584Ile Gly Lys Gln Val Leu Glu Leu Leu Lys Glu
Tyr Tyr Glu Glu Phe 515 520
525 aaa cag aat aat ctc gag taa
1605Lys Gln Asn Asn Leu Glu
530
99534PRTLactobacillus acidophilus 99Met Asp
Asn Ser Glu Glu Lys Lys Leu Glu Ala Leu Gly Ala Phe Glu 1 5
10 15 Ile Ser Arg Lys Met Leu Ala
Leu Ala Gln Lys Asn Glu Lys Ser Asn 20 25
30 Ile Phe Leu Asn Ala Gly Arg Gly Asn Pro Asn Trp
Ile Gln Thr Leu 35 40 45
Ala Arg Leu Ala Phe Val Arg Leu Val Gln Phe Gly Val Thr Glu Ser
50 55 60 Lys Leu Thr
Ile Asn Asn Gly Ile Met Ala Gly Tyr Ile Asn Thr Asp 65
70 75 80 Gly Ile Arg Glu Arg Leu Phe
Ala Phe Leu Asp Pro Asp Lys Asn Asp 85
90 95 Glu Asp Lys Phe Leu Ile Asp Ala Val Asn Tyr
Cys His Thr Glu Leu 100 105
110 Gly Leu Asn Arg Asp Lys Val Val Ala Glu Trp Val Asn Gly Ala
Val 115 120 125 Ala
Asn Asn Tyr Pro Val Pro Asp Arg Cys Leu Val Asn Thr Glu Lys 130
135 140 Ile Ile Asn Tyr Phe Leu
Gln Glu Leu Ser Tyr Lys Asp Ala Asn Leu 145 150
155 160 Ala Glu Gln Thr Asp Leu Phe Pro Thr Glu Gly
Gly Thr Ala Ala Ile 165 170
175 Val Tyr Ala Phe His Ser Leu Ala Glu Asn His Leu Leu Lys Lys Gly
180 185 190 Asp Lys
Ile Ala Ile Asn Glu Pro Ile Phe Thr Pro Tyr Leu Arg Ile 195
200 205 Pro Glu Leu Lys Asp Tyr Glu
Leu Val Glu Val Asp Leu His Ser Tyr 210 215
220 Glu Lys Asn Asp Trp Glu Ile Glu Pro Asn Glu Ile
Glu Lys Leu Lys 225 230 235
240 Asp Pro Ser Ile Lys Ala Leu Ile Val Val Asn Pro Thr Asn Pro Thr
245 250 255 Ser Lys Glu
Phe Asp Thr Asn Ala Leu Asn Ala Ile Lys Gln Ala Val 260
265 270 Glu Lys Asn Pro Lys Leu Met Ile
Ile Ser Asp Glu Val Tyr Gly Ala 275 280
285 Phe Val Pro Asn Phe Lys Ser Ile Tyr Ser Val Val Pro
Tyr Asn Thr 290 295 300
Met Leu Val Tyr Ser Tyr Ser Lys Leu Phe Gly Cys Thr Gly Trp Arg 305
310 315 320 Leu Gly Val Ile
Ala Leu Asn Glu Lys Asn Val Phe Asp Asp Asn Ile 325
330 335 Ala His Leu Asp Lys Val Glu Leu Arg
Gln Leu His Lys Arg Tyr Ser 340 345
350 Ser Val Val Leu Asp Pro Asp Lys Met Lys Phe Ile Asp Arg
Leu Cys 355 360 365
Ala Asp Ser Arg Ser Ile Gly Leu Tyr His Thr Ala Gly Leu Ser Thr 370
375 380 Pro Gln Gln Ile Met
Glu Ala Leu Phe Ser Met Thr His Leu Leu Thr 385 390
395 400 Ser Thr Asn Gly Gly Ser Asp Asp Pro Tyr
Ile Asp Ile Ala Arg Lys 405 410
415 Leu Val Ser Glu Arg Tyr Asp Gln Leu His Asp Ala Met Gln Ala
Pro 420 425 430 Lys
Asp Glu Thr Asp Thr Asn Thr His Tyr Tyr Ser Leu Ile Asp Ile 435
440 445 Tyr Arg Leu Ala Glu Lys
Ile Tyr Gly Lys Glu Phe Arg Asp Tyr Leu 450 455
460 Thr Asn Asn Phe Glu Gln Val Asp Phe Leu Leu
Lys Leu Ala Glu Lys 465 470 475
480 Asn Gly Val Val Leu Val Asp Gly Val Gly Phe Gly Ala Lys Pro Gly
485 490 495 Glu Leu
Arg Val Ser Gln Ala Asn Leu Pro Thr Glu Asp Tyr Ala Leu 500
505 510 Ile Gly Lys Gln Val Leu Glu
Leu Leu Lys Glu Tyr Tyr Glu Glu Phe 515 520
525 Lys Gln Asn Asn Leu Glu 530
1001209DNASinorhizobium melilotiCDS(1)..(1209) 100atg gcc ttc ctg gcg gat
gca ctg agt cgt gtt aaa ccg tcg gca acc 48Met Ala Phe Leu Ala Asp
Ala Leu Ser Arg Val Lys Pro Ser Ala Thr 1 5
10 15 atc gct gtg tcg cag aaa
gcc cgt gaa ctg aaa gca aaa ggc cgt gat 96Ile Ala Val Ser Gln Lys
Ala Arg Glu Leu Lys Ala Lys Gly Arg Asp 20
25 30 gtg att ggc ctg ggt gcg
ggc gaa ccg gat ttt gac acc ccg gac aac 144Val Ile Gly Leu Gly Ala
Gly Glu Pro Asp Phe Asp Thr Pro Asp Asn 35
40 45 atc aaa aaa gcg gcc att
gat gcc atc gac cgc ggc gaa acc aaa tat 192Ile Lys Lys Ala Ala Ile
Asp Ala Ile Asp Arg Gly Glu Thr Lys Tyr 50
55 60 acg cct gtg agc ggt att
ccg gaa ctg cgt gaa gcg atc gcc aaa aaa 240Thr Pro Val Ser Gly Ile
Pro Glu Leu Arg Glu Ala Ile Ala Lys Lys 65 70
75 80 ttc aaa cgc gaa aac aac
ctg gat tac acc gca gct cag acg att gtt 288Phe Lys Arg Glu Asn Asn
Leu Asp Tyr Thr Ala Ala Gln Thr Ile Val 85
90 95 ggc acc ggc ggt aaa caa
atc ctg ttt aac gcg ttc atg gcc acc ctg 336Gly Thr Gly Gly Lys Gln
Ile Leu Phe Asn Ala Phe Met Ala Thr Leu 100
105 110 aat ccg ggt gat gaa gtg
gtt att ccg gca ccg tat tgg gtg tct tac 384Asn Pro Gly Asp Glu Val
Val Ile Pro Ala Pro Tyr Trp Val Ser Tyr 115
120 125 ccg gaa atg gtt gct ctg
tgc ggc ggt acg ccg gtg ttt gtt ccg acc 432Pro Glu Met Val Ala Leu
Cys Gly Gly Thr Pro Val Phe Val Pro Thr 130
135 140 cgt cag gaa aac aat ttc
aaa ctg aaa gca gaa gat ctg gac cgc gct 480Arg Gln Glu Asn Asn Phe
Lys Leu Lys Ala Glu Asp Leu Asp Arg Ala 145 150
155 160 atc acc ccg aaa acg aaa
tgg ttt gtt ttc aac agc ccg tct aat ccg 528Ile Thr Pro Lys Thr Lys
Trp Phe Val Phe Asn Ser Pro Ser Asn Pro 165
170 175 tca ggc gcg gcc tat tcg
cat gaa gaa ctg aaa gca ctg acc gat gtc 576Ser Gly Ala Ala Tyr Ser
His Glu Glu Leu Lys Ala Leu Thr Asp Val 180
185 190 ctg atg aaa cat ccg cac
gtc tgg gtg ctg acg gat gac atg tat gaa 624Leu Met Lys His Pro His
Val Trp Val Leu Thr Asp Asp Met Tyr Glu 195
200 205 cac ctg acc tac ggt gac
ttt cgt ttc gcc acg ccg gtt gaa gtc gaa 672His Leu Thr Tyr Gly Asp
Phe Arg Phe Ala Thr Pro Val Glu Val Glu 210
215 220 ccg ggc ctg tac gaa cgc
acc ctg acg atg aat ggt gtg tca aaa gcg 720Pro Gly Leu Tyr Glu Arg
Thr Leu Thr Met Asn Gly Val Ser Lys Ala 225 230
235 240 tat gcg atg acc ggt tgg
cgt att ggc tac gca gct ggt ccg ctg cat 768Tyr Ala Met Thr Gly Trp
Arg Ile Gly Tyr Ala Ala Gly Pro Leu His 245
250 255 ctg att aaa gcg atg gat
atg atc caa ggc cag caa acg agt ggt gcg 816Leu Ile Lys Ala Met Asp
Met Ile Gln Gly Gln Gln Thr Ser Gly Ala 260
265 270 gcc tcc atc gca cag tgg
gca gct gtt gaa gct ctg aac ggc ccg caa 864Ala Ser Ile Ala Gln Trp
Ala Ala Val Glu Ala Leu Asn Gly Pro Gln 275
280 285 gat ttc atc ggt cgc aac
aaa gaa atc ttc cag ggc cgt cgc gac ctg 912Asp Phe Ile Gly Arg Asn
Lys Glu Ile Phe Gln Gly Arg Arg Asp Leu 290
295 300 gtc gtg agc atg ctg aac
cag gcc aaa ggc att tct tgc ccg acc ccg 960Val Val Ser Met Leu Asn
Gln Ala Lys Gly Ile Ser Cys Pro Thr Pro 305 310
315 320 gaa ggt gca ttt tat gtc
tac ccg agt tgt gcg ggt ctg att ggc aaa 1008Glu Gly Ala Phe Tyr Val
Tyr Pro Ser Cys Ala Gly Leu Ile Gly Lys 325
330 335 acc gcc ccg tcc ggt aaa
gtc atc gaa acg gat gaa gac ttc gtg tcc 1056Thr Ala Pro Ser Gly Lys
Val Ile Glu Thr Asp Glu Asp Phe Val Ser 340
345 350 gaa ctg ctg gaa acc gaa
ggc gtt gcg gtt gtc cac ggt tca gcc ttt 1104Glu Leu Leu Glu Thr Glu
Gly Val Ala Val Val His Gly Ser Ala Phe 355
360 365 ggt ctg ggc ccg aat ttc
cgt att tcg tat gcg acg tcc gaa gct ctg 1152Gly Leu Gly Pro Asn Phe
Arg Ile Ser Tyr Ala Thr Ser Glu Ala Leu 370
375 380 ctg gaa gaa gcc tgc cgt
cgc att cag cgt ttt tgt gcc gcc tgt cgt 1200Leu Glu Glu Ala Cys Arg
Arg Ile Gln Arg Phe Cys Ala Ala Cys Arg 385 390
395 400 ctc gag taa
1209Leu Glu
101402PRTSinorhizobium
meliloti 101Met Ala Phe Leu Ala Asp Ala Leu Ser Arg Val Lys Pro Ser Ala
Thr 1 5 10 15 Ile
Ala Val Ser Gln Lys Ala Arg Glu Leu Lys Ala Lys Gly Arg Asp
20 25 30 Val Ile Gly Leu Gly
Ala Gly Glu Pro Asp Phe Asp Thr Pro Asp Asn 35
40 45 Ile Lys Lys Ala Ala Ile Asp Ala Ile
Asp Arg Gly Glu Thr Lys Tyr 50 55
60 Thr Pro Val Ser Gly Ile Pro Glu Leu Arg Glu Ala Ile
Ala Lys Lys 65 70 75
80 Phe Lys Arg Glu Asn Asn Leu Asp Tyr Thr Ala Ala Gln Thr Ile Val
85 90 95 Gly Thr Gly Gly
Lys Gln Ile Leu Phe Asn Ala Phe Met Ala Thr Leu 100
105 110 Asn Pro Gly Asp Glu Val Val Ile Pro
Ala Pro Tyr Trp Val Ser Tyr 115 120
125 Pro Glu Met Val Ala Leu Cys Gly Gly Thr Pro Val Phe Val
Pro Thr 130 135 140
Arg Gln Glu Asn Asn Phe Lys Leu Lys Ala Glu Asp Leu Asp Arg Ala 145
150 155 160 Ile Thr Pro Lys Thr
Lys Trp Phe Val Phe Asn Ser Pro Ser Asn Pro 165
170 175 Ser Gly Ala Ala Tyr Ser His Glu Glu Leu
Lys Ala Leu Thr Asp Val 180 185
190 Leu Met Lys His Pro His Val Trp Val Leu Thr Asp Asp Met Tyr
Glu 195 200 205 His
Leu Thr Tyr Gly Asp Phe Arg Phe Ala Thr Pro Val Glu Val Glu 210
215 220 Pro Gly Leu Tyr Glu Arg
Thr Leu Thr Met Asn Gly Val Ser Lys Ala 225 230
235 240 Tyr Ala Met Thr Gly Trp Arg Ile Gly Tyr Ala
Ala Gly Pro Leu His 245 250
255 Leu Ile Lys Ala Met Asp Met Ile Gln Gly Gln Gln Thr Ser Gly Ala
260 265 270 Ala Ser
Ile Ala Gln Trp Ala Ala Val Glu Ala Leu Asn Gly Pro Gln 275
280 285 Asp Phe Ile Gly Arg Asn Lys
Glu Ile Phe Gln Gly Arg Arg Asp Leu 290 295
300 Val Val Ser Met Leu Asn Gln Ala Lys Gly Ile Ser
Cys Pro Thr Pro 305 310 315
320 Glu Gly Ala Phe Tyr Val Tyr Pro Ser Cys Ala Gly Leu Ile Gly Lys
325 330 335 Thr Ala Pro
Ser Gly Lys Val Ile Glu Thr Asp Glu Asp Phe Val Ser 340
345 350 Glu Leu Leu Glu Thr Glu Gly Val
Ala Val Val His Gly Ser Ala Phe 355 360
365 Gly Leu Gly Pro Asn Phe Arg Ile Ser Tyr Ala Thr Ser
Glu Ala Leu 370 375 380
Leu Glu Glu Ala Cys Arg Arg Ile Gln Arg Phe Cys Ala Ala Cys Arg 385
390 395 400 Leu Glu
1021167DNAPyrococcus horikoshiiCDS(1)..(1167) 102atg gaa atc atg gaa ttc
gaa gaa gcg ttc aaa gaa gtc tac gaa atg 48Met Glu Ile Met Glu Phe
Glu Glu Ala Phe Lys Glu Val Tyr Glu Met 1 5
10 15 gtc aaa ccg aaa tac aaa
ctg ttc acg gca ggt ccg gtg gct tgc ttt 96Val Lys Pro Lys Tyr Lys
Leu Phe Thr Ala Gly Pro Val Ala Cys Phe 20
25 30 ccg gaa gtc ctg gaa att
atg aaa gtg cag atg ttc tcg cat cgt agc 144Pro Glu Val Leu Glu Ile
Met Lys Val Gln Met Phe Ser His Arg Ser 35
40 45 aaa gaa tat cgc aaa gtt
cac atg gat acc gtc gaa cgt ctg cgc gaa 192Lys Glu Tyr Arg Lys Val
His Met Asp Thr Val Glu Arg Leu Arg Glu 50
55 60 ttt ctg gaa gtc gaa aaa
ggt gaa gtt ctg ctg gtc ccg agc tct ggc 240Phe Leu Glu Val Glu Lys
Gly Glu Val Leu Leu Val Pro Ser Ser Gly 65 70
75 80 acc ggt att atg gaa gca
tcc atc cgt aac ggc gtt tca aaa ggc ggt 288Thr Gly Ile Met Glu Ala
Ser Ile Arg Asn Gly Val Ser Lys Gly Gly 85
90 95 aaa gtg ctg gtt acg att
atc ggc gcc ttc ggt aaa cgt tat aaa gaa 336Lys Val Leu Val Thr Ile
Ile Gly Ala Phe Gly Lys Arg Tyr Lys Glu 100
105 110 gtg gtt gaa tcc aat ggt
cgc aaa gca gtc gtg ctg gaa tac gaa ccg 384Val Val Glu Ser Asn Gly
Arg Lys Ala Val Val Leu Glu Tyr Glu Pro 115
120 125 ggc aaa gca gtg aaa ccg
gaa gat ctg gat gac gct ctg cgc aaa aac 432Gly Lys Ala Val Lys Pro
Glu Asp Leu Asp Asp Ala Leu Arg Lys Asn 130
135 140 ccg gac gtg gaa gcg gtt
acc att acg tac aac gaa acc tcg acg ggt 480Pro Asp Val Glu Ala Val
Thr Ile Thr Tyr Asn Glu Thr Ser Thr Gly 145 150
155 160 gtt ctg aat ccg ctg ccg
gaa ctg gcg aaa gtc gcc aaa gaa cat gat 528Val Leu Asn Pro Leu Pro
Glu Leu Ala Lys Val Ala Lys Glu His Asp 165
170 175 aaa ctg gtc ttt gtg gac
gca gtg agc gct atg ggc ggt gct gat atc 576Lys Leu Val Phe Val Asp
Ala Val Ser Ala Met Gly Gly Ala Asp Ile 180
185 190 aaa ttc gac aaa tgg ggc
ctg gat gtt gtc ttt agt tcc tca cag aaa 624Lys Phe Asp Lys Trp Gly
Leu Asp Val Val Phe Ser Ser Ser Gln Lys 195
200 205 gcg ttc ggt gtt ccg ccg
ggt ctg gca att ggt gcc ttt agc gaa cgt 672Ala Phe Gly Val Pro Pro
Gly Leu Ala Ile Gly Ala Phe Ser Glu Arg 210
215 220 ttc ctg gaa atc gcc gaa
aaa atg ccg gaa cgc ggt tgg tat ttt gac 720Phe Leu Glu Ile Ala Glu
Lys Met Pro Glu Arg Gly Trp Tyr Phe Asp 225 230
235 240 att ccg ctg tac gtg aaa
tac ctg aaa gaa aaa gaa tct acc ccg agt 768Ile Pro Leu Tyr Val Lys
Tyr Leu Lys Glu Lys Glu Ser Thr Pro Ser 245
250 255 acg ccg ccg atg ccg caa
gtg ttc ggc atc aat gtt gcc ctg cgt att 816Thr Pro Pro Met Pro Gln
Val Phe Gly Ile Asn Val Ala Leu Arg Ile 260
265 270 atc gaa aaa atg ggc ggt
aaa gaa aaa tgg ctg gaa atg tac gaa aaa 864Ile Glu Lys Met Gly Gly
Lys Glu Lys Trp Leu Glu Met Tyr Glu Lys 275
280 285 cgc gca aaa atg gtc cgt
gaa ggt gtg cgc gaa att ggc ctg gat atc 912Arg Ala Lys Met Val Arg
Glu Gly Val Arg Glu Ile Gly Leu Asp Ile 290
295 300 ctg gct gaa ccg ggt cat
gaa tct ccg acc att acg gcg gtg ctg acc 960Leu Ala Glu Pro Gly His
Glu Ser Pro Thr Ile Thr Ala Val Leu Thr 305 310
315 320 ccg ccg ggt atc aaa ggt
gac gaa gtt tat gaa gcc atg cgt aaa cgc 1008Pro Pro Gly Ile Lys Gly
Asp Glu Val Tyr Glu Ala Met Arg Lys Arg 325
330 335 ggc ttt gaa ctg gca aaa
ggc tac ggt tca gtt aaa gaa aaa acc ttt 1056Gly Phe Glu Leu Ala Lys
Gly Tyr Gly Ser Val Lys Glu Lys Thr Phe 340
345 350 cgt att ggc cac atg ggt
tat atg aaa ttc gaa gat atc caa gaa atg 1104Arg Ile Gly His Met Gly
Tyr Met Lys Phe Glu Asp Ile Gln Glu Met 355
360 365 ctg gac aat ctg cgt gaa
gtc atc aac gaa ctg aaa aaa caa aaa ggt 1152Leu Asp Asn Leu Arg Glu
Val Ile Asn Glu Leu Lys Lys Gln Lys Gly 370
375 380 atc aac ctc gag taa
1167Ile Asn Leu Glu
385
103388PRTPyrococcus
horikoshii 103Met Glu Ile Met Glu Phe Glu Glu Ala Phe Lys Glu Val Tyr Glu
Met 1 5 10 15 Val
Lys Pro Lys Tyr Lys Leu Phe Thr Ala Gly Pro Val Ala Cys Phe
20 25 30 Pro Glu Val Leu Glu
Ile Met Lys Val Gln Met Phe Ser His Arg Ser 35
40 45 Lys Glu Tyr Arg Lys Val His Met Asp
Thr Val Glu Arg Leu Arg Glu 50 55
60 Phe Leu Glu Val Glu Lys Gly Glu Val Leu Leu Val Pro
Ser Ser Gly 65 70 75
80 Thr Gly Ile Met Glu Ala Ser Ile Arg Asn Gly Val Ser Lys Gly Gly
85 90 95 Lys Val Leu Val
Thr Ile Ile Gly Ala Phe Gly Lys Arg Tyr Lys Glu 100
105 110 Val Val Glu Ser Asn Gly Arg Lys Ala
Val Val Leu Glu Tyr Glu Pro 115 120
125 Gly Lys Ala Val Lys Pro Glu Asp Leu Asp Asp Ala Leu Arg
Lys Asn 130 135 140
Pro Asp Val Glu Ala Val Thr Ile Thr Tyr Asn Glu Thr Ser Thr Gly 145
150 155 160 Val Leu Asn Pro Leu
Pro Glu Leu Ala Lys Val Ala Lys Glu His Asp 165
170 175 Lys Leu Val Phe Val Asp Ala Val Ser Ala
Met Gly Gly Ala Asp Ile 180 185
190 Lys Phe Asp Lys Trp Gly Leu Asp Val Val Phe Ser Ser Ser Gln
Lys 195 200 205 Ala
Phe Gly Val Pro Pro Gly Leu Ala Ile Gly Ala Phe Ser Glu Arg 210
215 220 Phe Leu Glu Ile Ala Glu
Lys Met Pro Glu Arg Gly Trp Tyr Phe Asp 225 230
235 240 Ile Pro Leu Tyr Val Lys Tyr Leu Lys Glu Lys
Glu Ser Thr Pro Ser 245 250
255 Thr Pro Pro Met Pro Gln Val Phe Gly Ile Asn Val Ala Leu Arg Ile
260 265 270 Ile Glu
Lys Met Gly Gly Lys Glu Lys Trp Leu Glu Met Tyr Glu Lys 275
280 285 Arg Ala Lys Met Val Arg Glu
Gly Val Arg Glu Ile Gly Leu Asp Ile 290 295
300 Leu Ala Glu Pro Gly His Glu Ser Pro Thr Ile Thr
Ala Val Leu Thr 305 310 315
320 Pro Pro Gly Ile Lys Gly Asp Glu Val Tyr Glu Ala Met Arg Lys Arg
325 330 335 Gly Phe Glu
Leu Ala Lys Gly Tyr Gly Ser Val Lys Glu Lys Thr Phe 340
345 350 Arg Ile Gly His Met Gly Tyr Met
Lys Phe Glu Asp Ile Gln Glu Met 355 360
365 Leu Asp Asn Leu Arg Glu Val Ile Asn Glu Leu Lys Lys
Gln Lys Gly 370 375 380
Ile Asn Leu Glu 385 1041194DNAThermoanaerobacter
tengcongensisCDS(1)..(1194) 104atg aac ctg agc caa aac gca ctg caa atc
acg ccg agt atg acc ctg 48Met Asn Leu Ser Gln Asn Ala Leu Gln Ile
Thr Pro Ser Met Thr Leu 1 5 10
15 gaa atc acc gcc aaa gcc cgc caa ctg aaa
gcc gaa ggc gtc gat gtg 96Glu Ile Thr Ala Lys Ala Arg Gln Leu Lys
Ala Glu Gly Val Asp Val 20 25
30 att gac ttt ggc gtg ggt gaa ccg gat ttc
gac acc ccg gat tat atc 144Ile Asp Phe Gly Val Gly Glu Pro Asp Phe
Asp Thr Pro Asp Tyr Ile 35 40
45 aaa gaa gcg gcc att gaa gcc atc aaa aaa
ggt tat acc aaa tac acg 192Lys Glu Ala Ala Ile Glu Ala Ile Lys Lys
Gly Tyr Thr Lys Tyr Thr 50 55
60 ccg gca tct ggc att ctg gaa ctg aaa aaa
gct atc tgc gaa aaa ctg 240Pro Ala Ser Gly Ile Leu Glu Leu Lys Lys
Ala Ile Cys Glu Lys Leu 65 70
75 80 aaa cgt gaa aac ggt ctg ttt tat gaa ccg
gaa cag att gtg gtt tct 288Lys Arg Glu Asn Gly Leu Phe Tyr Glu Pro
Glu Gln Ile Val Val Ser 85 90
95 aat ggc gca aaa cat agt att tac aac gca
ctg tcc gct atc ctg aat 336Asn Gly Ala Lys His Ser Ile Tyr Asn Ala
Leu Ser Ala Ile Leu Asn 100 105
110 ccg ggt gat gaa gtt att atc ccg gtc ccg
tat tgg ctg agc tac ccg 384Pro Gly Asp Glu Val Ile Ile Pro Val Pro
Tyr Trp Leu Ser Tyr Pro 115 120
125 gaa atg gtg cgc ctg gcg tat ggc aaa ccg
gtt ttt gtc cag acc aaa 432Glu Met Val Arg Leu Ala Tyr Gly Lys Pro
Val Phe Val Gln Thr Lys 130 135
140 gaa gaa aac aac ttc aaa atc acc gca gaa
gaa ctg acg gca gct att 480Glu Glu Asn Asn Phe Lys Ile Thr Ala Glu
Glu Leu Thr Ala Ala Ile 145 150
155 160 aac ccg aaa acg aaa gct ctg atc ctg aat
tca ccg aac aat ccg acc 528Asn Pro Lys Thr Lys Ala Leu Ile Leu Asn
Ser Pro Asn Asn Pro Thr 165 170
175 ggt gcg gtg tat acg cgt aaa gaa ctg caa
gat atc gcc gaa gtc gtg 576Gly Ala Val Tyr Thr Arg Lys Glu Leu Gln
Asp Ile Ala Glu Val Val 180 185
190 gaa gaa acc ggc att ttt gtc atc tcg gac
gaa gtg tat gaa aaa ctg 624Glu Glu Thr Gly Ile Phe Val Ile Ser Asp
Glu Val Tyr Glu Lys Leu 195 200
205 att tac gaa ggt gaa cat gtt agt atc gct
tcc ctg ggc gaa aaa att 672Ile Tyr Glu Gly Glu His Val Ser Ile Ala
Ser Leu Gly Glu Lys Ile 210 215
220 aaa gaa ctg acc atc gtt gtc aac ggt atg
agt aaa gcg tat gcc atg 720Lys Glu Leu Thr Ile Val Val Asn Gly Met
Ser Lys Ala Tyr Ala Met 225 230
235 240 acc ggc tgg cgt att ggt tac acg gcc agc
tct ctg gat gtc gcg aaa 768Thr Gly Trp Arg Ile Gly Tyr Thr Ala Ser
Ser Leu Asp Val Ala Lys 245 250
255 gtg atg gcc aat att cag tca cac acc acg
tcg aac ccg aat agc atc 816Val Met Ala Asn Ile Gln Ser His Thr Thr
Ser Asn Pro Asn Ser Ile 260 265
270 gcg caa tat gcc agc gtg acc gca ctg acg
ggt gac ggt gtt gcc att 864Ala Gln Tyr Ala Ser Val Thr Ala Leu Thr
Gly Asp Gly Val Ala Ile 275 280
285 aaa cgc atg gtc gaa gaa ttc aac aaa cgt
cgc ctg tac gcg gtg gaa 912Lys Arg Met Val Glu Glu Phe Asn Lys Arg
Arg Leu Tyr Ala Val Glu 290 295
300 cgt atc tct aaa atg aaa ggt ctg aaa gca
gtt cgc ccg caa ggc gct 960Arg Ile Ser Lys Met Lys Gly Leu Lys Ala
Val Arg Pro Gln Gly Ala 305 310
315 320 ttc tac gtg ttc gtt aac atc gaa gaa tac
gtg ggc aaa aaa gtt aac 1008Phe Tyr Val Phe Val Asn Ile Glu Glu Tyr
Val Gly Lys Lys Val Asn 325 330
335 ggt cgt aaa atc aaa ggc agt ctg gat ttt
gcg acc ctg ctg atc gaa 1056Gly Arg Lys Ile Lys Gly Ser Leu Asp Phe
Ala Thr Leu Leu Ile Glu 340 345
350 gaa gca aac gtt gct gtg gtt ccg gcc ctg
ccg ttc ggc atg gac aat 1104Glu Ala Asn Val Ala Val Val Pro Ala Leu
Pro Phe Gly Met Asp Asn 355 360
365 tat att cgc atc tcc tac gca acg agt atg
gaa aac att gaa aaa ggt 1152Tyr Ile Arg Ile Ser Tyr Ala Thr Ser Met
Glu Asn Ile Glu Lys Gly 370 375
380 ctg gat cgc att gaa aac ttc ctg aat aaa
atc ctc gag taa 1194Leu Asp Arg Ile Glu Asn Phe Leu Asn Lys
Ile Leu Glu 385 390
395 105397PRTThermoanaerobacter
tengcongensis 105Met Asn Leu Ser Gln Asn Ala Leu Gln Ile Thr Pro Ser Met
Thr Leu 1 5 10 15
Glu Ile Thr Ala Lys Ala Arg Gln Leu Lys Ala Glu Gly Val Asp Val
20 25 30 Ile Asp Phe Gly Val
Gly Glu Pro Asp Phe Asp Thr Pro Asp Tyr Ile 35
40 45 Lys Glu Ala Ala Ile Glu Ala Ile Lys
Lys Gly Tyr Thr Lys Tyr Thr 50 55
60 Pro Ala Ser Gly Ile Leu Glu Leu Lys Lys Ala Ile Cys
Glu Lys Leu 65 70 75
80 Lys Arg Glu Asn Gly Leu Phe Tyr Glu Pro Glu Gln Ile Val Val Ser
85 90 95 Asn Gly Ala Lys
His Ser Ile Tyr Asn Ala Leu Ser Ala Ile Leu Asn 100
105 110 Pro Gly Asp Glu Val Ile Ile Pro Val
Pro Tyr Trp Leu Ser Tyr Pro 115 120
125 Glu Met Val Arg Leu Ala Tyr Gly Lys Pro Val Phe Val Gln
Thr Lys 130 135 140
Glu Glu Asn Asn Phe Lys Ile Thr Ala Glu Glu Leu Thr Ala Ala Ile 145
150 155 160 Asn Pro Lys Thr Lys
Ala Leu Ile Leu Asn Ser Pro Asn Asn Pro Thr 165
170 175 Gly Ala Val Tyr Thr Arg Lys Glu Leu Gln
Asp Ile Ala Glu Val Val 180 185
190 Glu Glu Thr Gly Ile Phe Val Ile Ser Asp Glu Val Tyr Glu Lys
Leu 195 200 205 Ile
Tyr Glu Gly Glu His Val Ser Ile Ala Ser Leu Gly Glu Lys Ile 210
215 220 Lys Glu Leu Thr Ile Val
Val Asn Gly Met Ser Lys Ala Tyr Ala Met 225 230
235 240 Thr Gly Trp Arg Ile Gly Tyr Thr Ala Ser Ser
Leu Asp Val Ala Lys 245 250
255 Val Met Ala Asn Ile Gln Ser His Thr Thr Ser Asn Pro Asn Ser Ile
260 265 270 Ala Gln
Tyr Ala Ser Val Thr Ala Leu Thr Gly Asp Gly Val Ala Ile 275
280 285 Lys Arg Met Val Glu Glu Phe
Asn Lys Arg Arg Leu Tyr Ala Val Glu 290 295
300 Arg Ile Ser Lys Met Lys Gly Leu Lys Ala Val Arg
Pro Gln Gly Ala 305 310 315
320 Phe Tyr Val Phe Val Asn Ile Glu Glu Tyr Val Gly Lys Lys Val Asn
325 330 335 Gly Arg Lys
Ile Lys Gly Ser Leu Asp Phe Ala Thr Leu Leu Ile Glu 340
345 350 Glu Ala Asn Val Ala Val Val Pro
Ala Leu Pro Phe Gly Met Asp Asn 355 360
365 Tyr Ile Arg Ile Ser Tyr Ala Thr Ser Met Glu Asn Ile
Glu Lys Gly 370 375 380
Leu Asp Arg Ile Glu Asn Phe Leu Asn Lys Ile Leu Glu 385
390 395 1061284DNAClostridium
cellulolyticumCDS(1)..(1284) 106atg aaa agc tac aaa gac ctg agc aaa gaa
gaa ctg aaa tcc gaa atc 48Met Lys Ser Tyr Lys Asp Leu Ser Lys Glu
Glu Leu Lys Ser Glu Ile 1 5 10
15 gaa atc ctg gaa aaa cgc tac aac gaa ttc
aaa gca caa aac ctg aaa 96Glu Ile Leu Glu Lys Arg Tyr Asn Glu Phe
Lys Ala Gln Asn Leu Lys 20 25
30 ctg gat atg acc cgt ggt aaa ccg tgc gct
gaa cag ctg gac ctg tct 144Leu Asp Met Thr Arg Gly Lys Pro Cys Ala
Glu Gln Leu Asp Leu Ser 35 40
45 atg gat atg ctg gac att ccg gcg gtg gaa
ctg cgc aaa gcg gcc gat 192Met Asp Met Leu Asp Ile Pro Ala Val Glu
Leu Arg Lys Ala Ala Asp 50 55
60 ggc acc gac tgt ttt aat tat ggc gtt ctg
gat ggt att ccg gaa gca 240Gly Thr Asp Cys Phe Asn Tyr Gly Val Leu
Asp Gly Ile Pro Glu Ala 65 70
75 80 aaa gct ctg ttc gcc caa atg ctg gaa gtg
agc acg gat gaa atc atg 288Lys Ala Leu Phe Ala Gln Met Leu Glu Val
Ser Thr Asp Glu Ile Met 85 90
95 gtt ggc ggt aac agc tct ctg aat ctg atg
tat gac acc att gcg cgt 336Val Gly Gly Asn Ser Ser Leu Asn Leu Met
Tyr Asp Thr Ile Ala Arg 100 105
110 gcc atg tcg ctg ggc atc ctg ggt agc acg
ccg tgg tct aaa ctg aac 384Ala Met Ser Leu Gly Ile Leu Gly Ser Thr
Pro Trp Ser Lys Leu Asn 115 120
125 agt gtg aaa ttt ctg tgc ccg agc ccg ggc
tac gat cgc cat ttt gca 432Ser Val Lys Phe Leu Cys Pro Ser Pro Gly
Tyr Asp Arg His Phe Ala 130 135
140 att tgt gaa ctg ttc ggt atc gaa atg att
acc atc gat atg aaa cag 480Ile Cys Glu Leu Phe Gly Ile Glu Met Ile
Thr Ile Asp Met Lys Gln 145 150
155 160 gac ggc ccg gat atg gac acg gtt gaa aaa
ctg gtc tcc gaa gat gac 528Asp Gly Pro Asp Met Asp Thr Val Glu Lys
Leu Val Ser Glu Asp Asp 165 170
175 tca att aaa ggt atc tgg tgc gtg ccg aaa
tat tcc aat ccg gat ggc 576Ser Ile Lys Gly Ile Trp Cys Val Pro Lys
Tyr Ser Asn Pro Asp Gly 180 185
190 att acc tac acg gat gaa gtg gtt gac cgt
ttc tca aac ctg aaa ccg 624Ile Thr Tyr Thr Asp Glu Val Val Asp Arg
Phe Ser Asn Leu Lys Pro 195 200
205 aaa gcc aaa gat ttt cgc atc ttc tgg gac
aat gca tat tgc gtt cat 672Lys Ala Lys Asp Phe Arg Ile Phe Trp Asp
Asn Ala Tyr Cys Val His 210 215
220 cac ctg acc gaa aac ccg gat aaa ctg aaa
aac atc ctg aaa gct tgt 720His Leu Thr Glu Asn Pro Asp Lys Leu Lys
Asn Ile Leu Lys Ala Cys 225 230
235 240 aaa gat gcg ggt aac gac aat atg gtc tac
atc ttt agt tcc acg tca 768Lys Asp Ala Gly Asn Asp Asn Met Val Tyr
Ile Phe Ser Ser Thr Ser 245 250
255 aaa gtg tcg ttc ccg ggt gca ggt gtc gca
gtg atg gca acc tcg acg 816Lys Val Ser Phe Pro Gly Ala Gly Val Ala
Val Met Ala Thr Ser Thr 260 265
270 gaa aac atc aaa ggc att aaa aaa tct ctg
acc atc cag acg atc ggt 864Glu Asn Ile Lys Gly Ile Lys Lys Ser Leu
Thr Ile Gln Thr Ile Gly 275 280
285 cat gat aaa att aat caa ctg cgt cac gcc
aaa tac ttc aaa aac ctg 912His Asp Lys Ile Asn Gln Leu Arg His Ala
Lys Tyr Phe Lys Asn Leu 290 295
300 gat ggt atc aac agc cac atg aaa aaa cac
gca gac atc ctg aaa ccg 960Asp Gly Ile Asn Ser His Met Lys Lys His
Ala Asp Ile Leu Lys Pro 305 310
315 320 aaa ttt aac acc gtc ctg gaa att ttc gaa
ggc gaa ctg ggc ggt aaa 1008Lys Phe Asn Thr Val Leu Glu Ile Phe Glu
Gly Glu Leu Gly Gly Lys 325 330
335 gat atc gct tct tgg aac aaa ccg aat ggc
ggt tat ttt gtt agt ctg 1056Asp Ile Ala Ser Trp Asn Lys Pro Asn Gly
Gly Tyr Phe Val Ser Leu 340 345
350 aac acc atg gat aat tgt gcg aaa gaa gtt
gct aaa ctg gcg agt gaa 1104Asn Thr Met Asp Asn Cys Ala Lys Glu Val
Ala Lys Leu Ala Ser Glu 355 360
365 gcc ggc gtc gca ctg acc aaa gca ggc gct
acg ttc ccg tac ggt aac 1152Ala Gly Val Ala Leu Thr Lys Ala Gly Ala
Thr Phe Pro Tyr Gly Asn 370 375
380 gat ccg cgt gac cgc aat ctg cgc att gcc
ccg acc atg ccg ccg atc 1200Asp Pro Arg Asp Arg Asn Leu Arg Ile Ala
Pro Thr Met Pro Pro Ile 385 390
395 400 gaa gaa ctg aaa aaa gcc att gaa gtc ctg
gtc att tgt gtc caa ctg 1248Glu Glu Leu Lys Lys Ala Ile Glu Val Leu
Val Ile Cys Val Gln Leu 405 410
415 gtc tcc gct aat aaa ctg ctg aat caa ctc
gag taa 1284Val Ser Ala Asn Lys Leu Leu Asn Gln Leu
Glu 420 425
107427PRTClostridium cellulolyticum
107Met Lys Ser Tyr Lys Asp Leu Ser Lys Glu Glu Leu Lys Ser Glu Ile 1
5 10 15 Glu Ile Leu Glu
Lys Arg Tyr Asn Glu Phe Lys Ala Gln Asn Leu Lys 20
25 30 Leu Asp Met Thr Arg Gly Lys Pro Cys
Ala Glu Gln Leu Asp Leu Ser 35 40
45 Met Asp Met Leu Asp Ile Pro Ala Val Glu Leu Arg Lys Ala
Ala Asp 50 55 60
Gly Thr Asp Cys Phe Asn Tyr Gly Val Leu Asp Gly Ile Pro Glu Ala 65
70 75 80 Lys Ala Leu Phe Ala
Gln Met Leu Glu Val Ser Thr Asp Glu Ile Met 85
90 95 Val Gly Gly Asn Ser Ser Leu Asn Leu Met
Tyr Asp Thr Ile Ala Arg 100 105
110 Ala Met Ser Leu Gly Ile Leu Gly Ser Thr Pro Trp Ser Lys Leu
Asn 115 120 125 Ser
Val Lys Phe Leu Cys Pro Ser Pro Gly Tyr Asp Arg His Phe Ala 130
135 140 Ile Cys Glu Leu Phe Gly
Ile Glu Met Ile Thr Ile Asp Met Lys Gln 145 150
155 160 Asp Gly Pro Asp Met Asp Thr Val Glu Lys Leu
Val Ser Glu Asp Asp 165 170
175 Ser Ile Lys Gly Ile Trp Cys Val Pro Lys Tyr Ser Asn Pro Asp Gly
180 185 190 Ile Thr
Tyr Thr Asp Glu Val Val Asp Arg Phe Ser Asn Leu Lys Pro 195
200 205 Lys Ala Lys Asp Phe Arg Ile
Phe Trp Asp Asn Ala Tyr Cys Val His 210 215
220 His Leu Thr Glu Asn Pro Asp Lys Leu Lys Asn Ile
Leu Lys Ala Cys 225 230 235
240 Lys Asp Ala Gly Asn Asp Asn Met Val Tyr Ile Phe Ser Ser Thr Ser
245 250 255 Lys Val Ser
Phe Pro Gly Ala Gly Val Ala Val Met Ala Thr Ser Thr 260
265 270 Glu Asn Ile Lys Gly Ile Lys Lys
Ser Leu Thr Ile Gln Thr Ile Gly 275 280
285 His Asp Lys Ile Asn Gln Leu Arg His Ala Lys Tyr Phe
Lys Asn Leu 290 295 300
Asp Gly Ile Asn Ser His Met Lys Lys His Ala Asp Ile Leu Lys Pro 305
310 315 320 Lys Phe Asn Thr
Val Leu Glu Ile Phe Glu Gly Glu Leu Gly Gly Lys 325
330 335 Asp Ile Ala Ser Trp Asn Lys Pro Asn
Gly Gly Tyr Phe Val Ser Leu 340 345
350 Asn Thr Met Asp Asn Cys Ala Lys Glu Val Ala Lys Leu Ala
Ser Glu 355 360 365
Ala Gly Val Ala Leu Thr Lys Ala Gly Ala Thr Phe Pro Tyr Gly Asn 370
375 380 Asp Pro Arg Asp Arg
Asn Leu Arg Ile Ala Pro Thr Met Pro Pro Ile 385 390
395 400 Glu Glu Leu Lys Lys Ala Ile Glu Val Leu
Val Ile Cys Val Gln Leu 405 410
415 Val Ser Ala Asn Lys Leu Leu Asn Gln Leu Glu 420
425 1081299DNARhodococcus
erythropolisCDS(1)..(1299) 108atg ccg att caa acc cag att ggt ctg atg agc
cac gaa gaa ctg acg 48Met Pro Ile Gln Thr Gln Ile Gly Leu Met Ser
His Glu Glu Leu Thr 1 5 10
15 agc gaa cac gaa agc caa agc gca aaa tac acg
caa ctg caa gct caa 96Ser Glu His Glu Ser Gln Ser Ala Lys Tyr Thr
Gln Leu Gln Ala Gln 20 25
30 aaa ctg gcg ctg gat ctg acc cgt ggt aaa ccg
agc ccg gaa cag ctg 144Lys Leu Ala Leu Asp Leu Thr Arg Gly Lys Pro
Ser Pro Glu Gln Leu 35 40
45 gac ctg tct gcc gaa ctg ctg acg ctg ccg ggc
gat ggt gac ttt cgt 192Asp Leu Ser Ala Glu Leu Leu Thr Leu Pro Gly
Asp Gly Asp Phe Arg 50 55
60 gat ggc agc ggt acc gac tgc cgc aat tat ggt
ggt ctg acg ggt ctg 240Asp Gly Ser Gly Thr Asp Cys Arg Asn Tyr Gly
Gly Leu Thr Gly Leu 65 70 75
80 ccg gaa ctg cgt gcg att ttc ggc gaa ctg ctg
ggt atc ccg gtg gcc 288Pro Glu Leu Arg Ala Ile Phe Gly Glu Leu Leu
Gly Ile Pro Val Ala 85 90
95 aac ctg ctg gcc ggt aac aat gct tcc ctg gaa
atc atg cat gat aac 336Asn Leu Leu Ala Gly Asn Asn Ala Ser Leu Glu
Ile Met His Asp Asn 100 105
110 gtg gtt ttt agt ctg ctg cac ggt acc ccg gac
tcc gca cgt ccg tgg 384Val Val Phe Ser Leu Leu His Gly Thr Pro Asp
Ser Ala Arg Pro Trp 115 120
125 gcc cag gaa gaa aaa att aaa ttt ctg tgt ccg
gct ccg ggc tac gat 432Ala Gln Glu Glu Lys Ile Lys Phe Leu Cys Pro
Ala Pro Gly Tyr Asp 130 135
140 cgc cat ttc gcg atc acg gaa agt ctg ggt att
gaa atg atc gcc gtg 480Arg His Phe Ala Ile Thr Glu Ser Leu Gly Ile
Glu Met Ile Ala Val 145 150 155
160 ccg atg aac cac gat ggc ccg gac gtc gtg aaa
att gca gaa ctg gtt 528Pro Met Asn His Asp Gly Pro Asp Val Val Lys
Ile Ala Glu Leu Val 165 170
175 gct tct gat ccg caa atc aaa ggc atg tgg gcg
gtg ccg gtt tat gcc 576Ala Ser Asp Pro Gln Ile Lys Gly Met Trp Ala
Val Pro Val Tyr Ala 180 185
190 aat ccg acc ggc gca gtt tac tca gaa gaa att
gtc cgt acc ctg gca 624Asn Pro Thr Gly Ala Val Tyr Ser Glu Glu Ile
Val Arg Thr Leu Ala 195 200
205 tcg atg ccg acg gcg gcc ccg gat ttt cgt ctg
tat tgg gac aac gca 672Ser Met Pro Thr Ala Ala Pro Asp Phe Arg Leu
Tyr Trp Asp Asn Ala 210 215
220 tac gct gtc cat ccg ctg gtt ggc gaa acc gcg
ccg agt tat gat att 720Tyr Ala Val His Pro Leu Val Gly Glu Thr Ala
Pro Ser Tyr Asp Ile 225 230 235
240 ctg tcc atg gca gct gaa gca ggt cac ccg aac
cgt ccg ctg gtc ttt 768Leu Ser Met Ala Ala Glu Ala Gly His Pro Asn
Arg Pro Leu Val Phe 245 250
255 gca agt acc tcc aaa atc acg ttc gcg ggc gcc
ggt gtg agc ttt ttc 816Ala Ser Thr Ser Lys Ile Thr Phe Ala Gly Ala
Gly Val Ser Phe Phe 260 265
270 ggt agc tct gcg gaa aat ctg gcc tgg tac cag
aaa ttc ctg ggc aaa 864Gly Ser Ser Ala Glu Asn Leu Ala Trp Tyr Gln
Lys Phe Leu Gly Lys 275 280
285 aaa tct atc ggt ccg gat aaa gtt aac caa ctg
cgt cat ctg cgc ttt 912Lys Ser Ile Gly Pro Asp Lys Val Asn Gln Leu
Arg His Leu Arg Phe 290 295
300 ttc ggc aat gct gac ggt gtc cgt gcg cac atg
gaa aaa cac cgc gca 960Phe Gly Asn Ala Asp Gly Val Arg Ala His Met
Glu Lys His Arg Ala 305 310 315
320 ttt ctg gct ccg aaa ttc gaa ctg gtg ctg cgt
att ctg gaa gat cgc 1008Phe Leu Ala Pro Lys Phe Glu Leu Val Leu Arg
Ile Leu Glu Asp Arg 325 330
335 ctg ggt gca agc aaa gtt gct tct tgg acc gaa
ccg aaa ggc ggt tat 1056Leu Gly Ala Ser Lys Val Ala Ser Trp Thr Glu
Pro Lys Gly Gly Tyr 340 345
350 ttt atc agc ctg gat gtt gtc gac ggc acg gcg
aaa cgc gtg att gaa 1104Phe Ile Ser Leu Asp Val Val Asp Gly Thr Ala
Lys Arg Val Ile Glu 355 360
365 ctg gcg aaa aac gca ggt atc gca ctg acc gcg
gcc ggt tca gcg ttt 1152Leu Ala Lys Asn Ala Gly Ile Ala Leu Thr Ala
Ala Gly Ser Ala Phe 370 375
380 ccg tac tcg acg gat ccg gat gac cgt aat att
cgt ctg gca ccg tca 1200Pro Tyr Ser Thr Asp Pro Asp Asp Arg Asn Ile
Arg Leu Ala Pro Ser 385 390 395
400 ttc ccg tcg acc gcc gaa ctg gaa gtt gca atg
gat ggt gtc gca acc 1248Phe Pro Ser Thr Ala Glu Leu Glu Val Ala Met
Asp Gly Val Ala Thr 405 410
415 tgt gtc ctg ctg gcc gca acg gaa tca cgc ctg
tca gaa atc ctc gag 1296Cys Val Leu Leu Ala Ala Thr Glu Ser Arg Leu
Ser Glu Ile Leu Glu 420 425
430 taa
1299109432PRTRhodococcus erythropolis 109Met Pro
Ile Gln Thr Gln Ile Gly Leu Met Ser His Glu Glu Leu Thr 1 5
10 15 Ser Glu His Glu Ser Gln Ser
Ala Lys Tyr Thr Gln Leu Gln Ala Gln 20 25
30 Lys Leu Ala Leu Asp Leu Thr Arg Gly Lys Pro Ser
Pro Glu Gln Leu 35 40 45
Asp Leu Ser Ala Glu Leu Leu Thr Leu Pro Gly Asp Gly Asp Phe Arg
50 55 60 Asp Gly Ser
Gly Thr Asp Cys Arg Asn Tyr Gly Gly Leu Thr Gly Leu 65
70 75 80 Pro Glu Leu Arg Ala Ile Phe
Gly Glu Leu Leu Gly Ile Pro Val Ala 85
90 95 Asn Leu Leu Ala Gly Asn Asn Ala Ser Leu Glu
Ile Met His Asp Asn 100 105
110 Val Val Phe Ser Leu Leu His Gly Thr Pro Asp Ser Ala Arg Pro
Trp 115 120 125 Ala
Gln Glu Glu Lys Ile Lys Phe Leu Cys Pro Ala Pro Gly Tyr Asp 130
135 140 Arg His Phe Ala Ile Thr
Glu Ser Leu Gly Ile Glu Met Ile Ala Val 145 150
155 160 Pro Met Asn His Asp Gly Pro Asp Val Val Lys
Ile Ala Glu Leu Val 165 170
175 Ala Ser Asp Pro Gln Ile Lys Gly Met Trp Ala Val Pro Val Tyr Ala
180 185 190 Asn Pro
Thr Gly Ala Val Tyr Ser Glu Glu Ile Val Arg Thr Leu Ala 195
200 205 Ser Met Pro Thr Ala Ala Pro
Asp Phe Arg Leu Tyr Trp Asp Asn Ala 210 215
220 Tyr Ala Val His Pro Leu Val Gly Glu Thr Ala Pro
Ser Tyr Asp Ile 225 230 235
240 Leu Ser Met Ala Ala Glu Ala Gly His Pro Asn Arg Pro Leu Val Phe
245 250 255 Ala Ser Thr
Ser Lys Ile Thr Phe Ala Gly Ala Gly Val Ser Phe Phe 260
265 270 Gly Ser Ser Ala Glu Asn Leu Ala
Trp Tyr Gln Lys Phe Leu Gly Lys 275 280
285 Lys Ser Ile Gly Pro Asp Lys Val Asn Gln Leu Arg His
Leu Arg Phe 290 295 300
Phe Gly Asn Ala Asp Gly Val Arg Ala His Met Glu Lys His Arg Ala 305
310 315 320 Phe Leu Ala Pro
Lys Phe Glu Leu Val Leu Arg Ile Leu Glu Asp Arg 325
330 335 Leu Gly Ala Ser Lys Val Ala Ser Trp
Thr Glu Pro Lys Gly Gly Tyr 340 345
350 Phe Ile Ser Leu Asp Val Val Asp Gly Thr Ala Lys Arg Val
Ile Glu 355 360 365
Leu Ala Lys Asn Ala Gly Ile Ala Leu Thr Ala Ala Gly Ser Ala Phe 370
375 380 Pro Tyr Ser Thr Asp
Pro Asp Asp Arg Asn Ile Arg Leu Ala Pro Ser 385 390
395 400 Phe Pro Ser Thr Ala Glu Leu Glu Val Ala
Met Asp Gly Val Ala Thr 405 410
415 Cys Val Leu Leu Ala Ala Thr Glu Ser Arg Leu Ser Glu Ile Leu
Glu 420 425 430
1101356DNASaccharophagus degradansCDS(1)..(1356) 110atg caa gct ccg tac
acc aat ctg aaa acg cac cac aaa tgg gtc gaa 48Met Gln Ala Pro Tyr
Thr Asn Leu Lys Thr His His Lys Trp Val Glu 1 5
10 15 ttc tcc gcc gaa gac
acc acg ctg aac ctg agt aac gcc tca gtc gaa 96Phe Ser Ala Glu Asp
Thr Thr Leu Asn Leu Ser Asn Ala Ser Val Glu 20
25 30 cag ctg caa gaa tgg
aaa cag caa ctg tcg gcg gaa tat gat aac gtt 144Gln Leu Gln Glu Trp
Lys Gln Gln Leu Ser Ala Glu Tyr Asp Asn Val 35
40 45 ctg gcc cgt aaa ctg
aat ctg gac ctg acc cgc ggc aaa ccg agt gcg 192Leu Ala Arg Lys Leu
Asn Leu Asp Leu Thr Arg Gly Lys Pro Ser Ala 50
55 60 gaa cag ctg agt ctg
tcc gat gct atg gac ggc att ctg gcg ggt gat 240Glu Gln Leu Ser Leu
Ser Asp Ala Met Asp Gly Ile Leu Ala Gly Asp 65
70 75 80 tat att acg gcc agt
ggc atc gac gtg cgt aac tac ggc ggt ctg gaa 288Tyr Ile Thr Ala Ser
Gly Ile Asp Val Arg Asn Tyr Gly Gly Leu Glu 85
90 95 ggt atc ccg gaa gcg
cgt gcg att ggc tcc gat atc ctg ggt gtt ccg 336Gly Ile Pro Glu Ala
Arg Ala Ile Gly Ser Asp Ile Leu Gly Val Pro 100
105 110 gtc gaa aac gtt ctg
gcc ggc ggt aat agc tct ctg acc ctg atg tac 384Val Glu Asn Val Leu
Ala Gly Gly Asn Ser Ser Leu Thr Leu Met Tyr 115
120 125 cag acg atg gca att
gct cat caa ttc ggt ctg gct ggc gaa ggt agc 432Gln Thr Met Ala Ile
Ala His Gln Phe Gly Leu Ala Gly Glu Gly Ser 130
135 140 gcg tgg tct cag gaa
ggc acc gtg aaa ttt ctg tgc ccg gtt ccg ggt 480Ala Trp Ser Gln Glu
Gly Thr Val Lys Phe Leu Cys Pro Val Pro Gly 145
150 155 160 tat gat cgt cat tac
agc gtt tgt gaa cac ctg ggc atc gaa atg ctg 528Tyr Asp Arg His Tyr
Ser Val Cys Glu His Leu Gly Ile Glu Met Leu 165
170 175 acc gtc gcg atg acc
tct acg ggt ccg gat atg gac caa gtg gaa aaa 576Thr Val Ala Met Thr
Ser Thr Gly Pro Asp Met Asp Gln Val Glu Lys 180
185 190 atg att gcg gcc gat
ccg agc atc aaa ggc atg tgg tgc gtt ccg aaa 624Met Ile Ala Ala Asp
Pro Ser Ile Lys Gly Met Trp Cys Val Pro Lys 195
200 205 tat agt aat ccg acc
ggt gtg gtt tac tcc gac gaa acg gtc gaa cgt 672Tyr Ser Asn Pro Thr
Gly Val Val Tyr Ser Asp Glu Thr Val Glu Arg 210
215 220 att gca aac ctg ggc
aat atc gct ggt aaa aac ttt cgc gtg ttc tgg 720Ile Ala Asn Leu Gly
Asn Ile Ala Gly Lys Asn Phe Arg Val Phe Trp 225
230 235 240 gat aat gcg tat gcc
att cat gat ctg tca gac aac ccg gtt gca ctg 768Asp Asn Ala Tyr Ala
Ile His Asp Leu Ser Asp Asn Pro Val Ala Leu 245
250 255 gct aat atc ttt gaa
gcc tgt aaa gca gct ggc acc gaa gat tcg gtg 816Ala Asn Ile Phe Glu
Ala Cys Lys Ala Ala Gly Thr Glu Asp Ser Val 260
265 270 att cag ttc gca tca
acc tcg aaa gtc acg cac gcc ggc agc ggt gtg 864Ile Gln Phe Ala Ser
Thr Ser Lys Val Thr His Ala Gly Ser Gly Val 275
280 285 gca ttt atc gcg gcc
tcg gat acc aac ctg aaa ttt ttc aaa ctg gca 912Ala Phe Ile Ala Ala
Ser Asp Thr Asn Leu Lys Phe Phe Lys Leu Ala 290
295 300 ctg ggc ttc atg acg
att ggt ccg gat aaa gtg aat cag ctg cgt cat 960Leu Gly Phe Met Thr
Ile Gly Pro Asp Lys Val Asn Gln Leu Arg His 305
310 315 320 gcc aaa ttt ttc gca
gct gac ggt gca ctg tca gct cac atg gcg aaa 1008Ala Lys Phe Phe Ala
Ala Asp Gly Ala Leu Ser Ala His Met Ala Lys 325
330 335 cac gcg gcc att atc
aaa ccg cgc ttt gcg agt gtt ctg aaa cac ctg 1056His Ala Ala Ile Ile
Lys Pro Arg Phe Ala Ser Val Leu Lys His Leu 340
345 350 gaa gca gct ttc tcc
gat aac gac ctg ggc gaa tgg gaa agc gcg gat 1104Glu Ala Ala Phe Ser
Asp Asn Asp Leu Gly Glu Trp Glu Ser Ala Asp 355
360 365 ggc ggt tat ttt att
tct ttc gac acc cgt ccg ggt ctg gcc cag aaa 1152Gly Gly Tyr Phe Ile
Ser Phe Asp Thr Arg Pro Gly Leu Ala Gln Lys 370
375 380 gtc gtg aaa ctg gcc
ggc gat gca ggt gtg aaa ctg acc ccg gcg ggt 1200Val Val Lys Leu Ala
Gly Asp Ala Gly Val Lys Leu Thr Pro Ala Gly 385
390 395 400 gca acg ttt ccg tac
ggt aaa gat ccg cag gac tct aat att cgc atc 1248Ala Thr Phe Pro Tyr
Gly Lys Asp Pro Gln Asp Ser Asn Ile Arg Ile 405
410 415 gca ccg acc gtg ccg
acg gtt gat caa gtc gaa gaa gct atg caa gtc 1296Ala Pro Thr Val Pro
Thr Val Asp Gln Val Glu Glu Ala Met Gln Val 420
425 430 ttc gtc ctg tgt gtg
aaa ctg gcg tcg gtg gaa caa gca ctg gca aat 1344Phe Val Leu Cys Val
Lys Leu Ala Ser Val Glu Gln Ala Leu Ala Asn 435
440 445 tcg ctc gag taa
1356Ser Leu Glu
450
111451PRTSaccharophagus degradans 111Met Gln Ala Pro Tyr Thr Asn Leu Lys
Thr His His Lys Trp Val Glu 1 5 10
15 Phe Ser Ala Glu Asp Thr Thr Leu Asn Leu Ser Asn Ala Ser
Val Glu 20 25 30
Gln Leu Gln Glu Trp Lys Gln Gln Leu Ser Ala Glu Tyr Asp Asn Val
35 40 45 Leu Ala Arg Lys
Leu Asn Leu Asp Leu Thr Arg Gly Lys Pro Ser Ala 50
55 60 Glu Gln Leu Ser Leu Ser Asp Ala
Met Asp Gly Ile Leu Ala Gly Asp 65 70
75 80 Tyr Ile Thr Ala Ser Gly Ile Asp Val Arg Asn Tyr
Gly Gly Leu Glu 85 90
95 Gly Ile Pro Glu Ala Arg Ala Ile Gly Ser Asp Ile Leu Gly Val Pro
100 105 110 Val Glu Asn
Val Leu Ala Gly Gly Asn Ser Ser Leu Thr Leu Met Tyr 115
120 125 Gln Thr Met Ala Ile Ala His Gln
Phe Gly Leu Ala Gly Glu Gly Ser 130 135
140 Ala Trp Ser Gln Glu Gly Thr Val Lys Phe Leu Cys Pro
Val Pro Gly 145 150 155
160 Tyr Asp Arg His Tyr Ser Val Cys Glu His Leu Gly Ile Glu Met Leu
165 170 175 Thr Val Ala Met
Thr Ser Thr Gly Pro Asp Met Asp Gln Val Glu Lys 180
185 190 Met Ile Ala Ala Asp Pro Ser Ile Lys
Gly Met Trp Cys Val Pro Lys 195 200
205 Tyr Ser Asn Pro Thr Gly Val Val Tyr Ser Asp Glu Thr Val
Glu Arg 210 215 220
Ile Ala Asn Leu Gly Asn Ile Ala Gly Lys Asn Phe Arg Val Phe Trp 225
230 235 240 Asp Asn Ala Tyr Ala
Ile His Asp Leu Ser Asp Asn Pro Val Ala Leu 245
250 255 Ala Asn Ile Phe Glu Ala Cys Lys Ala Ala
Gly Thr Glu Asp Ser Val 260 265
270 Ile Gln Phe Ala Ser Thr Ser Lys Val Thr His Ala Gly Ser Gly
Val 275 280 285 Ala
Phe Ile Ala Ala Ser Asp Thr Asn Leu Lys Phe Phe Lys Leu Ala 290
295 300 Leu Gly Phe Met Thr Ile
Gly Pro Asp Lys Val Asn Gln Leu Arg His 305 310
315 320 Ala Lys Phe Phe Ala Ala Asp Gly Ala Leu Ser
Ala His Met Ala Lys 325 330
335 His Ala Ala Ile Ile Lys Pro Arg Phe Ala Ser Val Leu Lys His Leu
340 345 350 Glu Ala
Ala Phe Ser Asp Asn Asp Leu Gly Glu Trp Glu Ser Ala Asp 355
360 365 Gly Gly Tyr Phe Ile Ser Phe
Asp Thr Arg Pro Gly Leu Ala Gln Lys 370 375
380 Val Val Lys Leu Ala Gly Asp Ala Gly Val Lys Leu
Thr Pro Ala Gly 385 390 395
400 Ala Thr Phe Pro Tyr Gly Lys Asp Pro Gln Asp Ser Asn Ile Arg Ile
405 410 415 Ala Pro Thr
Val Pro Thr Val Asp Gln Val Glu Glu Ala Met Gln Val 420
425 430 Phe Val Leu Cys Val Lys Leu Ala
Ser Val Glu Gln Ala Leu Ala Asn 435 440
445 Ser Leu Glu 450 11211PRTCorynebacterium
ammoniagenesmisc_feature(3)..(3)Xaa can be any naturally occurring amino
acid 112Met Ser Xaa Ile Ala Gln Xaa Ile Leu Asp Gln 1 5
10
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