Patent application title: BETA-MANNANASE HAVING IMPROVED ENZYMATIC ACTIVITY
Inventors:
Rey-Ting Guo (Taipei, TW)
Jian-Wen Huang (Taipei, TW)
Ya-Shan Cheng (Taipei, TW)
Tzu-Hui Wu (Taipei, TW)
Hui-Lin Lai (Taipei, TW)
Cheng-Yen Lin (Taipei, TW)
Ting-Yung Huang (Taipei, TW)
Assignees:
Dongguan APAC Biotechnology CO., Ltd.
IPC8 Class: AC12N924FI
USPC Class:
435200
Class name: Enzyme (e.g., ligases (6. ), etc.), proenzyme; compositions thereof; process for preparing, activating, inhibiting, separating, or purifying enzymes hydrolase (3. ) acting on glycosyl compound (3.2)
Publication date: 2014-10-23
Patent application number: 20140315273
Abstract:
A β-mannanase having increased enzymaic activity is disclosed. The
β-mannanase has a modified amino acid sequence of SEQ ID NO: 2,
wherein the modification is a substitution of Tyrosine at position 216
with Tryptophan.Claims:
1. A β-mannanase comprising a modified amino acid sequence of SEQ ID
NO: 2, wherein the modification is a substitution of Tyrosine at position
216 with Tryptophan.
2. The β-mannanase according to claim 1 wherein the amino acid sequence of SEQ ID NO: 2 is encoded by ManBK gene isolated from Aspergillus niger BK01.
3. The β-mannanase according to claim 1 being an acidic and thermotolerant mannanase.
4. The β-mannanase according to claim 1 having a full length amino acid sequence of SEQ ID NO: 4.
5. (canceled)
6. (canceled)
7. The β-mannanase according to claim 1 wherein the β-mannanase is used in a food industry, a feed industry, or a paper pulp industry.
8. The β-mannanase according to claim 2 wherein the β-mannanase is used in a food industry, a feed industry, or a paper pulp industry.
9. The β-mannanase according to claim 3 wherein the β-mannanase is used in a food industry, a feed industry, or a paper pulp industry.
10. The β-mannanase according to claim 4 wherein the β-mannanase is used in a food industry, a feed industry, or a paper pulp industry.
Description:
FIELD OF THE INVENTION
[0001] The present invention relates to a β-mannanase, and more particularly to a β-mannanase having improved enzymatic activity.
BACKGROUND OF THE INVENTION
[0002] β-1,4 Mannans are major components of hemicellulose in plant cell wall of softwood, plant seeds and beans. Four types of polysaccharides including linear mannan, galactomannan, glucomannan, galactoglucomannan that are linked via β-1,4-glycosidic bonds compose mannans. Mannan hydrolysis provides wide array of biotechnological applications, such as feed manufacture, pulp and paper industries, and hydrolyzing coffee extract to reduce viscosity. A set of enzymes are required for complete degradation of mannans, including endo-β-1,4-mannanase (β-mannanase, EC 3.2.1.78), exo-β-mannosidase (EC 3.2.1.25) to cleave the main chain, and β-glucosidase (EC 3.2.1.21), α-galactosidase (EC 3.2.1.22), and acetyl mannan esterase to remove side chain decoration. Among them, β-mannanase which catalyzes random hydrolysis of manno-glycosidic bonds in the main chain is the key enzyme. More recently, major products of β-mannanase, mannotriose and mannobiose (mannooligosaccharides, MOS), have been proved beneficial as animal nutrition additive due to its prebiotic properties.
[0003] β-Mannanases are derived from various organisms including bacteria, yeasts, and filamentous fungi. According to the amino acid sequence homology, β-mannanases are mostly classified to glycoside hydrolase (GH) families 5, 26 and 113. These families share the same (β/α)8 folding and catalytic machinery, that two glutamate residues at active site serve as general acid/base and nucleophile to catalyze the cleavage of glycosidic bonds via a retaining double displacement mechanisms. Since industrial process is usually carried out at high temperatures, stable enzyme usage under a broad range of temperature is highly desirable. Therefore, β-mannanase needs to be modified to meet the requirement for different industrial usages. There are two ways to achieve these goals, one way is to screen suitable genes in nature, and the second way is modifying current enzyme genes based on their 3-D structural information.
[0004] In the present invention, the crystal structure of β-mannanase is analyzed and the enzyme activity of β-mannanase is improved by site-directed mutagenesis of the gene.
SUMMARY OF THE INVENTION
[0005] An object of the present invention is to modify β-mannanase by means of structural analysis and site-directed mutagenesis to efficiently increase the enzyme activity, and improve its economic value of industrial application.
[0006] According to an aspect of the present invention, there is provided a β-mannanase having increased enzymaic activity. The β-mannanase has a modified amino acid sequence of SEQ ID NO: 2, wherein the modification is a substitution of Tyrosine at position 216 with Tryptophan.
[0007] In an embodiment, the amino acid sequence of SEQ ID NO: 2 is encoded by ManBK gene isolated from Aspergillus niger BK01, and the β-mannanase is an acidic and thermotolerant mannanase.
[0008] In an embodiment, the β-mannanase has a full length amino acid sequence of SEQ ID NO: 4.
[0009] According to another aspect of the present invention, there is provided a nucleic acid encoding the aforesaid β-mannanase, and a recombinant plasmid comprising the aforesaid nucleic acid.
[0010] According to an additional aspect of the present invention, there is provided an industrial use of the aforesaid β-mannanase, wherein the industrial use comprises uses in food industry, feed industry, and paper pulp industry.
[0011] The above objects and advantages of the present invention will become more readily apparent to those ordinarily skilled in the art after reviewing the following detailed description and accompanying drawings, in which:
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows the gene sequence and the amino acid sequence of the wild-type ManBK;
[0013] FIG. 2 shows the protein structure of the wild-type ManBK, which was superimposed with Trichoderma reesei mannanase in complex with mannobiose;
[0014] FIG. 3 shows the sequence of the mutagenic primer for the Y216W mutant;
[0015] FIG. 4 shows the gene sequence and the amino acid sequence of the Y216W mutant;
[0016] FIG. 5 shows the β-mannanase activity analysis of the wild-type ManBK and the Y216W mutant; and
[0017] FIG. 6 shows the kinetic analysis of the wild-type ManBK and the Y216W mutant.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0018] The present invention will now be described more specifically with reference to the following embodiments. It is to be noted that the following descriptions of preferred embodiments of this invention are presented herein for purpose of illustration and description only; it is not intended to be exhaustive or to be limited to the precise form disclosed.
[0019] In the present invention, a gene of the β-mannanase ManBK was isolated from Aspergillus niger BK01, and ManBK is an acidic and thermotolerant β-mannanase. In order to improve the industrial application value of this enzyme, the protein structure of the apo-form ManBK was solved by X-ray crystallography, and the solved structure was superimposed with Trichoderma reesei mannanase (having 57% similarity in protein sequence compared with ManBK) in complex with mannobiose. Then, based on the structural information of the enzyme, the important amino acid residues in the active site were selected for site-directed mutagenesis to improve the enzymatic activity. The enzyme modification process of ManBK and the resulted mannanase protein are described in detail as follows.
[0020] First, the ManBK gene was obtained from Aspergillus niger BK01 (GenBank accession no. FJ268574), and as shown in FIG. 1, the full length of sequence of the ManBK gene is 1038 base pairs (SEQ ID NO: 1), which encodes a protein of 345 amino acids (SEQ ID NO: 2). The ManBK gene was constructed into pPICZαA vector by using EcoRI and NotI sites. The primers for polymerase chain reaction were 5'-GGTATTGAGGGTCGCGCGGCGGC GGCGGCGATGTCCTTCGCTTCCACTTCCG-3' (forward primer) and 5'-AGAGGAGAGTTAGAGCCTTAAGCGGAACCGATAGCAGC-3' (reverse primer). The constructed plasmid was transformed into a competent cell as a wild-type expression vector.
[0021] To solve the protein structure of ManBK by X-ray crystallography, the protein crystal was obtained by using sitting drop vapor diffusion method at room temperature by Hampton screen kit. The protein crystal of ManBK in apo form was prepared by mixing 2 μl mannanase solution (10 mg/ml in 25 mM Tris-HCl, pH 7.5) with equal amounts of mixture solution and mother liquor, and equilibrating with 500 μl of the mother liquor at room temperature. The wild-type ManBK crystal was obtained by a condition composed of 0.1M Bis-Tris pH 5.5, 0.4M magnesium chloride, and 29% PEG3350. The molecular replacement method was used for phasing X-ray diffraction data, and the protein structure of ManBK was subsequently determined by crystallographic software.
[0022] FIG. 2 shows the protein structure of ManBK solved by X-ray crystallography, and the solved structure was superimposed with Trichoderma reesei mannanase in complex with mannobiose in subsites +1 and +2. The protein structure of ManBK has (β/α)8 barrel fold, wherein 8 μ-sheets are located in the interior and 8 α-helixes pack around the exterior. By studying the structural information of ManBK, 30 amino acid residues were selected to be modified. Particularly, Tyr216 is located in the active site of the enzyme and may be important to the catalytic reaction of ManBK, and thus is targeted for site-directed mutagenesis, and it is found that the mutation of Tyr216 improves the enzymatic activity of ManBK, while other mutations do not show significant effects and are not redundantly described here. The following describes the processes for site-directed mutagenesis, protein expression and activity assay of Y216W mutant.
[0023] The Y216W mutant was prepared by using QuikChange site-directed mutagenesis kit with ManBK gene as a template. The sequence of the primer for Y216W mutant was shown in FIG. 3, wherein Y216W means Tyrosine at position 216 was mutated into Tryptophan; in other words, the modification is a substitution of Tyrosine at position 216 with Tryptophan. The original template was removed via DpnI digestion under 37° C., and then the plasmid with mutated gene was transformed into E. coli and screened with Ampicillin. Finally, the mutated gene was confirmed by DNA sequencing. Therefore, the Y216W mutant was constructed, and as shown in FIG. 4, the gene sequence was numbered as SEQ ID NO: 3, and the amino acid sequence was numbered as SEQ ID NO: 4.
[0024] The wild-type and mutant ManBK were expressed in Pichia. First, the plasmid DNA was linearized by PmeI and transformed into the P. pastoris X33 strain by electroporation. The transformants were selected on YPD (1% yeast extract, 2% peptone, 2% glucose, 2% agar) plates containing 100 μg/mL Zeocin and incubated at 30° C. for 2 days. The picked colonies were inoculated into 5 ml YPD medium at 30° C. overnight and further amplified into 50 ml BMGY medium at 30° C. overnight. After that, the cultured medium was changed to 20 ml BMMY with 0.5% methanol to induce the target protein expression. The samples were collected at different time points for every 24 hours, and meanwhile, the methanol was added into the flask to the final concentration of 0.5%. After induction for 4 days, the cells were harvested by centrifugation at 3500 rpm and the supernatant was collected for further purification.
[0025] The supernatant was purified by FPLC (fast protein liquid chromatography) using Ni2+ column and DEAE column. Finally, the wild-type and mutant ManBK peoteins, which had above 95% purity, were concentrated up to 5 mg/ml in protein buffer (25 mM Tris and 150 mM NaCl, pH 7.5) and then stored at -80° C.
[0026] To verify the difference between the wild-type and mutant ManBK, the β-mannanase activity assay and the kinetic analysis were performed. The β-mannanase activity was determined by dinitrosalicylic acid (DNS) method using mannose as a standard. The reaction was started by mixing 0.2 mL appropriately diluted enzyme sample with 1.8 mL of 3 mg/L locust bean gum (LBG) in 0.05 M citrate acid, pH 5.3. After 5-min incubation at 50° C., the reaction was stopped by adding 3 ml of DNS-reagent and boiled for 5 min to remove residual enzyme activity. After cooling in cold water bath for 5 min, the 540 nm absorbance of the reaction solution was measured. One unit of β-mannanase activity was defined as the amount of enzyme releasing 1 μmol of mannose equivalents per minute per mg of total soluble proteins under the assay conditions.
[0027] FIG. 5 shows the β-mannanase activity analysis of the wild-type ManBK and the Y216W mutant. The specific activity of the wild-type ManBK and the Y216W mutant are 646 and 784 U/mg. These results indicated that the specific activity of enzyme was increased 19% when Tyr216 was mutated to Tryptophan.
[0028] For the kinetic analysis, optimal protein concentration was first determined by using a series of 0.4-3.6 μg/ml protein solutions and 10 mg/ml LBG. The enzyme activity was then measured by using the optimal level of protein and a series of 0.5-10 mg/ml LBG solutions. Based on these data, the kinetic parameters were obtained by using the Michaelis-Menten model and curve-fitting analysis with a computer.
[0029] FIG. 6 shows the kinetic analysis of the wild-type ManBK and the Y216W mutant. The Y216W mutant had a higher catalytic rate (kcat) than the wild-type enzyme while the Km value of the Y216W mutant was also slightly higher than that of the wild-type enzyme. Higher Km of an enzyme indicates lower affinity to the substrate. However, it also indicates faster substrate release rate. Therefore, with the presence of sufficient substrate in general industrial application, the specific activity of Y216W mutant was higher than that of the wild-type enzyme.
[0030] From the above, in order to improve the enzymatic activity of ManBK, the present invention solved the protein structure of the apo-form ManBK by X-ray crystallography, and the ManBK structure was superimposed with Trichoderma reesei mannanase complex structure. According to the superimposed structure, Tyr216 which is located in the active site is selected for site-directed mutagenesis and the tyrosine at position 216 was mutated into tryptophan to construct the Y216W mutant. From the β-mannanase activity assay and the kinetic analysis, the Y216W mutant exhibited significantly increased specific activity when compared to the wild-type, so it can reduce the production cost and will has more industrial applications. In addition, since ManBK has thermostability and can be applied to many industries with thermal processes, once the enzymatic activity thereof is increased, the production cost will be reduced and the profit will be increased. Therefore, the present invention successfully modified ManBK to improve the enzymatic activity thereof, and thus, the present invention possesses high industrial value.
[0031] While the invention has been described in terms of what is presently considered to be the most practical and preferred embodiments, it is to be understood that the invention needs not be limited to the disclosed embodiment. On the contrary, it is intended to cover various modifications and similar arrangements included within the spirit and scope of the appended claims which are to be accorded with the broadest interpretation so as to encompass all such modifications and similar structures.
Sequence CWU
1
1
411038DNAAspergillus niger BK01 1tccttcgctt ccacttccgg attgcagttc
actattgacg gtgagactgg ttacttcgct 60ggaactaact cctactggat cggtttcttg
actgacaacg ctgacgttga cttggttatg 120ggtcacttga agtcctccgg tttgaagatc
ttgagagttt ggggtttcaa cgacgttact 180tcccaaccat cctccggtac tgtttggtat
caattgcacc aggacggaaa gtccactatc 240aacactggtg ctgacggatt gcagagattg
gactacgttg tttcctccgc tgagcagcac 300gacatcaagc ttatcatcaa cttcgttaac
tactggactg actacggtgg tatgtccgct 360tacgtttctg cttatggtgg ttctggtgag
actgacttct acacttccga cactatgcag 420tccgcttacc agacttacat caagactgtt
gttgagagat actccaactc ctccgctgtt 480ttcgcttggg aattggctaa cgagccaaga
tgtccttcct gtgacacttc cgtcttgtac 540aactggatcg aaaagacttc caagttcatc
aagggtttgg acgctgacag aatggtctgt 600attggtgacg agggtttcgg tttgaacatt
gactctgacg gttcctaccc ataccaattc 660tccgagggtt tgaacttcac tatgaacttg
gacatcgaca ctatcgactt cggtacattg 720cacttgtacc cagactcttg gggtacttct
gatgattggg gtaacggttg gatcactgct 780catggtgctg cttgtaaggc tgctggtaag
ccatgtttgt tggaagagta cggtgttact 840tccaaccact gttctgttga gggtgcttgg
caaaagactg ctttgtccac tactggtgtt 900ggtgctgact tgttctggca atacggtgac
gacttgtcca ctggtaagtc tccagatgac 960ggtaacacta tctactacgg tacttccgac
taccagtgtt tggttactga ccacgttgct 1020gctatcggtt ccgcttaa
10382345PRTAspergillus niger BK01 2Ser
Phe Ala Ser Thr Ser Gly Leu Gln Phe Thr Ile Asp Gly Glu Thr 1
5 10 15 Gly Tyr Phe Ala Gly Thr Asn
Ser Tyr Trp Ile Gly Phe Leu Thr Asp 20 25
30 Asn Ala Asp Val Asp Leu Val Met Gly His Leu Lys Ser
Ser Gly Leu 35 40 45 Lys Ile
Leu Arg Val Trp Gly Phe Asn Asp Val Thr Ser Gln Pro Ser 50
55 60 Ser Gly Thr Val Trp Tyr Gln Leu His Gln Asp
Gly Lys Ser Thr Ile65 70 75
80Asn Thr Gly Ala Asp Gly Leu Gln Arg Leu Asp Tyr Val Val Ser Ser
85 90 95Ala Glu Gln His Asp
Ile Lys Leu Ile Ile Asn Phe Val Asn Tyr Trp 100
105 110Thr Asp Tyr Gly Gly Met Ser Ala Tyr Val Ser Ala
Tyr Gly Gly Ser 115 120 125 Gly
Glu Thr Asp Phe Tyr Thr Ser Asp Thr Met Gln Ser Ala Tyr Gln 130
135 140Thr Tyr Ile Lys Thr Val Val Glu Arg Tyr
Ser Asn Ser Ser Ala Val145 150 155
160Phe Ala Trp Glu Leu Ala Asn Glu Pro Arg Cys Pro Ser Cys Asp
Thr 165 170 175Ser Val Leu
Tyr Asn Trp Ile Glu Lys Thr Ser Lys Phe Ile Lys Gly 180
185 190Leu Asp Ala Asp Arg Met Val Cys Ile Gly
Asp Glu Gly Phe Gly Leu 195 200
205 Asn Ile Asp Ser Asp Gly Ser Tyr Pro Tyr Gln Phe Ser Glu Gly Leu
210 215 220 Asn Phe Thr Met Asn Leu Asp
Ile Asp Thr Ile Asp Phe Gly Thr Leu225 230
235 240 His Leu Tyr Pro Asp Ser Trp Gly Thr Ser Asp
Asp Trp Gly Asn Gly 245 250
255 Trp Ile Thr Ala His Gly Ala Ala Cys Lys Ala Ala Gly Lys Pro Cys
260 265 270Leu Leu Glu Glu Tyr Gly
Val Thr Ser Asn His Cys Ser Val Glu Gly 275 280
285Ala Trp Gln Lys Thr Ala Leu Ser Thr Thr Gly Val Gly Ala
Asp Leu 290 295 300Phe Trp Gln Tyr Gly
Asp Asp Leu Ser Thr Gly Lys Ser Pro Asp Asp305 310
315 320Gly Asn Thr Ile Tyr Tyr Gly Thr Ser Asp
Tyr Gln Cys Leu Val Thr 325 330
335Asp His Val Ala Ala Ile Gly Ser Ala 340
345 31038DNAArtificial SequenceSynthetically generated DNA encoding a
modified enzyme 3tccttcgctt ccacttccgg attgcagttc actattgacg
gtgagactgg ttacttcgct 60ggaactaact cctactggat cggtttcttg actgacaacg
ctgacgttga cttggttatg 120ggtcacttga agtcctccgg tttgaagatc ttgagagttt
ggggtttcaa cgacgttact 180tcccaaccat cctccggtac tgtttggtat caattgcacc
aggacggaaa gtccactatc 240aacactggtg ctgacggatt gcagagattg gactacgttg
tttcctccgc tgagcagcac 300gacatcaagc ttatcatcaa cttcgttaac tactggactg
actacggtgg tatgtccgct 360tacgtttctg cttatggtgg ttctggtgag actgacttct
acacttccga cactatgcag 420tccgcttacc agacttacat caagactgtt gttgagagat
actccaactc ctccgctgtt 480ttcgcttggg aattggctaa cgagccaaga tgtccttcct
gtgacacttc cgtcttgtac 540aactggatcg aaaagacttc caagttcatc aagggtttgg
acgctgacag aatggtctgt 600attggtgacg agggtttcgg tttgaacatt gactctgacg
gttcctggcc ataccaattc 660tccgagggtt tgaacttcac tatgaacttg gacatcgaca
ctatcgactt cggtacattg 720cacttgtacc cagactcttg gggtacttct gatgattggg
gtaacggttg gatcactgct 780catggtgctg cttgtaaggc tgctggtaag ccatgtttgt
tggaagagta cggtgttact 840tccaaccact gttctgttga gggtgcttgg caaaagactg
ctttgtccac tactggtgtt 900ggtgctgact tgttctggca atacggtgac gacttgtcca
ctggtaagtc tccagatgac 960ggtaacacta tctactacgg tacttccgac taccagtgtt
tggttactga ccacgttgct 1020gctatcggtt ccgcttaa
10384345PRTArtificial SequenceSequence
synthetically translated from SEQ ID NO 3 4Ser Phe Ala Ser Thr Ser
Gly Leu Gln Phe Thr Ile Asp Gly Glu Thr 1 5
10 15 Gly Tyr Phe Ala Gly Thr Asn Ser Tyr Trp Ile Gly
Phe Leu Thr Asp 20 25 30
Asn Ala Asp Val Asp Leu Val Met Gly His Leu Lys Ser Ser Gly Leu
35 40 45 Lys Ile Leu Arg Val Trp Gly
Phe Asn Asp Val Thr Ser Gln Pro Ser 50 55
60 Ser Gly Thr Val Trp Tyr Gln Leu His Gln Asp Gly Lys Ser Thr
Ile65 70 75 80Asn Thr
Gly Ala Asp Gly Leu Gln Arg Leu Asp Tyr Val Val Ser Ser 85
90 95Ala Glu Gln His Asp Ile Lys Leu
Ile Ile Asn Phe Val Asn Tyr Trp 100 105
110Thr Asp Tyr Gly Gly Met Ser Ala Tyr Val Ser Ala Tyr Gly Gly
Ser 115 120 125 Gly Glu Thr Asp
Phe Tyr Thr Ser Asp Thr Met Gln Ser Ala Tyr Gln 130
135 140Thr Tyr Ile Lys Thr Val Val Glu Arg Tyr Ser Asn
Ser Ser Ala Val145 150 155
160Phe Ala Trp Glu Leu Ala Asn Glu Pro Arg Cys Pro Ser Cys Asp Thr
165 170 175Ser Val Leu Tyr Asn
Trp Ile Glu Lys Thr Ser Lys Phe Ile Lys Gly 180
185 190Leu Asp Ala Asp Arg Met Val Cys Ile Gly Asp Glu
Gly Phe Gly Leu 195 200 205 Asn
Ile Asp Ser Asp Gly Ser Trp Pro Tyr Gln Phe Ser Glu Gly Leu 210
215 220 Asn Phe Thr Met Asn Leu Asp Ile Asp Thr
Ile Asp Phe Gly Thr Leu225 230 235
240 His Leu Tyr Pro Asp Ser Trp Gly Thr Ser Asp Asp Trp Gly
Asn Gly 245 250 255 Trp
Ile Thr Ala His Gly Ala Ala Cys Lys Ala Ala Gly Lys Pro Cys
260 265 270Leu Leu Glu Glu Tyr Gly Val
Thr Ser Asn His Cys Ser Val Glu Gly 275 280
285Ala Trp Gln Lys Thr Ala Leu Ser Thr Thr Gly Val Gly Ala Asp
Leu 290 295 300Phe Trp Gln Tyr Gly Asp
Asp Leu Ser Thr Gly Lys Ser Pro Asp Asp305 310
315 320Gly Asn Thr Ile Tyr Tyr Gly Thr Ser Asp Tyr
Gln Cys Leu Val Thr 325 330
335Asp His Val Ala Ala Ile Gly Ser Ala 340
345
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