Method for Producing a Dairy Product

Abstract

ates to a method for producing a dairy product using an enzyme having lactase activity.

Description

SEQUENCE LISTING

[0001]The present invention comprises a sequence listing.

TECHNICAL FIELD

[0002]The present invention relates to a method for producing a dairy product using an enzyme having lactase activity.

BACKGROUND OF THE INVENTION

[0003]Lactose intolerance is perhaps the best-known food sensitivity in the United States and other parts of the world. It is estimated that about 70% of the world's population has a genetically controlled limited ability to digest lactose. Therefore, to help dairy maldigesters keep dairy foods in their diet, there is a growing demand for dairy food products that contain no or only low levels of lactose.

[0004]Lactase is used commercially to break down lactose in milk to produce dairy products which are suitable for people with lactose intolerance and/or have a sweeter taste. Because glucose and galactose are sweeter than lactose, lactase produces a more pleasant taste. Lactase is also used in the manufacture of ice cream. Lactose crystallises at the low temperatures of ice cream, whereas glucose and galactose stay liquid and contribute to a smoother texture. Lactase is also used in the conversion of whey into syrup. Lactase is also used for production of condensed milk.

[0005]Lactases have been isolated from a large variety of organisms, including microorganisms. Lactase is often an intracellular component of microorganisms like Kluyveromyces and Bacillus. Kluyveromyces, especially K. fragilis and K. lactis, and other fungi such as those of the genera Candida, Torula and Torulopsis, are a common source of fungal lactases, whereas B. coagulans and B circulans are well known sources for bacterial lactases. Several commercial lactase preparations derived from these organisms are available such as Lactozym® (available from Novozymes, Denmark), HA-Lactase (available from Chr. Hansen, Denmark) and Maxilact® (available from DSM, the Netherlands), all from K. lactis. All these lactases are so called neutral lactases having a pH optimum between pH 6 and pH 8. When such lactases are used in the production of, e.g., low-lactose yoghurt, the enzyme treatment will either have to be done in a separate step before fermentation or rather high enzyme dosages have to be used, because their activity drop as the pH decreases during fermentation. Also, these lactases are not suitable for hydrolysis of lactose in milk performed at high temperature, which would in some cases be beneficial to keep the microbial count low and thus ensure good milk quality.

[0006]Several extracellular lactases have been described having a lower pH optimum, see, e.g., U.S. Pat. No. 5,736,374 which describes an example of such lactase, produced by Aspergillus oryzae.

[0007]A lactase from Bifidobacterium bifidum has been described having a high transgalactosylating activity, both in the full-length form and especially when truncated from the C-terminal end (see, e.g., Jorgensen et al. (2001), Appl. Microbiol. Biotechnol., 57: 647-652 or EP patent 1,283,876).

[0008]It is an object of the present invention to provide a method for production of dairy products, e.g. fermented dairy products, such as yoghurt, having a low level of lactose by using a lactase. It is also an object to provide a method for production of low-lactose beverage milk having an extended shelf-life by using a lactase, where the method gives rise to low formation of off-flavour and/or low formation of brown colour as compared to known methods. Lactase to be used according to the invention should hydrolyse lactose efficiently and optimally allow for almost complete lactose hydrolysis. Especially, such lactase should have a high ratio of lactase to transgalactosylase activity. For use in the production of fermented dairy products, the lactase should be active over a broad pH range.

SUMMARY OF THE INVENTION

[0009]The present inventors have surprisingly found that a C-terminally truncated fragment of the extracellular lactase from Bifidobacterium bifidum, which was originally isolated and patented for its ability to make high amounts of galactooligosaccharides from lactose, can be used very successfully for hydrolysis of lactose in milk. When tested in water+100 g/l lactose at 37° C., the enzyme makes galactooligosaccharides with high efficiency as described in the prior art. However, when tested in milk, the ratio of hydrolytic to transgalactosylating activity has changed markedly, resulting in efficient hydrolysis and very low production of galactooligosaccharides.

[0010]Consequently, the present invention relates to a method for producing a dairy product comprising

[0011]a) providing a milk-based substrate comprising lactose; and

[0012]b) treating said substrate with an enzyme having lactase activity and having an amino acid sequence which is at least 70% identical to amino acids 28-1931 of SEQ ID NO: 1 or a fragment thereof.

[0013]In a preferred aspect, the invention relates to a method for producing a dairy product comprising

[0014]a) providing a milk-based substrate comprising lactose; and

[0015]b) treating said substrate with an enzyme having lactase activity and having an amino acid sequence which is at least 70% identical to amino acids 28-1331 of SEQ ID NO: 2.

[0016]Further, the inventors have surprisingly found that very low levels of lactose can be achieved when using the lactase from Bifidobacterium bifidum as compared to other lactases typically used for treatment of milk. Another unexpected advantage of using the lactase from Bifidobacterium bifidum is that the enzyme is active at high temperatures, allowing for treatment of the milk at, e.g., 52° C., thus reducing the microbial count and thereby improving the quality of the milk.

[0017]Therefore, in another aspect, the present invention relates to a method for producing a dairy product comprising

[0018]a) providing a milk-based substrate comprising lactose, and

[0019]b) treating said substrate with an enzyme having lactase activity,

[0020]where step b) takes place at a temperature of at least 50° C.

[0021]In a preferred embodiment, step b) takes place at a temperature of at least 52° C.

[0022]Also, the inventors have surprisingly found that the lactase from Bifidobacterium bifidum is active over a broad pH range.

[0023]Therefore, in another aspect, the present invention relates to a method for producing a dairy product comprising

[0024]a) providing a milk-based substrate comprising lactose, and

[0025]b) treating said substrate with an enzyme having lactase activity, where the pH optimum of the lactase activity at 37° C. is above pH 5, and where the lactase activity of the enzyme at pH 5 is at least 50% of its lactase activity at pH 6 when measured at 37° C.

[0026]Use of a lactase enzyme being active over a broad pH spectrum is especially useful for the production of fermented dairy products, where it allows for low enzyme dosage, since the enzyme is still active during and after fermentation. Also, very low levels of lactose in the fermented dairy product can be reached using such enzyme.

[0027]Therefore, in a preferred aspect, the present invention relates to a method for producing a low-lactose fermented dairy product comprising

[0028]a) providing a milk-based substrate comprising lactose,

[0029]b) treating said substrate with an enzyme having lactase activity, where the pH optimum of the lactase activity at 37° C. is above pH 5, and where the lactase activity of the enzyme at pH 5 is at least 50% of its lactase activity at pH 6 when measured at 37° C., and

[0030]c) fermenting said substrate with a microorganism.

[0031]The present inventors have also surprisingly found that in the manufacture of low-lactose beverage milk having an extended shelf life, the lactose hydrolysis can preferentially be carried out at high temperature, such as at a temperature of at least 60° C. Preferentially, such manufacture may comprise simultaneous low-pasteurization and lactase treatment.

[0032]Therefore, in a preferred aspect, the present invention relates to a method for producing a dairy product comprising

[0033]a) providing a milk-based substrate comprising lactose, and

[0034]b) treating said substrate with an enzyme having lactase activity,

[0035]wherein step b) is performed for between 10 minutes and 4 hours at a temperature of between 62° C. and 64° C.

[0036]In a more preferred aspect, step b) in such method is followed by cooling to below 10° C. without further heat treatment. This will allow for the enzyme to be still active after the milk has been cooled, i.e. during its storage. In another more preferred aspect, step b) in such method is followed by UHT treatment.

[0037]Preferably, in the methods of the invention, at least 70% of the lactose in the milk-based substrate is hydrolysed. More preferably, at least 80%, such as at least 85%, at least 90%, at least 95% or at least 98%, of the lactose in the milk-based substrate is hydrolysed.

[0038]In another aspect, the present invention relates to a method for producing a dairy product comprising

[0039]a) providing a milk-based substrate comprising lactose; and

[0040]b) treating said substrate with an enzyme having lactase activity and having an amino acid sequence which is at least 70% identical to SEQ ID NO: 3 or a fragment thereof.

[0041]In another aspect, the present invention relates to a method for producing a dairy product comprising

[0042]a) providing a milk-based substrate comprising lactose; and

[0043]b) treating said substrate with an enzyme having lactase activity and having an amino acid sequence which is at least 70% identical to SEQ ID NO: 4 or a fragment thereof.

[0044]In another aspect, the present invention relates to a method for producing a dairy product comprising

[0045]a) providing a milk-based substrate comprising lactose; and

[0046]b) treating said substrate with an enzyme having lactase activity and having an amino acid sequence which is at least 70% identical to SEQ ID NO: 5 or a fragment thereof.

[0047]In another aspect, the present invention relates to a method for producing a dairy product comprising

[0048]a) providing a milk-based substrate comprising lactose; and

[0049]b) treating said substrate with an enzyme having lactase activity and having an amino acid sequence which is at least 70% identical to SEQ ID NO: 6 or a fragment thereof.

[0050]In yet another aspect, the present invention relates to a method for producing a dairy product comprising

[0051]a) providing a milk-based substrate comprising lactose; and

[0052]b) treating said substrate with an enzyme having lactase activity and having an amino acid sequence which is at least 70% identical to SEQ ID NO: 7 or a fragment thereof.

DETAILED DISCLOSURE OF THE INVENTION

[0053]Milk-Based Substrate

[0054]The term "milk", in the context of the present invention, is to be understood as the lacteal secretion obtained by milking any mammal, such as cows, sheep, goats, buffaloes or camels.

[0055]"Milk-based substrate", in the context of the present invention, may be any raw and/or processed milk material. Useful milk-based substrates include, but are not limited to solutions/suspensions of any milk or milk like products comprising lactose, such as whole or low fat milk, skim milk, buttermilk, reconstituted milk powder, condensed milk, solutions of dried milk, UHT milk, whey, whey permeate, acid whey, or cream.

[0056]Preferably, the milk-based substrate is milk or an aqueous solution of skim milk powder.

[0057]The milk-based substrate may be more concentrated than raw milk.

[0058]In one embodiment, the milk-based substrate has a ratio of protein to lactose of at least 0.2, preferably at least 0.3, at least 0.4, at least 0.5, at least 0.6 or, most preferably, at least 0.7.

[0059]The milk-based substrate may be homogenized and pasteurized according to methods known in the art.

[0060]"Homogenizing" as used herein means intensive mixing to obtain a soluble suspension or emulsion. It may be performed so as to break up the milk fat into smaller sizes so that it no longer separates from the milk. This may be accomplished by forcing the milk at high pressure through small orifices.

[0061]"Pasteurizing" as used herein means reducing or eliminating the presence of live organisms, such as microorganisms, in the milk-based substrate. Preferably, pasteurization is attained by maintaining a specified temperature for a specified period of time. The specified temperature is usually attained by heating. The temperature and duration may be selected in order to kill or inactivate certain bacteria, such as harmful bacteria, and/or to inactivate enzymes in the milk. A rapid cooling step may follow.

[0062]Dairy Product

[0063]A "dairy product" in the context of the present invention may be any food product wherein one of the major constituents is milk-based. Preferable, the major constituent is milk-based. More preferably, the major constituent is a milk-based substrate which has been treated with an enzyme having lactase activity according to a method of the invention. In the context of the present invention "one of the major constituents" means a constituent having a dry matter which constitutes more than 20%, preferably more than 30% or more than 40% of the total dry matter of the dairy product, whereas "the major constituent" means a constituent having a dry matter which constitutes more than 50%, preferably more than 60% or more than 70% of the total dry matter of the dairy product.

[0064]A dairy product according to the invention may be, e.g., skim milk, low fat milk, whole milk, cream, UHT milk, milk having an extended shelf life, a fermented milk product, cheese, yoghurt, butter, dairy spread, butter milk, acidified milk drink, sour cream, whey based drink, ice cream, condensed milk, dulce de leche or a flavoured milk drink. A dairy product may be manufactured by any method known in the art.

[0065]A dairy product may additionally comprise non-milk components, e.g. vegetable components such as, e.g., vegetable oil, vegetable protein, and/or vegetable carbohydrates. Dairy products may also comprise further additives such as, e.g., enzymes, flavouring agents, microbial cultures such as probiotic cultures, salts, sweeteners, sugars, acids, fruit, fruit juices, or any other component known in the art as a component of, or additive to, a dairy product.

[0066]In one embodiment of the invention, one or more milk components and/or milk fractions account for at least 50% (weight/weight), such as at least 70%, e.g. at least 80%, preferably at least 90%, of the dairy product.

[0067]In one embodiment of the invention, one or more milk-based substrates having been treated with an enzyme having lactase activity according to a method of the invention account for at least 50% (weight/weight), such as at least 70%, e.g. at least 80%, preferably at least 90%, of the dairy product.

[0068]In one embodiment of the invention, the dairy product is a dairy product which is not enriched by addition of galactooligosaccharides.

[0069]In one embodiment of the invention, the enzyme-treated milk-based substrate is not dried before being used as an ingredient in the dairy product.

[0070]In one embodiment of the invention, the dairy product is ice cream. In the present context, ice cream may be any kind of ice cream such as full fat ice cream, low fat ice cream, or ice cream based on yoghurt or other fermented milk products. Ice cream may be manufactured by any method known in the art.

[0071]In one embodiment of the invention, the dairy product is milk or condensed milk.

[0072]In one preferred embodiment of the invention, the dairy product is UHT milk. UHT milk in the context of the present invention is milk which has been subjected to a sterilization procedure which is intended to kill all microorganisms, including the bacterial spores. UHT (ultra high temperature) treatment may be, e.g., heat treatment for 30 seconds at 130° C., or heat treatment for one second at 145° C.

[0073]In one preferred embodiment of the invention, the dairy product is ESL milk. ESL milk in the context of the present invention is milk which has an extended shelf life due to microfiltration and/or heat treatment and which is able to stay fresh for at least 15 days, preferably for at least 20 days, on the store shelf at 2-5° C.

[0074]In another preferred embodiment of the invention, the dairy product is a fermented dairy product, e.g., yoghurt.

[0075]Fermented Dairy Product

[0076]A "fermented dairy product" in the context of the present invention is to be understood as any dairy product wherein any type of fermentation forms part of the production process. Examples of fermented dairy products are products like yoghurt, buttermilk, creme fraiche, quark and fromage frais. A fermented dairy product may be produced by any method known in the art.

[0077]"Fermentation" in the method of the present invention means the conversion of carbohydrates into alcohols or acids through the action of a microorganism. Preferably, fermentation in the method of the present invention comprises conversion of lactose to lactic acid.

[0078]In the context of the present invention, "microorganism" may include any bacterium or fungus being able to ferment the milk substrate.

[0079]The microorganisms used for most fermented milk products are selected from the group of bacteria generally referred to as lactic acid bacteria. As used herein, the term "lactic acid bacterium" designates a gram-positive, microaerophilic or anaerobic bacterium, which ferments sugars with the production of acids including lactic acid as the predominantly produced acid, acetic acid and propionic acid. The industrially most useful lactic acid bacteria are found within the order "Lactobacillales" which includes Lactococcus spp., Streptococcus spp., Lactobacillus spp., Leuconostoc spp., Pseudoleuconostoc spp., Pediococcus spp., Brevibacterium spp., Enterococcus spp. and Propionibacterium spp. Additionally, lactic acid producing bacteria belonging to the group of anaerobic bacteria, bifidobacteria, i.e. Bifidobacterium spp., which are frequently used as food cultures alone or in combination with lactic acid bacteria, are generally included in the group of lactic acid bacteria.

[0080]Lactic acid bacteria are normally supplied to the dairy industry either as frozen or freeze-dried cultures for bulk starter propagation or as so-called "Direct Vat Set" (DVS) cultures, intended for direct inoculation into a fermentation vessel or vat for the production of a fermented dairy product. Such cultures are in general referred to as "starter cultures" or "starters".

[0081]Commonly used starter culture strains of lactic acid bacteria are generally divided into mesophilic organisms having optimum growth temperatures at about 30° C. and thermophilic organisms having optimum growth temperatures in the range of about 40 to about 45° C. Typical organisms belonging to the mesophilic group include Lactococcus lactis, Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides subsp. cremoris, Pseudoleuconostoc mesenteroides subsp. cremoris, Pediococcus pentosaceus, Lactococcus lactis subsp. lactis biovar. diacetylactis, Lactobacillus casei subsp. casei and Lactobacillus paracasei subsp. paracasei. Thermophilic lactic acid bacterial species include as examples Streptococcus thermophilus, Enterococcus faecium, Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus.

[0082]Also the anaerobic bacteria belonging to the genus Bifidobacterium including Bifidobacterium bifidum, Bifidobacterium animalis and Bifidobacterium longum are commonly used as dairy starter cultures and are generally included in the group of lactic acid bacteria. Additionally, species of Propionibacteria are used as dairy starter cultures, in particular in the manufacture of cheese. Additionally, organisms belonging to the Brevibacterium genus are commonly used as food starter cultures.

[0083]Another group of microbial starter cultures are fungal cultures, including yeast cultures and cultures of filamentous fungi, which are particularly used in the manufacture of certain types of cheese and beverage. Examples of fungi include Penicillium roqueforti, Penicillium candidum, Geotrichum candidum, Torula kefir, Saccharomyces kefir and Saccharomyces cerevisiae.

[0084]In one embodiment of the present invention, the microorganism used for fermentation of the milk-based substrate is Lactobacillus casei or a mixture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.

[0085]Fermentation processes to be used in a method of the present invention are well known and the person of skill in the art will know how to select suitable process conditions, such as temperature, oxygen, amount and characteristics of microorganism/s, additives such as e.g. carbohydrates, flavours, minerals, enzymes, and process time. Obviously, fermentation conditions are selected so as to support the achievement of the present invention.

[0086]As a result of fermentation, pH of the milk-based substrate will be lowered. The pH of a fermented dairy product of the invention may be, e.g., in the range 3.5-6, such as in the range 3.5-5, preferably in the range 3.8-4.8.

[0087]In a preferred embodiment, the fermented dairy product is yoghurt.

[0088]Method for Producing a Dairy Product

[0089]As mentioned above, the present invention in one aspect relates to a method for producing a dairy product comprising:

[0090]a) providing a milk-based substrate comprising lactose; and

[0091]b) treating said substrate with an enzyme having lactase activity and having an amino acid sequence which is at least 70% identical to amino acids 28-1931 of SEQ ID NO: 1 or a fragment thereof.

[0092]In a preferred aspect, the invention relates to a method for producing a dairy product comprising

[0093]a) providing a milk-based substrate comprising lactose; and

[0094]b) treating said substrate with an enzyme having lactase activity and having an amino acid sequence which is at least 70% identical to amino acids 28-1331 of SEQ ID NO: 2.

[0095]In another aspect, the invention relates to a method for producing a dairy product comprising

[0096]a) providing a milk-based substrate comprising lactose; and

[0097]b) treating said substrate with an enzyme having lactase activity and having an amino acid sequence which is at least 70% identical to SEQ ID NO: 3 or a fragment thereof.

[0098]In another aspect, the invention relates to a method for producing a dairy product comprising

[0099]a) providing a milk-based substrate comprising lactose; and

[0100]b) treating said substrate with an enzyme having lactase activity and having an amino acid sequence which is at least 70% identical to SEQ ID NO: 4 or a fragment thereof.

[0101]In another aspect, the invention relates to a method for producing a dairy product comprising

[0102]a) providing a milk-based substrate comprising lactose; and

[0103]b) treating said substrate with an enzyme having lactase activity and having an amino acid sequence which is at least 70% identical to SEQ ID NO: 5 or a fragment thereof.

[0104]In another aspect, the invention relates to a method for producing a dairy product comprising

[0105]a) providing a milk-based substrate comprising lactose; and

[0106]b) treating said substrate with an enzyme having lactase activity and having an amino acid sequence which is at least 70% identical to SEQ ID NO: 6 or a fragment thereof.

[0107]In another aspect, the invention relates to a method for producing a dairy product comprising

[0108]a) providing a milk-based substrate comprising lactose; and

[0109]b) treating said substrate with an enzyme having lactase activity and having an amino acid sequence which is at least 70% identical to SEQ ID NO: 7 or a fragment thereof.

[0110]In another aspect, the invention relates to a method for producing a dairy product comprising

[0111]a) providing a milk-based substrate comprising lactose, and

[0112]b) treating said substrate with an enzyme having lactase activity,

[0113]wherein step b) takes place at a temperature of at least 50° C.

[0114]In yet another aspect, the invention relates to a method for producing a dairy product comprising

[0115]a) providing a milk-based substrate comprising lactose,

[0116]b) treating said substrate with an enzyme having lactase activity, where the pH optimum of the lactase activity at 37° C. is above pH 5, and where the lactase activity of the enzyme at pH 5 is at least 50% of its lactase activity at pH 6 when measured at 37° C.

[0117]The skilled person will know how to determine the lactase activity at different pH and thereby determine the pH optimum for the enzyme. The lactase activity at different pH may be determined by measuring hydrolysis of lactose at 37° C. for 30 minutes, preferably in a buffer comprising succinate, HEPES, CHES, KCl, CaCl₂ and MgCl₂, e.g., by using a method as described in the Examples of the present application. For the avoidance of doubt, HEPES is a buffering agent, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and CHES is a buffering agent, N-Cyclohexyl-2-aminoethanesulfonic acid.

[0118]The enzyme-treated milk-based substrate may optionally be mixed with other ingredients to obtain the dairy product. In one embodiment of the invention, the enzyme-treated milk-based substrate is mixed with other ingredients to obtain the dairy product.

[0119]In one embodiment of the invention, the dairy product is milk. In another embodiment, the dairy product is condensed milk. In another embodiment, the dairy product is ice cream. In another embodiment, the dairy product is UHT milk. In another embodiment, the dairy product is ESL milk. In yet another embodiment, the dairy product is a fermented dairy product, e.g., yoghurt.

[0120]Preferably, the dairy product is a low-lactose dairy product. "Low-lactose", in the context of the present invention, means that the amount of lactose in the dairy product, such as in the fermented dairy product, has been reduced by at least 70%, preferably 80%, 90%, 95%, 98%, 99% or 99,5%.

[0121]Method for Producing a Low-Lactose Fermented Dairy Product

[0122]One embodiment of the present invention relates to a method for producing a low-lactose fermented dairy product comprising

[0123]a) providing a milk-based substrate comprising lactose,

[0124]b) treating said substrate with an enzyme having lactase activity, where the lactase activity of the enzyme at pH 5 is at least 50% of its lactase activity at pH 6 when measured at 37° C., and

[0125]c) fermenting said substrate with a microorganism.

[0126]Another embodiment of the invention relates to a method for producing a low-lactose fermented dairy product comprising

[0127]a) providing a milk-based substrate comprising lactose,

[0128]b) treating said substrate with an enzyme having lactase activity and having an amino acid sequence which is at least 70% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or a fragment of any of these, and

[0129]c) fermenting said substrate with a microorganism.

[0130]Preferably, in these embodiments, step b) and step c) are performed essentially at the same time.

[0131]In the context of the method of the invention, "essentially at the same time" means that enzyme treatment and fermentation are not performed as separate steps, i.e., incubation of the milk-based substrate with the enzyme is not performed as a separate step before inoculation with the microorganism. Instead, the enzyme and the microorganism may be added to the milk-based substrate at essentially the same time. I.e., the enzyme may be added to the milk-based substrate immediately before inoculation with the microorganism. "Immediately before" in this context means without a separate incubation step for the enzymatic hydrolysis. Alternatively, the microorganism may be added immediately before the enzyme, or the microorganism and the enzyme may be added at the same time. "Essentially at the same time" in the context of the method of the invention may mean that the enzyme is active throughout the whole of step c).

[0132]"Essentially at the same time" does not mean that the enzymatic hydrolysis of the lactose in the milk-based substrate is completed when the fermentation is completed, i.e. when the pH has dropped to a level preventing further fermenting activity of the microbial starter culture.

[0133]In a preferred embodiment of the invention, the enzyme is still active after completion of step

[0134]c). In a more preferred embodiment, the enzyme has retained at least 20%, such as at least 30%, at least 40%, at least 50%, at least 60% or at least 70%, of its lactase activity after completion of step c) as compared to its activity when added to the milk-based substrate.

[0135]After completion of step c) may mean when at least one of the below is true: [0136]temperature is lowered to below 30° C. [0137]pH has reached 4.55 [0138]pH does no longer decrease by more than 0.2 units per hour.

[0139]In another preferred embodiment, less than 80%, such as less than 70%, less than 60%, less than 50%, less than 40%, less than 30% or less than 20%, of the lactose has been hydrolyzed after two hours of fermentation. "After two hours of fermentation" in the context of the invention means that the milk-based substrate, after having been inoculated with the microorganism, has been incubated for two hours at a temperature which is appropriate for the fermentation process.

[0140]In another preferred embodiment, less than 80%, such as less than 70%, less than 60%, less than 50%, less than 40%, less than 30% or less than 20%, of the lactose has been hydrolyzed when pH of the milk-based substrate has dropped to pH 5.

[0141]In yet another preferred embodiment, less than 80%, such as less than 70%, less than 60%, less than 50%, less than 40%, less than 30% or less than 20%, of the lactose has been hydrolyzed when step c) is completed.

[0142]In a preferred embodiment, more than 70%, such as more than 80%, more than 90%, more than 95%, more than 97%, more than 98% or more than 99%, of the lactose in the milk-based substrate has been hydrolyzed two days, i.e. 48 hours, after start of the fermentation. "Start of the fermentation" is when the milk-based substrate has been inoculated with the microorganism and incubated at a temperature which is appropriate for the fermentation process.

[0143]In another preferred embodiment, more than 70%, such as more than 80%, more than 90%, more than 95%, more than 97%, more than 98% or more than 99%, of the lactose in the milk-based substrate has been hydrolyzed two days, i.e. 48 hours, after completion of step c).

[0144]In another preferred embodiment, more than 70%, such as more than 80%, more than 90%, more than 95%, more than 97%, more than 98% or more than 99%, of the lactose in the milk-based substrate has been hydrolyzed in the final fermented dairy product. The "final fermented dairy product" is the fermented dairy product as sold to the consumer of the product.

[0145]In the embodiments of the invention where the milk-based substrate is being fermented, the enzyme treatment is preferably conducted at the natural pH of the milk-based substrate during the fermentation process, i.e. the pH will gradually decrease from the natural pH of the unfermented milk-based substrate to the pH of the fermented milk-based substrate. In such aspect, the enzyme treatment is preferably conducted at an appropriate temperature for the fermentation process.

[0146]Method for Producing a Low-Lactose beverage Milk Product Having an Extended Shelf Life

[0147]One embodiment of the present invention relates to a method for producing a low-lactose milk product comprising

[0148]a) providing a milk-based substrate comprising lactose, and

[0149]b) treating said substrate with an enzyme having lactase activity,

[0150]wherein step b) takes place at a temperature of at least 60° C.

[0151]In a preferred embodiment, step b) takes place at a temperature of at least 62° C., such as at least 63° C., more preferred at a temperature of at least 64° C., such as at least 65° C., at least 67° C. or at least 70° C., and most preferred at a temperature of at least 75° C.

[0152]The low-lactose milk product may be beverage milk having a longer shelf life than fresh milk which normally has a shelf life of 4-7 days. It may have an improved quality as compared to other low-lactose beverage milk products having a longer shelf life. It may, e.g., have a lower microbial count, a less bitter taste and/or a less brown colour.

[0153]Preferably, the milk product is ESL milk. More preferably, the milk product is UHT milk.

[0154]In a preferred aspect, the milk-based substrate is raw milk. In another preferred aspect, the milk-based substrate, preferably raw milk, has not been pasteurized before step b).

[0155]In a preferred aspect, no pasteurization of the enzyme treated milk-based substrate is performed after step b).

[0156]In a preferred aspect, microfiltration of the milk-based substrate is performed before step b). In that case, the enzyme should preferentially be sterile. In another preferred aspect, microfiltration of the enzyme treated milk-based substrate is performed after step b).

[0157]In a preferred aspect, step b) is performed for between 10 minutes and 4 hours at a temperature of between 62° C. and 64° C. In a more preferred aspect, step b) is performed for between 20 minutes and 2 hours at a temperature of between 62° C. and 64° C. In an even more preferred aspect, step b) is performed for between 20 and 60 minutes, such as for about 30 minutes, at a temperature of about 63° C. Such simultaneous low pasteurization and lactase treatment of, e.g., raw milk will give rise to low-lactose beverage milk having a higher quality as compared to low-lactose beverage milk where lactase treatment has been performed at low temperature, e.g., at 5° C. for up to 24 hours as is often used in the dairy industry.

[0158]In a more preferred aspect, step b) is followed by cooling to below 10° C. without further heat treatment. This will allow for the enzyme to be still active after the milk has been cooled, i.e. during its storage. Preferably, less than 80% of the lactose has been hydrolyzed when step b) is completed, and more than 90% of the lactose has been hydrolyzed after one week. More preferably, less than 60% of the lactose has been hydrolyzed when step b) is completed, and more than 95% of the lactose has been hydrolyzed after one week

[0159]In another more preferred aspect, step b) is followed by UHT treatment.

[0160]Enzyme Having Lactase Activity

[0161]In step b) in the methods of the present invention, the milk-based substrate is treated with an enzyme having lactase activity.

[0162]A lactase in the context of the present invention is any glycoside hydrolase having the ability to hydrolyse the disaccharide lactose into constituent galactose and glucose monomers. The group of lactases comprises but is not limited to enzymes assigned to subclass EC 3.2.1.108. Enzymes assigned to other subclasses, such as, e.g., EC 3.2.1.23, may also be lactases in the context of the present invention. A lactase in the context of the invention may have other activities than the lactose hydrolysing activity, such as for example a transgalactosylating activity. In the context of the invention, the lactose hydrolysing activity of the lactase may be referred to as its lactase activity or its beta-galactosidase activity.

[0163]Enzymes having lactase activity to be used in a method of the present invention may be of animal, of plant or of microbial origin. Preferred enzymes are obtained from microbial sources, in particular from a filamentous fungus or yeast, or from a bacterium.

[0164]The enzyme may, e.g., be derived from a strain of Agaricus, e.g. A. bisporus; Ascovaginospora; Aspergillus, e.g. A. niger, A. awamori, A. foetidus, A. japonicus, A. oryzae; Candida; Chaetomium; Chaetotomastia; Dictyostelium, e.g. D. discoideum; Kluveromyces, e.g. K. fragilis, K. lactis; Mucor, e.g. M. javanicus, M. mucedo, M. subtilissimus; Neurospora, e.g. N. crassa; Rhizomucor, e.g. R. pusillus; Rhizopus, e.g. R. arrhizus, R. japonicus, R. stolonifer; Sclerotinia, e.g. S. libertiana; Torula; Torulopsis; Trichophyton, e.g. T. rubrum; Whetzelinia, e.g. W. sclerotiorum; Bacillus, e.g. B. coagulans, B. circulans, B. megaterium, B. novalis, B. subtilis, B. pumilus, B. stearothermophilus, B. thuringiensis; Bifidobacterium, e.g. B. longum, B. bifidum, B. animalis; Chryseobacterium; Citrobacter, e.g. C. freundii; Clostridium, e.g. C. perfringens; Diplodia, e.g. D. gossypina; Enterobacter, e.g. E. aerogenes, E. cloacae Edwardsiella, E. tarda; Erwinia, e.g. E. herbicola; Escherichia, e.g. E. coli; Klebsiella, e.g. K. pneumoniae; Miriococcum; Myrothesium; Mucor; Neurospora, e.g. N. crassa; Proteus, e.g. P. vulgaris; Providencia, e.g. P. stuartii; Pycnoporus, e.g. Pycnoporus cinnabarinus, Pycnoporus sanguineus; Ruminococcus, e.g. R. torques; Salmonella, e.g. S. typhimurium; Serratia, e.g. S. liquefasciens, S. marcescens; Shigella, e.g. S. flexneri; Streptomyces, e.g. S. antibioticus, S. castaneoglobisporus, S. violeceoruber; Trametes; Trichoderma, e.g. T. reesei, T. viride; Yersinia, e.g. Y. enterocolitica.

[0165]In a preferred embodiment, the enzyme is a lactase from a bacterium, e.g. from the family Bifidobacteriaceae, such as from the genus Bifidobacterium, such as from a strain of B. bifidum, B. animalis or B. longum. In a more preferred embodiment, the enzyme is a lactase from Bifidobacterium bifidum. A preferred enzyme is a lactase having a sequence which is at least 50%, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to amino acids 28-1931 of SEQ ID NO: 1 or to a lactase active fragment thereof. Such lactase active fragment of SEQ ID NO: 1 may be any fragment of SEQ ID NO: 1 having lactase activity. A lactase active fragment of SEQ ID NO: 1 may be, e.g., amino acids 28-979, amino acids 28-1170, amino acids 28-1323, amino acids 28-1331, or amino acids 28-1600 of SEQ ID NO: 1.

[0166]In a preferred embodiment, an enzyme having lactase activity to be used in a method of the present invention comprises an amino acid sequence which is at least 50% identical to amino acids 28-1331 of SEQ ID NO: 2. In a more preferred embodiment, the enzyme comprises an amino acid sequence which is at least 60%, such as at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to amino acids 28-1331 of SEQ ID NO: 2.

[0167]In another preferred embodiment, an enzyme having lactase activity to be used in a method of the present invention has an amino acid sequence which is at least 50% identical to amino acids 28-1331 of SEQ ID NO: 2. In a more preferred embodiment, the enzyme has an amino acid sequence which is at least 60%, such as at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to amino acids 28-1331 of SEQ ID NO: 2.

[0168]In another embodiment, an enzyme having lactase activity to be used in a method of the present invention has an amino acid sequence which is at least 50% identical to SEQ ID NO: 3. Preferably, the enzyme has an amino acid sequence which is at least 60%, such as at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to SEQ ID NO: 3.

[0169]In another embodiment, an enzyme having lactase activity to be used in a method of the present invention has an amino acid sequence which is at least 50% identical to SEQ ID NO: 4. Preferably, the enzyme has an amino acid sequence which is at least 60%, such as at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to SEQ ID NO: 4.

[0170]In another embodiment, an enzyme having lactase activity to be used in a method of the present invention has an amino acid sequence which is at least 50% identical to SEQ ID NO: 5. Preferably, the enzyme has an amino acid sequence which is at least 60%, such as at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to SEQ ID NO: 5.

[0171]In another embodiment, an enzyme having lactase activity to be used in a method of the present invention has an amino acid sequence which is at least 50% identical to SEQ ID NO: 6. Preferably, the enzyme has an amino acid sequence which is at least 60%, such as at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to SEQ ID NO: 6.

[0172]In another embodiment, an enzyme having lactase activity to be used in a method of the present invention has an amino acid sequence which is at least 50% identical to SEQ ID NO: 7. Preferably, the enzyme has an amino acid sequence which is at least 60%, such as at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identical to SEQ ID NO: 7.

[0173]For purposes of the present invention, the degree of identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al. (2000) Trends in Genetics 16: 276-277), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labelled "longest identity" (obtained using the-no brief option) is used as the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment-Total Number of Gaps in Alignment)

[0174]Lactases to be used in a method of the present invention may be extracellular. They may have a signal sequence at their N-terminus, which is cleaved off during secretion.

[0175]Lactases to be used in a method of the present invention may be derived from any of the sources mentioned herein. The term "derived" means in this context that the enzyme may have been isolated from an organism where it is present natively, i.e. the identity of the amino acid sequence of the enzyme are identical to a native enzyme. The term "derived" also means that the enzymes may have been produced recombinantly in a host organism, the recombinantly produced enzyme having either an identity identical to a native enzyme or having a modified amino acid sequence, e.g. having one or more amino acids which are deleted, inserted and/or substituted, i.e. a recombinantly produced enzyme which is a mutant and/or a fragment of a native amino acid sequence. Within the meaning of a native enzyme are included natural variants. Furthermore, the term "derived" includes enzymes produced synthetically by, e.g., peptide synthesis. The term "derived" also encompasses enzymes which have been modified e.g. by glycosylation, phosphorylation etc., whether in vivo or in vitro. With respect to recombinantly produced enzyme the term "derived from" refers to the identity of the enzyme and not the identity of the host organism in which it is produced recombinantly.

[0176]The lactase may be obtained from a microorganism by use of any suitable technique. For instance, a lactase enzyme preparation may be obtained by fermentation of a suitable microorganism and subsequent isolation of a lactase preparation from the resulting fermented broth or microorganism by methods known in the art. The lactase may also be obtained by use of recombinant DNA techniques. Such method normally comprises cultivation of a host cell transformed with a recombinant DNA vector comprising a DNA sequence encoding the lactase in question and the DNA sequence being operationally linked with an appropriate expression signal such that it is capable of expressing the lactase in a culture medium under conditions permitting the expression of the enzyme and recovering the enzyme from the culture. The DNA sequence may also be incorporated into the genome of the host cell. The DNA sequence may be of genomic, cDNA or synthetic origin or any combinations of these, and may be isolated or synthesized in accordance with methods known in the art.

[0177]Lactases to be used in a method of the present invention may be purified. The term "purified" as used herein covers lactase enzyme protein essentially free from insoluble components from the production organism. The term "purified" also covers lactase enzyme protein essentially free from insoluble components from the native organism from which it is obtained. Preferably, it is also separated from some of the soluble components of the organism and culture medium from which it is derived. More preferably, it is separated by one or more of the unit operations: filtration, precipitation, or chromatography.

[0178]Accordingly, the enzyme having lactase activity may be purified, viz. only minor amounts of other proteins being present. The expression "other proteins" relate in particular to other enzymes. The term "purified" as used herein also refers to removal of other components, particularly other proteins and most particularly other enzymes present in the cell of origin of the lactase. The lactase may be "substantially pure", i.e. free from other components from the organism in which it is produced, i.e., e.g., a host organism for recombinantly produced lactase. Preferably, the lactase is an at least 40% (w/w) pure enzyme protein preparation, more preferably at least 50%, 60%, 70%, 80% or even at least 90% pure.

[0179]The term enzyme having lactase activity includes whatever auxiliary compounds that may be necessary for the enzyme's catalytic activity, such as, e.g., an appropriate acceptor or cofactor, which may or may not be naturally present in the reaction system.

[0180]The enzyme may be in any form suited for the use in question, such as, e.g., in the form of a dry powder or granulate, a non-dusting granulate, a liquid, a stabilized liquid, or a protected enzyme.

[0181]The enzyme is added in a suitable amount to achieve the desired degree of lactose hydrolysis under the chosen reaction conditions. The enzyme may be added at a concentration of between 100 and 5000 LAU per litre milk-based substrate, preferably less than 3000, such as less than 1500, less than 1000, less than 750 or less than 500, LAU per litre milk-based substrate.

[0182]In a preferred embodiment, the enzyme is added at a concentration of between 5 and 100 LAU per g lactose in the milk-based substrate, preferably less than 50, such as less than 40, less than 30, less than 20 or less than 10, LAU per g lactose in the milk-based substrate.

[0183]In the context of the present application, 1 lactase unit (1 LAU) is the amount of enzyme which releases 1 micromole glucose per minute in M-buffer at pH 6.5 and 37° C. with a lactose concentration of 4.75% w/v. M-buffer is prepared by dissolving 3.98 g C₆H₅Na₃O₇-2H₂O, 8.31 g citric acid, 0.9 g K₂SO₄, 2.6 g K₂HPO₄, 7.35 g KH₂PO₄, 5.45 g KOH, 4.15 g MgCl₂-6H₂O, 3.75 g CaCl₂-2H₂O and 1.4 g NaHCO₃ in 4 litre water, adding 12.5 ml 4N NaOH, adjusting to pH 6.5 using HCl, and adding water up to a total volume of 5 litre.

[0184]The activity in LAU of a specific lactase may be determined by direct measurement of glucose released from lactose under the conditions described above. The skilled person will know how to determine such activity. Alternatively, the activity may be determined by using the lactase activity assay described in Example 1 of the present application. Here, the activity is obtained by comparing to a standard curve run with a lactase of known activity, and the activity of the unknown sample calculated from this. The lactase of known activity may, e.g., be Lactozym obtained from Novozymes A/S, Denmark.

[0185]In a preferred embodiment, the enzyme having lactase activity to be used in a method of the present invention has a lactase activity at 37° C. and pH 5 which is at least 55%, such as at least 60%, at least 65%, at least 70% or at least 75%, of its lactase activity at 37° C. and pH 6.

[0186]In another preferred embodiment, the enzyme having lactase activity to be used in a method of the present invention has a lactase activity at 37° C. and pH 4.5 which is at least 10%, such as at least 20%, at least 30%, at least 35% or at least 40%, of its lactase activity at 37° C. and pH 6.

[0187]In another preferred embodiment, the enzyme having lactase activity to be used in a method of the present invention has a pH optimum of the lactase activity at 37° C. which is above pH 5.5.

[0188]In another preferred embodiment, the enzyme having lactase activity to be used in a method of the present invention has a lactase activity at a temperature of 52° C. and a pH of 6.5 which is at least 50%, such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80%, of its lactase activity at a temperature of 38° C. and a pH of 6.5.

[0189]The skilled person will know how to determine the lactase activity at different pH and temperature. The lactase activity at different pH and temperature is preferably determined by using a method as described in the Examples of the present application.

[0190]In a preferred embodiment of the present invention, Km of the enzyme having lactase activity at 5° C. is below 25 mM, such as below 20 mM, below 15 mM or below 10 mM. In another preferred embodiment, Km of the enzyme having lactase activity at 37° C. is below 25 mM, such as below 20 mM or below 15 mM. The skilled person will know how to determine Km for the lactase activity at a specific temperature. Km may be determined by the method used in the Examples of the present application.

[0191]In another preferred embodiment, the enzyme when hydrolysing the lactose in the milk-based substrate has a ratio of lactase to transgalactosylase activity of more than 1:1, such as more than 2:1 or more than 3:1. In another preferred embodiment, the enzyme treatment is performed under conditions where the lactase activity of the enzyme is higher than the transgalactosylase activity, such as at least two times higher or at least three times higher. The ratio of lactase to transgalactosylase activity in the milk-based substrate may, e.g., be determined by HPLC analysis. In another preferred embodiment, the enzyme treatment is performed under conditions where at least 50% (w/w %) of the hydrolyzed lactose is converted into free galactose. In another preferred embodiment, the enzyme treatment is performed under conditions where the hydrolyzed lactose is converted into equal amounts of free glucose and free galactose.

Example 1

[0192]Lactase Activity-Assay in Eppendorf Tubes at 37° C., pH 6.5

[0193]Principle:

[0194]Lactase hydrolyses lactose into glucose and galactose. Glucose is measured after a modified version of the common glucose oxidase/peroxidase assay (Werner, W. et al. (1970) Z. analyt. Chem. 252: 224.).

[0195]LAU is defined as the amount of enzyme liberating 1 micromole of glucose per min at 37° C., pH 6.5 in M-buffer (M-buffer is defined in the description of the present patent application). Alternatively, the activity in LAU for a specific lactase may be determined by the method described here. The value obtained is compared to a standard curve run with a lactase of known activity, and the activity of the unknown sample calculated from this. The lactase of known activity may, e.g., be Lactozym obtained from Novozymes A/S, Denmark.

[0196]Solutions:

[0197]Assay buffer: 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 0.01% Triton X100, pH 6.5

[0198]GOD-Perid solution: 65 mM sodium phosphate, pH 7, 0.4 g/l Glucose oxidase, 0.013 g/l HRP (Horse Radish Peroxidase), 0.65 g/l ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)).

[0199]Substrate:

[0200]160 mM lactose, 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mM CaCl₂, 1 mom MgCl₂, pH 6.5.

[0201]Standard:

[0202]Lactozym (available from Novozymes A/S, Denmark) with a known activity in LAU/g is used as standard, diluted in assay buffer in the range from 0.09-0.7 LAU/g.

[0203]Samples:

[0204]Enzyme samples are diluted appropriately in assay buffer.

[0205]Procedure:

[0206]1. 375 ul substrate is incubated 5 minutes at 37° C.

[0207]2. 25 ul enzyme diluted in assay buffer is added.

[0208]3. The reaction is stopped after 30 minutes by adding 60 ul 1 M NaOH

[0209]4. 20 ul is transferred to a 96 well microtiter plate and 200 ul GOD-Perid solution is added.

[0210]After 30 minutes at room temperature, the absorbance is measured at 420 nm.

Example 2

[0211]100 ml 9% skimmed milk solution having approximately 5% lactose was made by mixing 9 g skimmed milk powder (Kerry) in 91 ml ionic water. 10 ml of the solution was transferred to a test tube containing a magnetic stirring bar and placed in a water bath at 37° C. After 15 min enzyme was added. Enzymes tested were Lactozym, a commercially available lactase from Novozymes A/S, Denmark, having an activity of 3060 LAU/g, and an experimental lactase from Bifidobacterium bifidum having the encoded sequence shown in SEQ ID NO: 2 and an activity of 295 LAU/g. Amino acids 1 to 27 of SEQ ID NO: 2 is a signal sequence which is presumably cleaved off and amino acids 1332 to 1341 is a tag used for purification of the experimental enzyme.

[0212]Dosages were 5640 LAU/I milk of Lactozym and 2700 LAU/l milk of the Bifidobacterium lactase. Milk samples were taken at regular intervals up till 4 hrs. and the enzyme inactivated by heating to 99° C. for 10 min in a thermomixer. Samples were diluted appropriately and filtered through a 0.20 um filter.

[0213]Lactose hydrolysis was measured using a Dionex BioLC equipped with a Dionex PA1 column and a Pulsed Amperiometrisk Detektor (PAD). Peaks were identified and quantified by comparing with known standards of lactose, glucose and galactose. Results are given below.

TABLE-US-00001 TABLE 1 Lactose, glucose and galactose in reconstituted skimmed milk after treatment with Lactozym or Bifidobacterium lactase. Lactozym Bifidobacterium lactase Time Lactose Glucose Galactose Lactose Glucose Galactose min mM mM mM mM mM mM 5 152 6 5 156 3 3 30 64 92 76 91 71 70 60 35 118 99 45 117 114 120 19 131 111 8 144 142 180 15 141 119 1 155 153 240 14 150 128 1 162 160

[0214]When tested in milk with 5% lactose, no transferase activity is observed when using the Bifidobacterium lactase. Glucose and galactose production are equal and total monosaccharide production match that expected from the lactose hydrolyzed. For comparison, Lactozym produces less galactose than glucose clearly showing that galactooligosaccharides have been produced. Also, final lactose levels are significantly lower when using the Bifidobacterium lactase illustrating the lower Km value of this enzyme.

Example 3

[0215]100 ml 15 or 30% (w/w) whey permeate containing primarily lactose and ions was made by mixing 15 or 30 g spray-dried whey permeate powder (Variolac, Arla) in 85 or 70 ml ionic water respectively. The solution was poured in a flask containing a magnetic stirring bar and placed in a water bath at 37° C. After 15 min, enzyme was added. Enzymes tested were Lactozym, a commercially available lactase from Novozymes A/S, Denmark, having an activity of 3060 LAU/g, and an experimental lactase from Bifidobacterium bifidum having the encoded sequence shown in SEQ ID NO: 2 and an activity of 295 LAU/g.

[0216]Dosages were 4225 LAU/l milk of Lactozym and 2025 LAU/l milk of the Bifidobacterium lactase. Milk samples were taken at regular intervals up till 5.5 hrs. and the enzyme inactivated by heating to 99° C. for 10 min in a thermomixer. Samples were diluted appropriately and filtered through a 0.20 um filter.

[0217]Lactose hydrolysis was measured using a Dionex BioLC equipped with a Dionex PA1 column and a Pulsed Amperiometrisk Detektor (PAD). Peaks were identified and quantified by comparing with known standards of lactose, glucose and galactose.

[0218]Results are given below.

TABLE-US-00002 TABLE 2 Lactose, glucose and galactose in 15% DS whey permeate after treatment with Lactozym or Bifidobacterium lactase. Lactozym Bifidobacterium lactase Time Lactose Glucose Galactose Lactose Glucose Galactose min mM mM mM mM mM mM 0 499 1 2 499 1 2 30 312 135 106 410 61 63 60 211 224 155 349 119 122 120 110 295 221 220 199 202 180 66 324 249 149 281 290 240 50 346 279 84 336 348 330 37 372 312 31 350 368

TABLE-US-00003 TABLE 3 Lactose, glucose and galactose in 30% DS whey permeate after treatment with Lactozym or Bifidobacterium lactase. Lactozym Bifidobacterium lactase Time Lactose Glucose Galactose Lactose Glucose Galactose min mM mM mM mM mM mM 0 848 1 4 848 1 4 30 824 109 75 819 43 45 60 615 253 150 788 86 83 120 420 370 242 651 159 158 180 291 459 300 625 232 230 240 246 559 373 501 283 273 330 154 544 367 391 333 324 1440 54 649 545 20 727 739

[0219]Also when tested at higher lactose concentrations as in 15% or 30% whey permeate no or very little galactooligosaccharides are produced. Again, the produced galactose and glucose levels are equal and match the amount of lactose hydrolyzed. For comparison, Lactozym produces less galactose than glucose, clearly showing that galactooligosaccharides have been produced.

Example 4

[0220]pH Profile (at 37° C.) and Temperature Profile (at pH 6.5) of Experimental Lactase from Bifidobacterium bifidum using Lactose as Substrate.

[0221]Principle:

[0222]Lactase hydrolyses lactose and glucose+galactose is formed. Glucose is measured after a modified version of the common glucose oxidase/peroxidase assay (Werner, W. et al. (1970) Z. analyt. Chem. 252: 224.) pH profile

[0223]Substrate:

[0224]167 mM lactose, 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂ and pH adjusted to pH 3, 4, 5, 6, 7, 8, 9 and 10 with NaOH.

[0225]Enzyme Sample:

[0226]Experimental lactase from Bifidobacterium bifidum having the encoded sequence shown in SEQ ID NO: 2 was diluted appropriately in 150 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 0.01% Triton X100.

[0227]Procedure: [0228]10 ul enzyme sample diluted in enzyme dilution buffer was added to PCR tubes at room temp. [0229]90 ul substrate was added at room temp. and quickly placed in a Peltier Thermal Cycler (PCT-200, MJ research) at 37° C. and incubated for 30 min and then placed on ice. [0230]The reaction was stopped by adding 100 ul 0.25 M NaOH. [0231]20 ul was transferred to a 96 well microtitre plate and 230 ul 65 mM sodium phosphate, pH 7, 0.4 g/l Glucose oxidase, 0.013 g/l HRP, 0.65 g/l ABTS solution was added. After 30 minutes at room temperature, the absorbance was measured at 420 nm.

TABLE-US-00004 [0231] TABLE 4 B. bifidum lactase relative pH activity (% of activity at pH 6) 3 0 4 4 5 75 6 100 7 85 8 39 9 10 10 4

[0232]Temperature Profile

[0233]Substrate:

[0234]167 mM lactose, 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂ and pH adjusted to pH 6.5 with NaOH.

[0235]Enzyme Sample:

[0236]Experimental lactase from Bifidobacterium bifidum having the encoded sequence shown in SEQ ID NO: 2 was diluted appropriately in 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 0.01% Triton X100 and pH adjusted to pH 6.5.

[0237]Procedure: [0238]10 ul enzyme sample diluted in enzyme dilution buffer was added to PCR tubes at room temp. [0239]90 ul preheated (in a Peltier Thermal Cycler 30-70° C.) substrate was added and incubation was performed with a temp. gradient from 30-70° C. for 30 min. and then placed on ice. [0240]The reaction was stopped by adding 100 ul 0.25 M NaOH. [0241]20 ul was transferred to a 96 well microtitre plate and 230 ul 65 mM sodium phosphate, pH 7, 0.4 g/l Glucose oxidase, 0.013 g/l HRP, 0.65 g/l ABTS solution was added. After 30 minutes at room temperature, the absorbance was measured at 420 nm.

TABLE-US-00005 [0241] TABLE 5 Temp. B. bifidum lactase relative ° C. activity (% of activity at 38.1° C.) 20 54 21 63 22 64 24 68 26 73 29 81 31 88 34 94 36 96 38 100 43 96 48 91 52 83 57 76 62 58 66 32 69 20 70 17

Example 5

[0242]Determination of Km for Lactase Enzymes at 5° C.

[0243]Principle:

[0244]Lactase hydrolyses lactose and glucose +galactose is formed. Glucose is measured after a modified version of the common glucose oxidase/peroxidase assay (Werner, W. et al. (1970) Z. analyt. Chem. 252: 224.)

[0245]Substrate:

[0246]Different lactose concentrations ranging from Km/5 to 10*Km, 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂ and pH adjusted to pH 6.5 with NaOH.

[0247]Enzyme Sample:

[0248]Experimental lactase from Bifidobacterium bifidum having the encoded sequence shown in SEQ ID NO: 2 was diluted appropriately in 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 0.01% Triton X100, pH adjusted to pH 6.5 with NaOH.

[0249]12 g/l Lactozym (commercially available lactase from Novozymes A/S, Denmark) was diluted 6000 times in the same buffer.

[0250]Procedure: [0251]10 ul enzyme sample (5° C.) was added to a 96 well microtitre plate on ice. [0252]90 ul substrate (5° C.) was added and incubated for 2 hours at 5° C. [0253]The reaction was stopped by adding 100 ul 0.25 M NaOH. [0254]20 ul was transferred to a 96 well microtitre plate and 230 ul 65 mM sodium phosphate, pH 7, 0.4 g/l Glucose oxidase, 0.013 g/l HRP, 0.65 g/l ABTS solution was added. After 30 minutes at room temperature, the absorbance was measured at 420 nm.

[0255]Km Determination:

[0256]Computerized nonlinear least-squares fitting and the Michaelis-Menten equation:

v=(Vmax*S)/(Km+S)

[0257]was used. Km for the Bifidobacterium lactase and Lactozym were determined to be 8 mM and 30 mM, respectively.

[0258]In a similar test performed at 37° C., Km for the Bifidobacterium lactase and Lactozym were determined to be 13 mM and 30 mM, respectively.

Example 6

[0259]Yoghurt Trials

[0260]Commercial homogenized milk with 1.5% fat was pasteurized at 90° C. for 20 min. 200 ml of the milk was transferred into baby bottles and tempered to 43° C. The milk was inoculated with a frozen probiotic yoghurt culture from Chr. Hansen, Denmark, (F-DVS ABY-3) using an inoculation level of 0.02%. At the same time, enzyme was added to the milk. Enzyme products tested were Ha-lactase, a commercially available lactase from Chr. Hansen, Denmark, having an activity of 8021 LAU/g and an experimental lactase from Bifidobacterium bifidum having the encoded sequence shown in SEQ ID No. 2 and an activity of 295 LAU/g.

[0261]Dosages were 1500, 3000 and 3750 LAU/L milk of Ha-lactase and 710, 1420, and 1780 LAU/L of the Bifidobacterium lactase. The milk samples were fermented at 43° C. until pH reached 4.55 within approximately five hours. The yoghurts were then stirred, cooled to 25° C. and placed at 8° C. for storage. Samples were collected 2 hours after addition of culture and enzyme, at end pH (pH 4.55) and after 20-24 hours (Day 1) of storage at 8° C. The biological activity was stopped by addition of sulphuric acid. Proteins were precipitated adding perchloric acid and MQW containing standards were then added.

[0262]Lactose hydrolysis was measured using a Dionex ICS-3000 system equipped with a Carbopac20 connected with an electrochemical detector (ED). Peaks were identified and quantified by comparing with known standards of lactose, glucose and galactose. Results for lactose hydrolysis are given below.

TABLE-US-00006 TABLE 6 Lactose in yoghurt treated with different dosages of Ha-lactase or Bifidobacterium lactase. A reference sample with no addition of enzyme was also tested Lactose (mg/g) Refer- ence HA-lactase Bifidobacterium lactase No 1500 3000 3750 710 1420 1780 Time lactase LAU/L LAU/L LAU/L LAU/L LAU/L LAU/L Ini- 56.0 tial 2 h 48.4 13.3 3.4 2.6 29.6 10.2 5.7 End 39.0 10.9 2.5 1.9 8.7 0.6 0.6 pH Day 39.3 10.5 2.4 1.8 3.4 0.5 0.5 1

[0263]The level of lactose in the yoghurt samples show that Ha-lactase has highest activity in the beginning of the fermentation, during the first two hours of fermentation. After two hours Ha-lactase is clearly inactivated, due to the lowering of pH. The Bifidobacterium lactase, on the other hand, stays active during the whole fermentation and also to some extent during cold storage. At the lowest tested dosage of 710 LAU/L, the lactose level is significantly reduced during cold storage when using the Bifidobacterium lactase.

Example 7

[0264]Yoghurt Trials

[0265]Commercial homogenized milk with 1.5% fat was pasteurized at 90° C. for 20 min. 200 ml of the milk was transferred into baby bottles and tempered to 43° C. The milk was inoculated with a frozen probiotic yoghurt culture from Chr. Hansen, Denmark, (F-DVS ABY-3) using an inoculation level if 0.02%. At the same time enzyme was added to the milk. Enzyme products tested were Ha-lactase, a commercially available lactase from Chr. Hansen, Denmark, having an activity of 8021 LAU/g and an experimental lactase from Bifidobacterium bifidum having the encoded sequence shown in SEQ ID No. 2 and an activity of 295 LAU/g.

[0266]Dosage was 1500 LAU/L milk of Ha-lactase and 710, 530 and 360 LAU/L of the Bifidobacterium lactase. The milk samples were fermented at 43° C. until pH reached 4.55 within approximately five hours. The yoghurts were then stirred, cooled to 25° C. and placed at 8° C. for storage. Samples were collected 2 hours after addition of culture and enzyme, at end pH (pH4.55) and after 1, 2, 3 and 7 days of storage at 8° C. The biological activity was stopped by addition of sulphuric acid. Proteins were precipitated adding perchloric acid and MQW containing standards was then added.

[0267]Lactose hydrolysis was measured using a Dionex ICS-3000 system equipped with a Carbopac20 connected with an electrochemical detector (ED). Peaks were identified and quantified by comparing with known standards of lactose, glucose and galactose. Results for lactose hydrolysis are given below.

TABLE-US-00007 TABLE 7 Lactose (mg/g) Ha-lactase Bifidobacterium lactase 1500 710 530 360 Time No lactase LAU/L LAU/L LAU/L LAU/L Initial 47.9 2 h 45.8 4.7 21.2 24.2 29.6 End pH 35.5 2.3 0.8 3.8 9.6 Day 1 33.7 3.1 0.2 0.8 4.7 Day 2 2.7 0.5 0.7 2.9 Day 3 2.6 0.3 0.2 1.5 Day 7 2.6 0.1 0.2 0.3

[0268]As described in the previous example, the activity period of the two enzymes tested differs. Ha-lactase shows high activity at the start of fermentation whereas the Bifidobacterium lactase stays active during the whole fermentation time and also during cold storage. Hence, after two days of storage the lactose level is similar or lower in samples with the Bifidobacterium lactase compared to the Ha-lactase.

[0269]Similar degrees of lactose hydrolysis are obtained day 2 in the yoghurts samples with 1500 LAU/L Ha-lactase and yoghurt samples with 360 LAU/L Bifidobacterium lactase.

Example 8

[0270]Milk Trials

[0271]Commercial homogenized milk with 1.5% fat was transferred to tubes (10 ml) and heated in water baths to 40° C., 50° C. and 55° C., respectively. Enzyme was then added to the milk samples. Enzyme products tested were Ha-lactase, a commercially available lactase from Chr. Hansen, Denmark, having an activity of 8040 LAU/g and an experimental lactase from Bifidobacterium bifidum having the encoded sequence shown in SEQ ID No. 2 and an activity of 295 LAU/g.

[0272]Dosage was 1500 LAU/L milk of Ha-lactase and 710 LAU/L of the Bifidobacterium lactase. Samples were collected 2 hours and 4 hours after addition of the enzyme. The biological activity was stopped by addition of sulphuric acid. Proteins were precipitated adding perchloric acid and MQW containing standards was then added.

[0273]Lactose hydrolysis was measured using a Dionex ICS-3000 system equipped with a Carbopac20 connected with an electrochemical detector (ED). Peaks were identified and quantified by comparing with known standards of lactose, glucose and galactose. Results for lactose hydrolysis are given below.

TABLE-US-00008 TABLE 8 Lactose (mg/g) Refer- ence HA-lactase - 1500 Bifidobacterium No LAU/L lactase - 710 LAU/L Time lactase 40° C. 50° C. 55° C. 40° C. 50° C. 55° C. 2 h 46.5 24.0 34.9 40.6 29.3 21.0 31.7 4 h 46.5 19.8 37.3 39.6 12.5 11.2 25.6

[0274]At the highest temperatures, 50° C. and 55° C., the Bifidobacterium lactase shows significantly higher activity compared to the Ha-lactase. Furthermore, the Bifidobacterium lactase stays active during the 4 hour reaction time, whereas no or only very low activity is observed for the Ha-lactase.

Example 9

[0275]Milk Trials-High Temperature

[0276]Commercial homogenized milk with 1.5% fat was transferred to tubes (10 ml) and tempered to 63° C. Enzyme was added to the milk samples. Enzyme products tested were Ha-lactase 5200, a commercially available lactase from Chr. Hansen (Denmark) having an activity of 8040 LAU/g and Lactoles, a commercial Bacillus lactase from Daiwa Kasei (Japan) having an activity of approximately 1500 LAU/g.

[0277]Applied dosages were 1500 LAU/L milk of Ha-lactase and Lactoles, respectively. At 63° C. samples were collected 15 minutes, 30 minutes, 2 hours and 4 hours after addition of the enzyme. The enzymatic activity in the samples was stopped by addition of sulphuric acid and proteins precipitated by addition of perchloric acid before HPLC analysis.

[0278]Lactose hydrolysis was measured using a Dionex ICS-3000 system equipped with a Carbopac20 connected with an electrochemical detector (ED). Peaks were identified and quantified by comparing with known standards of lactose, glucose and galactose. Results for lactose hydrolysis are given below.

TABLE-US-00009 TABLE 9 Lactose (mg/g) Reference- HA-lactase 5200 - Lactoles - Time No lactase 1500 LAU/L 1500 LAU/L 15 min. 48.9 44.6 22.7 30 min. 48.9 44.9 22.1 2 h 48.9 44.0 6.7 4 h 48.9 43.2 3.2

[0279]At 63° C. Ha-lactase 5200 is inactivated as no hydrolysis takes place during 4 hours of reaction. On the other hand, Lactoles shows high activity at this temperature during the whole reaction time. After 4 hours, a degree of hydrolysis of 93.4% is obtained.

Example 10

[0280]100 ml 9% skimmed milk solution having approximately 5% lactose was made by mixing 9 g skimmed milk powder (Kerry) in 91 ml ionic water. 10 ml of the solution was transferred to a test tube containing a magnetic stirring bar and placed in a water bath at 5° C. After 15 min enzyme was added. Enzymes tested were Lactozym, a commercially available lactase from Novozymes A/S, Denmark, having an activity of 3060 LAU/g, and an experimental lactase from Bifidobacterium bifidum having the encoded sequence shown in SEQ ID NO: 2 and an activity of 295 LAU/g. Amino acids 1 to 27 of SEQ ID NO: 2 is a signal sequence which is presumably cleaved off and amino acids 1332 to 1341 is a tag used for purification of the experimental enzyme.

[0281]Dosages were 3000 LAU/l milk of Lactozym and 1420 LAU/l milk of the Bifidobacterium lactase. Milk samples were taken at regular intervals up till 48 hrs. and the enzyme inactivated by heating to 99° C. for 10 min in a thermomixer. Samples were diluted appropriately and filtered through a 0.20 um filter.

[0282]Lactose hydrolysis was measured using a Dionex BioLC equipped with a Dionex PA1 column and a Pulsed Amperiometrisk Detektor (PAD). Peaks were identified and quantified by comparing with known standards of lactose, glucose and galactose. Results are given below.

TABLE-US-00010 TABLE 10 Lactose, glucose and galactose in reconstituted skimmed milk after treatment with Lactozym or Bifidobacterium lactase at 5° C. Lactozym Bifidobacterium lactase Time Lactose Glucose Galactose Lactose Glucose Galactose min mM mM mM mM mM mM 125 -- -- -- 110 28 31 240 87 64 60 -- -- -- 360 -- -- -- 70 65 67 460 62 104 91 55 74 76 1410 18 152 134 13 149 148 1620 13 137 125 7 139 140 1865 10 -- -- 5 167 167 2870 6 141 132 0.7 139 140

[0283]When tested in milk with 5% lactose at 5° C. again no transferase activity is observed when using the Bifidobacterium lactase. Glucose and galactose production are equal and total monosaccharide production match that expected from the lactose hydrolyzed. For comparison, Lactozym produces less galactose than glucose clearly showing that galactooligosaccharides have been produced. Also, final lactose levels are significantly lower when using the Bifidobacterium lactase further illustrating the lower Km value of this enzyme.

Sequence CWU 1

711931PRTBifidobacterium bifidum 1Met Lys Lys Pro Leu Gly Lys Ile Val Ala Ser Thr Ala Leu Leu Ile1 5 10 15Ser Val Ala Phe Ser Ser Ser Ile Ala Ser Ala Ala Val Glu Asp Ala 20 25 30Thr Arg Ser Asp Ser Thr Thr Gln Met Ser Ser Thr Pro Glu Val Ala 35 40 45Tyr Ser Ser Ala Val Asp Ser Lys Gln Asn Arg Thr Ser Asp Phe Asp 50 55 60Ala Asn Trp Lys Phe Met Leu Ser Asp Ser Val Gln Ala Gln Asp Pro65 70 75 80Ala Phe Asp Asp Ser Ala Trp Gln Gln Val Asp Leu Pro His Asp Tyr 85 90 95Ser Ile Thr Gln Lys Tyr Ser Gln Ser Asn Glu Ala Glu Ser Ala Tyr 100 105 110Leu Pro Gly Gly Thr Gly Trp Tyr Arg Lys Ser Phe Thr Ile Asp Arg 115 120 125Asp Leu Ala Gly Lys Arg Ile Ala Ile Asn Phe Asp Gly Val Tyr Met 130 135 140Asn Ala Thr Val Trp Phe Asn Gly Val Lys Leu Gly Thr His Pro Tyr145 150 155 160Gly Tyr Ser Pro Phe Ser Phe Asp Leu Thr Gly Asn Ala Lys Phe Gly 165 170 175Gly Glu Asn Thr Ile Val Val Lys Val Glu Asn Arg Leu Pro Ser Ser 180 185 190Arg Trp Tyr Ser Gly Ser Gly Ile Tyr Arg Asp Val Thr Leu Thr Val 195 200 205Thr Asp Gly Val His Val Gly Asn Asn Gly Val Ala Ile Lys Thr Pro 210 215 220Ser Leu Ala Thr Gln Asn Gly Gly Asp Val Thr Met Asn Leu Thr Thr225 230 235 240Lys Val Ala Asn Asp Thr Glu Ala Ala Ala Asn Ile Thr Leu Lys Gln 245 250 255Thr Val Phe Pro Lys Gly Gly Lys Thr Asp Ala Ala Ile Gly Thr Val 260 265 270Thr Thr Ala Ser Lys Ser Ile Ala Ala Gly Ala Ser Ala Asp Val Thr 275 280 285Ser Thr Ile Thr Ala Ala Ser Pro Lys Leu Trp Ser Ile Lys Asn Pro 290 295 300Asn Leu Tyr Thr Val Arg Thr Glu Val Leu Asn Gly Gly Lys Val Leu305 310 315 320Asp Thr Tyr Asp Thr Glu Tyr Gly Phe Arg Trp Thr Gly Phe Asp Ala 325 330 335Thr Ser Gly Phe Ser Leu Asn Gly Glu Lys Val Lys Leu Lys Gly Val 340 345 350Ser Met His His Asp Gln Gly Ser Leu Gly Ala Val Ala Asn Arg Arg 355 360 365Ala Ile Glu Arg Gln Val Glu Ile Leu Gln Lys Met Gly Val Asn Ser 370 375 380Ile Arg Thr Thr His Asn Pro Ala Ala Lys Ala Leu Ile Asp Val Cys385 390 395 400Asn Glu Lys Gly Val Leu Val Val Glu Glu Val Phe Asp Met Trp Asn 405 410 415Arg Ser Lys Asn Gly Asn Thr Glu Asp Tyr Gly Lys Trp Phe Gly Gln 420 425 430Ala Ile Ala Gly Asp Asn Ala Val Leu Gly Gly Asp Lys Asp Glu Thr 435 440 445Trp Ala Lys Phe Asp Leu Thr Ser Thr Ile Asn Arg Asp Arg Asn Ala 450 455 460Pro Ser Val Ile Met Trp Ser Leu Gly Asn Glu Met Met Glu Gly Ile465 470 475 480Ser Gly Ser Val Ser Gly Phe Pro Ala Thr Ser Ala Lys Leu Val Ala 485 490 495Trp Thr Lys Ala Ala Asp Ser Thr Arg Pro Met Thr Tyr Gly Asp Asn 500 505 510Lys Ile Lys Ala Asn Trp Asn Glu Ser Asn Thr Met Gly Asp Asn Leu 515 520 525Thr Ala Asn Gly Gly Val Val Gly Thr Asn Tyr Ser Asp Gly Ala Asn 530 535 540Tyr Asp Lys Ile Arg Thr Thr His Pro Ser Trp Ala Ile Tyr Gly Ser545 550 555 560Glu Thr Ala Ser Ala Ile Asn Ser Arg Gly Ile Tyr Asn Arg Thr Thr 565 570 575Gly Gly Ala Gln Ser Ser Asp Lys Gln Leu Thr Ser Tyr Asp Asn Ser 580 585 590Ala Val Gly Trp Gly Ala Val Ala Ser Ser Ala Trp Tyr Asp Val Val 595 600 605Gln Arg Asp Phe Val Ala Gly Thr Tyr Val Trp Thr Gly Phe Asp Tyr 610 615 620Leu Gly Glu Pro Thr Pro Trp Asn Gly Thr Gly Ser Gly Ala Val Gly625 630 635 640Ser Trp Pro Ser Pro Lys Asn Ser Tyr Phe Gly Ile Val Asp Thr Ala 645 650 655Gly Phe Pro Lys Asp Thr Tyr Tyr Phe Tyr Gln Ser Gln Trp Asn Asp 660 665 670Asp Val His Thr Leu His Ile Leu Pro Ala Trp Asn Glu Asn Val Val 675 680 685Ala Lys Gly Ser Gly Asn Asn Val Pro Val Val Val Tyr Thr Asp Ala 690 695 700Ala Lys Val Lys Leu Tyr Phe Thr Pro Lys Gly Ser Thr Glu Lys Arg705 710 715 720Leu Ile Gly Glu Lys Ser Phe Thr Lys Lys Thr Thr Ala Ala Gly Tyr 725 730 735Thr Tyr Gln Val Tyr Glu Gly Ser Asp Lys Asp Ser Thr Ala His Lys 740 745 750Asn Met Tyr Leu Thr Trp Asn Val Pro Trp Ala Glu Gly Thr Ile Ser 755 760 765Ala Glu Ala Tyr Asp Glu Asn Asn Arg Leu Ile Pro Glu Gly Ser Thr 770 775 780Glu Gly Asn Ala Ser Val Thr Thr Thr Gly Lys Ala Ala Lys Leu Lys785 790 795 800Ala Asp Ala Asp Arg Lys Thr Ile Thr Ala Asp Gly Lys Asp Leu Ser 805 810 815Tyr Ile Glu Val Asp Val Thr Asp Ala Asn Gly His Ile Val Pro Asp 820 825 830Ala Ala Asn Arg Val Thr Phe Asp Val Lys Gly Ala Gly Lys Leu Val 835 840 845Gly Val Asp Asn Gly Ser Ser Pro Asp His Asp Ser Tyr Gln Ala Asp 850 855 860Asn Arg Lys Ala Phe Ser Gly Lys Val Leu Ala Ile Val Gln Ser Thr865 870 875 880Lys Glu Ala Gly Glu Ile Thr Val Thr Ala Lys Ala Asp Gly Leu Gln 885 890 895Ser Ser Thr Val Lys Ile Ala Thr Thr Ala Val Pro Gly Thr Ser Thr 900 905 910Glu Lys Thr Val Arg Ser Phe Tyr Tyr Ser Arg Asn Tyr Tyr Val Lys 915 920 925Thr Gly Asn Lys Pro Ile Leu Pro Ser Asp Val Glu Val Arg Tyr Ser 930 935 940Asp Gly Thr Ser Asp Arg Gln Asn Val Thr Trp Asp Ala Val Ser Asp945 950 955 960Asp Gln Ile Ala Lys Ala Gly Ser Phe Ser Val Ala Gly Thr Val Ala 965 970 975Gly Gln Lys Ile Ser Val Arg Val Thr Met Ile Asp Glu Ile Gly Ala 980 985 990Leu Leu Asn Tyr Ser Ala Ser Thr Pro Val Gly Thr Pro Ala Val Leu 995 1000 1005Pro Gly Ser Arg Pro Ala Val Leu Pro Asp Gly Thr Val Thr Ser 1010 1015 1020Ala Asn Phe Ala Val His Trp Thr Lys Pro Ala Asp Thr Val Tyr 1025 1030 1035Asn Thr Ala Gly Thr Val Lys Val Pro Gly Thr Ala Thr Val Phe 1040 1045 1050Gly Lys Glu Phe Lys Val Thr Ala Thr Ile Arg Val Gln Arg Ser 1055 1060 1065Gln Val Thr Ile Gly Ser Ser Val Ser Gly Asn Ala Leu Arg Leu 1070 1075 1080Thr Gln Asn Ile Pro Ala Asp Lys Gln Ser Asp Thr Leu Asp Ala 1085 1090 1095Ile Lys Asp Gly Ser Thr Thr Val Asp Ala Asn Thr Gly Gly Gly 1100 1105 1110Ala Asn Pro Ser Ala Trp Thr Asn Trp Ala Tyr Ser Lys Ala Gly 1115 1120 1125His Asn Thr Ala Glu Ile Thr Phe Glu Tyr Ala Thr Glu Gln Gln 1130 1135 1140Leu Gly Gln Ile Val Met Tyr Phe Phe Arg Asp Ser Asn Ala Val 1145 1150 1155Arg Phe Pro Asp Ala Gly Lys Thr Lys Ile Gln Ile Ser Ala Asp 1160 1165 1170Gly Lys Asn Trp Thr Asp Leu Ala Ala Thr Glu Thr Ile Ala Ala 1175 1180 1185Gln Glu Ser Ser Asp Arg Val Lys Pro Tyr Thr Tyr Asp Phe Ala 1190 1195 1200Pro Val Gly Ala Thr Phe Val Lys Val Thr Val Thr Asn Ala Asp 1205 1210 1215Thr Thr Thr Pro Ser Gly Val Val Cys Ala Gly Leu Thr Glu Ile 1220 1225 1230Glu Leu Lys Thr Ala Thr Ser Lys Phe Val Thr Asn Thr Ser Ala 1235 1240 1245Ala Leu Ser Ser Leu Thr Val Asn Gly Thr Lys Val Ser Asp Ser 1250 1255 1260Val Leu Ala Ala Gly Ser Tyr Asn Thr Pro Ala Ile Ile Ala Asp 1265 1270 1275Val Lys Ala Glu Gly Glu Gly Asn Ala Ser Val Thr Val Leu Pro 1280 1285 1290Ala His Asp Asn Val Ile Arg Val Ile Thr Glu Ser Glu Asp His 1295 1300 1305Val Thr Arg Lys Thr Phe Thr Ile Asn Leu Gly Thr Glu Gln Glu 1310 1315 1320Phe Pro Ala Asp Ser Asp Glu Arg Asp Tyr Pro Ala Ala Asp Met 1325 1330 1335Thr Val Thr Val Gly Ser Glu Gln Thr Ser Gly Thr Ala Thr Glu 1340 1345 1350Gly Pro Lys Lys Phe Ala Val Asp Gly Asn Thr Ser Thr Tyr Trp 1355 1360 1365His Ser Asn Trp Thr Pro Thr Thr Val Asn Asp Leu Trp Ile Ala 1370 1375 1380Phe Glu Leu Gln Lys Pro Thr Lys Leu Asp Ala Leu Arg Tyr Leu 1385 1390 1395Pro Arg Pro Ala Gly Ser Lys Asn Gly Ser Val Thr Glu Tyr Lys 1400 1405 1410Val Gln Val Ser Asp Asp Gly Thr Asn Trp Thr Asp Ala Gly Ser 1415 1420 1425Gly Thr Trp Thr Thr Asp Tyr Gly Trp Lys Leu Ala Glu Phe Asn 1430 1435 1440Gln Pro Val Thr Thr Lys His Val Arg Leu Lys Ala Val His Thr 1445 1450 1455Tyr Ala Asp Ser Gly Asn Asp Lys Phe Met Ser Ala Ser Glu Ile 1460 1465 1470Arg Leu Arg Lys Ala Val Asp Thr Thr Asp Ile Ser Gly Ala Thr 1475 1480 1485Val Thr Val Pro Ala Lys Leu Thr Val Asp Arg Val Asp Ala Asp 1490 1495 1500His Pro Ala Thr Phe Ala Thr Lys Asp Val Thr Val Thr Leu Gly 1505 1510 1515Asp Ala Thr Leu Arg Tyr Gly Val Asp Tyr Leu Leu Asp Tyr Ala 1520 1525 1530Gly Asn Thr Ala Val Gly Lys Ala Thr Val Thr Val Arg Gly Ile 1535 1540 1545Asp Lys Tyr Ser Gly Thr Val Ala Lys Thr Phe Thr Ile Glu Leu 1550 1555 1560Lys Asn Ala Pro Ala Pro Glu Pro Thr Leu Thr Ser Val Ser Val 1565 1570 1575Lys Thr Lys Pro Ser Lys Leu Thr Tyr Val Val Gly Asp Ala Phe 1580 1585 1590Asp Pro Ala Gly Leu Val Leu Gln Leu Asn Tyr Asp Asp Asp Ser 1595 1600 1605Thr Gly Thr Val Thr Trp Asn Thr Gln Thr Ala Gly Asp Phe Thr 1610 1615 1620Phe Lys Pro Ala Leu Asp Ala Lys Leu Lys Val Thr Asp Lys Thr 1625 1630 1635Val Thr Val Thr Tyr Gln Gly Lys Ser Ala Val Ile Asp Ile Thr 1640 1645 1650Val Ser Gln Pro Ala Pro Thr Val Ser Lys Thr Asp Leu Asp Lys 1655 1660 1665Ala Ile Lys Ala Ile Glu Ala Lys Asn Pro Asp Ser Ser Lys Tyr 1670 1675 1680Thr Ala Asp Ser Trp Lys Thr Phe Ala Asp Ala Met Ala His Ala 1685 1690 1695Lys Ala Val Ile Ala Asp Asp Ser Ala Thr Gln Gln Asp Val Asp 1700 1705 1710Asn Ala Leu Lys Ala Leu Thr Asp Ala Tyr Ala Gly Leu Thr Glu 1715 1720 1725Lys Thr Pro Glu Pro Ala Pro Val Ser Lys Ser Glu Leu Asp Lys 1730 1735 1740Lys Ile Lys Ala Ile Glu Ala Glu Lys Leu Asp Gly Ser Lys Tyr 1745 1750 1755Thr Ala Glu Ser Trp Lys Ala Phe Glu Thr Ala Leu Ala His Ala 1760 1765 1770Lys Ala Val Ile Ala Ser Asp Ser Ala Thr Gln Gln Asn Val Asp 1775 1780 1785Ala Ala Leu Gly Ala Leu Thr Ser Ala Arg Asp Gly Leu Thr Glu 1790 1795 1800Lys Gly Glu Val Lys Pro Asp Pro Lys Pro Glu Pro Gly Thr Val 1805 1810 1815Asp Lys Ala Ala Leu Asp Lys Ala Val Lys Lys Val Glu Ala Glu 1820 1825 1830Lys Leu Asp Gly Ser Lys Tyr Thr Ala Asp Ser Trp Lys Ala Phe 1835 1840 1845Glu Thr Ala Leu Ala His Ala Lys Ala Val Ile Gly Asn Ala Asn 1850 1855 1860Ser Thr Gln Phe Asp Ile Asp Asn Ala Leu Ser Met Leu Asn Asp 1865 1870 1875Ala Arg Ala Ala Leu Lys Glu Lys Pro Gly Arg Ile Ile Ala Ile 1880 1885 1890Ile Asp Gly Ser Ala Leu Ser Lys Thr Gly Ala Ser Val Ala Ile 1895 1900 1905Ile Ala Ser Val Ala Ala Ala Met Leu Ala Val Gly Ala Gly Val 1910 1915 1920Met Ala Leu Arg Arg Lys Arg Ser 1925 193021341PRTBifidobacterium bifidum 2Met Lys Lys Pro Leu Gly Lys Ile Val Ala Ser Thr Ala Leu Leu Ile1 5 10 15Ser Val Ala Phe Ser Ser Ser Ile Ala Ser Ala Ile Glu Asp Ala Thr 20 25 30Arg Ser Asp Ser Thr Thr Gln Met Ser Ser Thr Pro Glu Val Ala Tyr 35 40 45Ser Ser Ala Val Asp Ser Lys Gln Asn Arg Thr Ser Asp Phe Asp Ala 50 55 60Asn Trp Lys Phe Met Leu Ser Asp Ser Val Gln Ala Gln Asp Pro Ala65 70 75 80Phe Asp Asp Ser Ala Trp Gln Gln Val Asp Leu Pro His Asp Tyr Ser 85 90 95Ile Thr Gln Lys Tyr Ser Gln Ser Asn Glu Ala Glu Ser Ala Tyr Leu 100 105 110Pro Gly Gly Thr Gly Trp Tyr Arg Lys Ser Phe Thr Ile Asp Arg Asp 115 120 125Leu Ala Gly Lys Arg Ile Ala Ile Asn Phe Asp Gly Val Tyr Met Asn 130 135 140Ala Thr Val Trp Phe Asn Gly Val Lys Leu Gly Thr His Pro Tyr Gly145 150 155 160Tyr Ser Pro Phe Ser Phe Asp Leu Thr Gly Asn Ala Lys Phe Gly Gly 165 170 175Glu Asn Thr Ile Val Val Lys Val Glu Asn Arg Leu Pro Ser Ser Arg 180 185 190Trp Tyr Ser Gly Ser Gly Ile Tyr Arg Asp Val Thr Leu Thr Val Thr 195 200 205Asp Gly Val His Val Gly Asn Asn Gly Val Ala Ile Lys Thr Pro Ser 210 215 220Leu Ala Thr Gln Asn Gly Gly Asp Val Thr Met Asn Leu Thr Thr Lys225 230 235 240Val Ala Asn Asp Thr Glu Ala Ala Ala Asn Ile Thr Leu Lys Gln Thr 245 250 255Val Phe Pro Lys Gly Gly Lys Thr Asp Ala Ala Ile Gly Thr Val Thr 260 265 270Thr Ala Ser Lys Ser Ile Ala Ala Gly Ala Ser Ala Asp Val Thr Ser 275 280 285Thr Ile Thr Ala Ala Ser Pro Lys Leu Trp Ser Ile Lys Asn Pro Asn 290 295 300Leu Tyr Thr Val Arg Thr Glu Val Leu Asn Gly Gly Lys Val Leu Asp305 310 315 320Thr Tyr Asp Thr Glu Tyr Gly Phe Arg Trp Thr Gly Phe Asp Ala Thr 325 330 335Ser Gly Phe Ser Leu Asn Gly Glu Lys Val Lys Leu Lys Gly Val Ser 340 345 350Met His His Asp Gln Gly Ser Leu Gly Ala Val Ala Asn Arg Arg Ala 355 360 365Ile Glu Arg Gln Val Glu Ile Leu Gln Lys Met Gly Val Asn Ser Ile 370 375 380Arg Thr Thr His Asn Pro Ala Ala Lys Ala Leu Ile Asp Val Cys Asn385 390 395 400Glu Lys Gly Val Leu Val Val Glu Glu Val Phe Asp Met Trp Asn Arg 405 410 415Ser Lys Asn Gly Asn Thr Glu Asp Tyr Gly Lys Trp Phe Gly Gln Ala 420 425 430Ile Ala Gly Asp Asn Ala Val Leu Gly Gly Asp Lys Asp Glu Thr Trp 435 440 445Ala Lys Phe Asp Leu Thr Ser Thr Ile Asn Arg Asp Arg Asn Ala Pro 450 455 460Ser Val Ile Met Trp Ser Leu Gly Asn Glu Met Met Glu Gly Ile Ser465 470 475 480Gly Ser Val Ser Gly Phe Ser Ala Thr Ser Ala Lys Leu Val Ala Trp 485 490 495Thr Lys Ala Ala Asp Ser Thr Arg Pro Met Thr Tyr Gly Asp Asn Lys 500 505 510Ile Lys Ala Asn Trp Asn Glu Ser Asn Thr Met

Gly Asp Asn Leu Thr 515 520 525Ala Asn Gly Gly Val Val Gly Thr Asn Tyr Ser Asp Gly Ala Asn Tyr 530 535 540Asp Lys Ile Arg Thr Thr His Pro Ser Trp Ala Ile Tyr Gly Ser Glu545 550 555 560Thr Ala Ser Ala Ile Asn Ser Arg Gly Ile Tyr Asn Arg Thr Thr Gly 565 570 575Gly Ala Gln Ser Ser Asp Lys Gln Leu Thr Ser Tyr Asp Asn Ser Ala 580 585 590Val Gly Trp Gly Ala Val Ala Ser Ser Ala Trp Tyr Asp Val Val Gln 595 600 605Arg Asp Phe Val Ala Gly Thr Tyr Val Trp Thr Gly Phe Asp Tyr Leu 610 615 620Gly Glu Pro Thr Pro Trp Asn Gly Thr Gly Ser Gly Ala Val Gly Ser625 630 635 640Trp Pro Ser Pro Lys Asn Ser Tyr Phe Gly Ile Val Asp Thr Ala Gly 645 650 655Phe Pro Lys Asp Thr Tyr Tyr Phe Tyr Gln Ser Gln Trp Asn Asp Asp 660 665 670Val His Thr Leu His Ile Leu Pro Ala Trp Asn Glu Asn Val Val Ala 675 680 685Lys Gly Ser Gly Asn Asn Val Pro Val Val Val Tyr Thr Asp Ala Ala 690 695 700Lys Val Lys Leu Tyr Phe Thr Pro Lys Gly Ser Thr Glu Gln Arg Leu705 710 715 720Ile Gly Glu Lys Ser Phe Thr Lys Lys Thr Thr Ala Ala Gly Tyr Thr 725 730 735Tyr Gln Val Tyr Glu Gly Ser Asp Lys Asp Ser Thr Ala His Lys Asn 740 745 750Met Tyr Leu Thr Trp Asn Val Pro Trp Ala Glu Gly Thr Ile Ser Ala 755 760 765Glu Ala Tyr Asp Glu Asn Asn Arg Leu Ile Pro Glu Gly Ser Thr Glu 770 775 780Gly Asn Ala Ser Val Thr Thr Thr Gly Lys Ala Ala Lys Leu Lys Ala785 790 795 800Asp Ala Asp Arg Lys Thr Ile Thr Ala Asp Gly Lys Asp Leu Ser Tyr 805 810 815Ile Glu Val Asp Val Thr Asp Ala Asn Gly His Ile Val Pro Asp Ala 820 825 830Ala Asn Arg Val Thr Phe Asp Val Lys Gly Ala Gly Lys Leu Val Gly 835 840 845Val Asp Asn Gly Ser Ser Pro Asp His Asp Ser Tyr Gln Ala Asp Asn 850 855 860Arg Lys Ala Phe Ser Gly Lys Val Leu Ala Ile Val Gln Ser Thr Lys865 870 875 880Glu Ala Gly Glu Ile Thr Val Thr Ala Lys Ala Asp Gly Leu Gln Ser 885 890 895Ser Thr Val Lys Ile Ala Thr Thr Ala Val Pro Gly Thr Ser Thr Glu 900 905 910Lys Thr Val Arg Ser Phe Tyr Tyr Ser Arg Asn Tyr Tyr Val Lys Thr 915 920 925Gly Asn Lys Pro Ile Leu Pro Ser Asp Val Glu Val Arg Tyr Ser Asp 930 935 940Gly Thr Ser Asp Arg Gln Asn Val Thr Trp Asp Ala Val Ser Asp Asp945 950 955 960Gln Ile Ala Lys Ala Gly Ser Phe Ser Val Ala Gly Thr Val Ala Gly 965 970 975Gln Lys Ile Ser Val Arg Val Thr Met Ile Asp Glu Ile Gly Ala Leu 980 985 990Leu Asn Tyr Ser Ala Ser Thr Pro Val Gly Thr Pro Ala Val Leu Pro 995 1000 1005Gly Ser Arg Pro Ala Val Leu Pro Asp Gly Thr Val Thr Ser Ala 1010 1015 1020Asn Phe Ala Val His Trp Thr Lys Pro Ala Asp Thr Val Tyr Asn 1025 1030 1035Thr Ala Gly Thr Val Lys Val Pro Gly Thr Ala Thr Val Phe Gly 1040 1045 1050Lys Glu Phe Lys Val Thr Ala Thr Ile Arg Val Gln Arg Ser Gln 1055 1060 1065Val Thr Ile Gly Ser Ser Val Ser Gly Asn Ala Leu Arg Leu Thr 1070 1075 1080Gln Asn Ile Pro Ala Asp Lys Gln Ser Asp Thr Leu Asp Ala Ile 1085 1090 1095Lys Asp Gly Ser Thr Thr Val Asp Ala Asn Thr Gly Gly Gly Ala 1100 1105 1110Asn Pro Ser Ala Trp Thr Asn Trp Ala Tyr Ser Lys Ala Gly His 1115 1120 1125Asn Thr Ala Glu Ile Thr Phe Glu Tyr Ala Thr Glu Gln Gln Leu 1130 1135 1140Gly Gln Ile Val Met Tyr Phe Phe Arg Asp Ser Asn Ala Val Arg 1145 1150 1155Phe Pro Asp Ala Gly Lys Thr Lys Ile Gln Ile Ser Ala Asp Gly 1160 1165 1170Lys Asn Trp Thr Asp Leu Ala Ala Thr Glu Thr Ile Ala Ala Gln 1175 1180 1185Glu Ser Ser Asp Arg Val Lys Pro Tyr Thr Tyr Asp Phe Ala Pro 1190 1195 1200Val Gly Ala Thr Phe Val Arg Val Thr Val Thr Asn Ala Asp Thr 1205 1210 1215Thr Thr Pro Ser Gly Val Val Cys Ala Gly Leu Thr Glu Ile Glu 1220 1225 1230Leu Lys Thr Ala Thr Ser Lys Phe Val Ala Asn Thr Ser Ala Ala 1235 1240 1245Leu Ser Ser Leu Thr Val Asn Gly Thr Lys Val Ser Asp Ser Val 1250 1255 1260Leu Ala Ala Gly Ser Tyr Asn Thr Pro Ala Ile Ile Ala Asp Val 1265 1270 1275Lys Ala Glu Gly Glu Gly Asn Ala Ser Val Thr Val Leu Pro Ala 1280 1285 1290His Asp Asn Val Ile Arg Val Ile Thr Glu Ser Glu Asp His Val 1295 1300 1305Thr Arg Lys Thr Phe Thr Ile Asn Leu Gly Thr Glu Gln Glu Phe 1310 1315 1320Pro Ala Asp Ser Asp Glu Arg Asp Gln His Gln His Gln His Gln 1325 1330 1335His Gln Gln 134031752PRTBifidobacterium bifidum 3Met Ala Val Arg Arg Leu Gly Gly Arg Ile Val Ala Phe Ala Ala Thr1 5 10 15Val Ala Leu Ser Ile Pro Leu Gly Leu Leu Thr Asn Ser Ala Trp Ala 20 25 30Val Glu Asp Ala Thr Arg Ser Asp Ser Thr Thr Gln Met Ser Ser Thr 35 40 45Pro Glu Val Val Tyr Ser Ser Ala Val Asp Ser Lys Gln Asn Arg Thr 50 55 60Ser Asp Phe Asp Ala Asn Trp Lys Phe Met Leu Ser Asp Ser Val Gln65 70 75 80Ala Gln Asp Pro Ala Phe Asp Asp Ser Ala Trp Gln Gln Val Asp Leu 85 90 95Pro His Asp Tyr Ser Ile Thr Gln Lys Tyr Ser Gln Ser Asn Glu Ala 100 105 110Glu Ser Ala Tyr Leu Pro Gly Gly Thr Gly Trp Tyr Arg Lys Ser Phe 115 120 125Thr Ile Asp Arg Asp Leu Ala Gly Lys Arg Ile Ala Ile Asn Phe Asp 130 135 140Gly Val Tyr Met Asn Ala Thr Val Trp Phe Asn Gly Val Lys Leu Gly145 150 155 160Thr His Pro Tyr Gly Tyr Ser Pro Phe Ser Phe Asp Leu Thr Gly Asn 165 170 175Ala Lys Phe Gly Gly Glu Asn Thr Ile Val Val Lys Val Glu Asn Arg 180 185 190Leu Pro Ser Ser Arg Trp Tyr Ser Gly Ser Gly Ile Tyr Arg Asp Val 195 200 205Thr Leu Thr Val Thr Asp Gly Val His Val Gly Asn Asn Gly Val Ala 210 215 220Ile Lys Thr Pro Ser Leu Ala Thr Gln Asn Gly Gly Asp Val Thr Met225 230 235 240Asn Leu Thr Thr Lys Val Ala Asn Asp Thr Glu Ala Ala Ala Asn Ile 245 250 255Thr Leu Lys Gln Thr Val Phe Pro Lys Gly Gly Lys Thr Asp Ala Ala 260 265 270Ile Gly Thr Val Thr Thr Ala Ser Lys Ser Ile Ala Ala Gly Ala Ser 275 280 285Ala Asp Val Thr Ser Thr Ile Thr Ala Ala Ser Pro Lys Leu Trp Ser 290 295 300Ile Lys Asn Pro Asn Leu Tyr Thr Val Arg Thr Glu Val Leu Asn Gly305 310 315 320Gly Lys Val Leu Asp Thr Tyr Asp Thr Glu Tyr Gly Phe Arg Trp Thr 325 330 335Gly Phe Asp Ala Thr Ser Gly Phe Ser Leu Asn Gly Glu Lys Val Lys 340 345 350Leu Lys Gly Val Ser Met His His Asp Gln Gly Ser Leu Gly Ala Val 355 360 365Ala Asn Arg Arg Ala Ile Glu Arg Gln Val Glu Ile Leu Gln Lys Met 370 375 380Gly Val Asn Ser Ile Arg Thr Thr His Asn Pro Ala Ala Lys Ala Leu385 390 395 400Ile Asp Val Cys Asn Glu Lys Gly Val Leu Val Val Glu Glu Val Phe 405 410 415Asp Met Trp Asn Arg Ser Lys Asn Gly Asn Thr Glu Asp Tyr Gly Lys 420 425 430Trp Phe Gly Gln Ala Ile Ala Gly Asp Asn Ala Val Leu Gly Gly Asp 435 440 445Lys Asp Glu Thr Trp Ala Lys Phe Asp Leu Thr Ser Thr Ile Asn Arg 450 455 460Asp Arg Asn Ala Pro Ser Val Ile Met Trp Ser Leu Gly Asn Glu Met465 470 475 480Met Glu Gly Ile Ser Gly Ser Val Ser Gly Phe Pro Ala Thr Ser Ala 485 490 495Lys Leu Val Ala Trp Thr Lys Ala Ala Asp Ser Thr Arg Pro Met Thr 500 505 510Tyr Gly Asp Asn Lys Ile Lys Ala Asn Trp Asn Glu Ser Asn Thr Met 515 520 525Gly Asp Asn Leu Thr Ala Asn Gly Gly Val Val Gly Thr Asn Tyr Ser 530 535 540Asp Gly Ala Asn Tyr Asp Lys Ile Arg Thr Thr His Pro Ser Trp Ala545 550 555 560Ile Tyr Gly Ser Glu Thr Ala Ser Ala Ile Asn Ser Arg Gly Ile Tyr 565 570 575Asn Arg Thr Thr Gly Gly Ala Gln Ser Ser Asp Lys Gln Leu Thr Ser 580 585 590Tyr Asp Asn Ser Ala Val Gly Trp Gly Ala Val Ala Ser Ser Ala Trp 595 600 605Tyr Asp Val Val Gln Arg Asp Phe Val Ala Gly Thr Tyr Val Trp Thr 610 615 620Gly Phe Asp Tyr Leu Gly Glu Pro Thr Pro Trp Asn Gly Thr Gly Ser625 630 635 640Gly Ala Val Gly Ser Trp Pro Ser Pro Lys Asn Ser Tyr Phe Gly Ile 645 650 655Val Asp Thr Ala Gly Phe Pro Lys Asp Thr Tyr Tyr Phe Tyr Gln Ser 660 665 670Gln Trp Asn Asp Asp Val His Thr Leu His Ile Leu Pro Ala Trp Asn 675 680 685Glu Asn Val Val Ala Lys Gly Ser Gly Asn Asn Val Pro Val Val Val 690 695 700Tyr Thr Asp Ala Ala Lys Val Lys Leu Tyr Phe Thr Pro Lys Gly Ser705 710 715 720Thr Glu Lys Arg Leu Ile Gly Glu Lys Ser Phe Thr Lys Lys Thr Thr 725 730 735Ala Ala Gly Tyr Thr Tyr Gln Val Tyr Glu Gly Ser Asp Lys Asp Ser 740 745 750Thr Ala His Lys Asn Met Tyr Leu Thr Trp Asn Val Pro Trp Ala Glu 755 760 765Gly Thr Ile Ser Ala Glu Ala Tyr Asp Glu Asn Asn Arg Leu Ile Pro 770 775 780Glu Gly Ser Thr Glu Gly Asn Ala Ser Val Thr Thr Thr Gly Lys Ala785 790 795 800Ala Lys Leu Lys Ala Asp Ala Asp Arg Lys Thr Ile Thr Ala Asp Gly 805 810 815Lys Asp Leu Ser Tyr Ile Glu Val Asp Val Thr Asp Ala Asn Gly His 820 825 830Ile Val Pro Asp Ala Ala Asn Arg Val Thr Phe Asp Val Lys Gly Ala 835 840 845Gly Lys Leu Val Gly Val Asp Asn Gly Ser Ser Pro Asp His Asp Ser 850 855 860Tyr Gln Ala Asp Asn Arg Lys Ala Phe Ser Gly Lys Val Leu Ala Ile865 870 875 880Val Gln Ser Thr Lys Glu Ala Gly Glu Ile Thr Val Thr Ala Lys Ala 885 890 895Asp Gly Leu Gln Ser Ser Thr Val Lys Ile Ala Thr Thr Ala Val Pro 900 905 910Gly Thr Ser Thr Glu Lys Thr Val Arg Ser Phe Tyr Tyr Ser Arg Asn 915 920 925Tyr Tyr Val Lys Thr Gly Asn Lys Pro Ile Leu Pro Ser Asp Val Glu 930 935 940Val Arg Tyr Ser Asp Gly Thr Ser Asp Arg Gln Asn Val Thr Trp Asp945 950 955 960Ala Val Ser Asp Asp Gln Ile Ala Lys Ala Gly Ser Phe Ser Val Ala 965 970 975Gly Thr Val Ala Gly Gln Lys Ile Ser Val Arg Val Thr Met Ile Asp 980 985 990Glu Ile Gly Ala Leu Leu Asn Tyr Ser Ala Ser Thr Pro Val Gly Thr 995 1000 1005Pro Ala Val Leu Pro Gly Ser Arg Pro Ala Val Leu Pro Asp Gly 1010 1015 1020Thr Val Thr Ser Ala Asn Phe Ala Val His Trp Thr Lys Pro Ala 1025 1030 1035Asp Thr Val Tyr Asn Thr Ala Gly Thr Val Lys Val Pro Gly Thr 1040 1045 1050Ala Thr Val Phe Gly Lys Glu Phe Lys Val Thr Ala Thr Ile Arg 1055 1060 1065Val Gln Arg Ser Gln Val Thr Ile Gly Ser Ser Val Ser Gly Asn 1070 1075 1080Ala Leu Arg Leu Thr Gln Asn Ile Pro Ala Asp Lys Gln Ser Asp 1085 1090 1095Thr Leu Asp Ala Ile Lys Asp Gly Ser Thr Thr Val Asp Ala Asn 1100 1105 1110Thr Gly Gly Gly Ala Asn Pro Ser Ala Trp Thr Asn Trp Ala Tyr 1115 1120 1125Ser Lys Ala Gly His Asn Thr Ala Glu Ile Thr Phe Glu Tyr Ala 1130 1135 1140Thr Glu Gln Gln Leu Gly Gln Ile Val Met Tyr Phe Phe Arg Asp 1145 1150 1155Ser Asn Ala Val Arg Phe Pro Asp Ala Gly Lys Thr Lys Ile Gln 1160 1165 1170Ile Ser Ala Asp Gly Lys Asn Trp Thr Asp Leu Ala Ala Thr Glu 1175 1180 1185Thr Ile Ala Ala Gln Glu Ser Ser Asp Arg Val Lys Pro Tyr Thr 1190 1195 1200Tyr Asp Phe Ala Pro Val Gly Ala Thr Phe Val Lys Val Thr Val 1205 1210 1215Thr Asn Ala Asp Thr Thr Thr Pro Ser Gly Val Val Cys Ala Gly 1220 1225 1230Leu Thr Glu Ile Glu Leu Lys Thr Ala Thr Ser Lys Phe Val Thr 1235 1240 1245Asn Thr Ser Ala Ala Leu Ser Ser Leu Thr Val Asn Gly Thr Lys 1250 1255 1260Val Ser Asp Ser Val Leu Ala Ala Gly Ser Tyr Asn Thr Pro Ala 1265 1270 1275Ile Ile Ala Asp Val Lys Ala Glu Gly Glu Gly Asn Ala Ser Val 1280 1285 1290Thr Val Leu Pro Ala His Asp Asn Val Ile Arg Val Ile Thr Glu 1295 1300 1305Ser Glu Asp His Val Thr Arg Lys Thr Phe Thr Ile Asn Leu Gly 1310 1315 1320Thr Glu Gln Glu Phe Pro Ala Asp Ser Asp Glu Arg Asp Tyr Pro 1325 1330 1335Ala Ala Asp Met Thr Val Thr Val Gly Ser Glu Gln Thr Ser Gly 1340 1345 1350Thr Ala Thr Glu Gly Pro Lys Lys Phe Ala Val Asp Gly Asn Thr 1355 1360 1365Ser Thr Tyr Trp His Ser Asn Trp Thr Pro Thr Thr Val Asn Asp 1370 1375 1380Leu Trp Ile Ala Phe Glu Leu Gln Lys Pro Thr Lys Leu Asp Ala 1385 1390 1395Leu Arg Tyr Leu Pro Arg Pro Ala Gly Ser Lys Asn Gly Ser Val 1400 1405 1410Thr Glu Tyr Lys Val Gln Val Ser Asp Asp Gly Thr Asn Trp Thr 1415 1420 1425Asp Ala Gly Ser Gly Thr Trp Thr Thr Asp Tyr Gly Trp Lys Leu 1430 1435 1440Ala Glu Phe Asn Gln Pro Val Thr Thr Lys His Val Arg Leu Lys 1445 1450 1455Ala Val His Thr Tyr Ala Asp Ser Gly Asn Asp Lys Phe Met Ser 1460 1465 1470Ala Ser Glu Ile Arg Leu Arg Lys Ala Val Asp Thr Thr Asp Ile 1475 1480 1485Ser Gly Ala Thr Val Thr Val Pro Ala Lys Leu Thr Val Asp Arg 1490 1495 1500Val Asp Ala Asp His Pro Ala Thr Phe Ala Thr Lys Asp Val Thr 1505 1510 1515Val Thr Leu Gly Asp Ala Thr Leu Arg Tyr Gly Val Asp Tyr Leu 1520 1525 1530Leu Asp Tyr Ala Gly Asn Thr Ala Val Gly Lys Ala Thr Val Thr 1535 1540 1545Val Arg Gly Ile Asp Lys Tyr Ser Gly Thr Val Ala Lys Thr Phe 1550 1555 1560Thr Ile Glu Leu Lys Asn Ala Pro Ala Pro Glu Pro Thr Leu Thr 1565 1570 1575Ser Val Ser Val Lys Thr Lys Pro Ser Lys Leu Thr Tyr Val Val 1580 1585 1590Gly Asp Ala Phe Asp Pro Ala Gly Leu Val Leu Gln His Asp Arg 1595 1600 1605Gln Ala Asp Arg Pro Pro Gln Pro Leu Val Gly Glu Gln Ala Asp 1610 1615 1620Glu Arg Gly Leu Thr Cys Gly Thr Arg Cys Asp Arg Val Glu

Gln 1625 1630 1635Leu Arg Lys His Glu Asn Arg Glu Ala His Arg Thr Gly Leu Asp 1640 1645 1650His Leu Glu Phe Val Gly Ala Ala Asp Gly Ala Val Gly Glu Gln 1655 1660 1665Ala Thr Phe Lys Val His Val His Ala Asp Gln Gly Asp Gly Arg 1670 1675 1680His Asp Asp Ala Asp Glu Arg Asp Ile Asp Pro His Val Pro Val 1685 1690 1695Asp His Ala Val Gly Glu Leu Ala Arg Ala Ala Cys His His Val 1700 1705 1710Ile Gly Leu Arg Val Asp Thr His Arg Leu Lys Ala Ser Gly Phe 1715 1720 1725Gln Ile Pro Ala Asp Asp Met Ala Glu Ile Asp Arg Ile Thr Gly 1730 1735 1740Phe His Arg Phe Glu Arg His Val Gly 1745 175041935PRTBifidobacterium bifidum 4Met Ala Val Arg Arg Leu Gly Gly Arg Ile Val Ala Phe Ala Ala Thr1 5 10 15Val Ala Leu Ser Ile Pro Leu Gly Leu Leu Thr Asn Ser Ala Trp Ala 20 25 30Val Glu Asp Ala Thr Arg Ser Asp Ser Thr Thr Gln Met Ser Ser Thr 35 40 45Pro Glu Val Val Tyr Ser Ser Ala Val Asp Ser Lys Gln Asn Arg Thr 50 55 60Ser Asp Phe Asp Ala Asn Trp Lys Phe Met Leu Ser Asp Ser Val Gln65 70 75 80Ala Gln Asp Pro Ala Phe Asp Asp Ser Ala Trp Gln Gln Val Asp Leu 85 90 95Pro His Asp Tyr Ser Ile Thr Gln Lys Tyr Ser Gln Ser Asn Glu Ala 100 105 110Glu Ser Ala Tyr Leu Pro Gly Gly Thr Gly Trp Tyr Arg Lys Ser Phe 115 120 125Thr Ile Asp Arg Asp Leu Ala Gly Lys Arg Ile Ala Ile Asn Phe Asp 130 135 140Gly Val Tyr Met Asn Ala Thr Val Trp Phe Asn Gly Val Lys Leu Gly145 150 155 160Thr His Pro Tyr Gly Tyr Ser Pro Phe Ser Phe Asp Leu Thr Gly Asn 165 170 175Ala Lys Phe Gly Gly Glu Asn Thr Ile Val Val Lys Val Glu Asn Arg 180 185 190Leu Pro Ser Ser Arg Trp Tyr Ser Gly Ser Gly Ile Tyr Arg Asp Val 195 200 205Thr Leu Thr Val Thr Asp Gly Val His Val Gly Asn Asn Gly Val Ala 210 215 220Ile Lys Thr Pro Ser Leu Ala Thr Gln Asn Gly Gly Asn Val Thr Met225 230 235 240Asn Leu Thr Thr Lys Val Ala Asn Asp Thr Lys Ala Ala Ala Asn Ile 245 250 255Thr Leu Lys Gln Thr Val Phe Pro Lys Gly Gly Lys Thr Asp Ala Ala 260 265 270Ile Gly Thr Val Thr Thr Ala Ser Lys Ser Ile Ala Ala Gly Ala Ser 275 280 285Ala Asp Val Thr Ser Thr Ile Thr Ala Ala Ser Pro Lys Leu Trp Ser 290 295 300Ile Lys Asn Pro Asn Leu Tyr Thr Val Arg Thr Glu Val Leu Asn Gly305 310 315 320Gly Lys Val Leu Asp Thr Tyr Asp Thr Glu Tyr Gly Phe Arg Trp Thr 325 330 335Gly Phe Asp Ala Thr Ser Gly Phe Ser Leu Asn Gly Glu Lys Val Lys 340 345 350Leu Lys Gly Val Ser Met His His Asp Gln Gly Ser Leu Gly Ala Val 355 360 365Ala Asn Arg Arg Ala Ile Glu Arg Gln Val Glu Ile Leu Gln Lys Met 370 375 380Gly Val Asn Ser Ile Arg Thr Thr His Asn Pro Ala Ala Lys Ala Leu385 390 395 400Ile Asp Val Cys Asn Glu Lys Gly Val Leu Val Val Glu Glu Val Phe 405 410 415Asp Met Trp Asn Arg Ser Lys Asn Gly Asn Thr Glu Asp Tyr Gly Lys 420 425 430Trp Phe Gly Gln Ala Ile Ala Gly Asp Asn Ala Val Leu Gly Gly Asp 435 440 445Lys Asp Glu Thr Trp Ala Lys Phe Asp Leu Thr Ser Thr Ile Asn Arg 450 455 460Asp Arg Asn Ala Pro Ser Val Ile Met Trp Ser Leu Gly Asn Glu Met465 470 475 480Met Glu Gly Ile Ser Gly Ser Val Ser Gly Phe Pro Ala Thr Ser Ala 485 490 495Lys Leu Val Ala Trp Thr Lys Ala Ala Asp Ser Thr Arg Pro Met Thr 500 505 510Tyr Gly Asp Asn Lys Ile Lys Ala Asn Trp Asn Glu Ser Asn Thr Met 515 520 525Gly Asp Asn Leu Thr Ala Asn Gly Gly Val Val Gly Thr Asn Tyr Ser 530 535 540Asp Gly Ala Asn Tyr Asp Lys Ile Arg Thr Thr His Pro Ser Trp Ala545 550 555 560Ile Tyr Gly Ser Glu Thr Ala Ser Ala Ile Asn Ser Arg Gly Ile Tyr 565 570 575Asn Arg Thr Thr Gly Gly Ala Gln Ser Ser Asp Lys Gln Leu Thr Ser 580 585 590Tyr Asp Asn Ser Ala Val Gly Trp Gly Ala Val Ala Ser Ser Ala Trp 595 600 605Tyr Asp Val Val Gln Arg Asp Phe Val Ala Gly Thr Tyr Val Trp Thr 610 615 620Gly Phe Asp Tyr Leu Gly Glu Pro Thr Pro Trp Asn Gly Thr Gly Ser625 630 635 640Gly Ala Val Gly Ser Trp Pro Ser Pro Lys Asn Ser Tyr Phe Gly Ile 645 650 655Val Asp Thr Ala Gly Phe Pro Lys Asp Thr Tyr Tyr Phe Tyr Gln Ser 660 665 670Gln Trp Asn Asp Asp Val His Thr Leu His Ile Leu Pro Ala Trp Asn 675 680 685Glu Asn Val Val Ala Lys Gly Ser Gly Asn Asn Val Pro Val Val Val 690 695 700Tyr Thr Asp Ala Ala Lys Val Lys Leu Tyr Phe Thr Pro Lys Gly Ser705 710 715 720Thr Glu Lys Arg Leu Ile Gly Glu Lys Ser Phe Thr Lys Lys Thr Thr 725 730 735Ala Ala Gly Tyr Thr Tyr Gln Val Tyr Glu Gly Ala Asp Lys Asp Ser 740 745 750Thr Ala His Lys Asn Met Tyr Leu Thr Trp Asn Val Pro Trp Ala Glu 755 760 765Gly Thr Ile Ser Ala Glu Ala Tyr Asp Glu Asn Asn Arg Leu Ile Pro 770 775 780Glu Gly Ser Thr Glu Gly Asn Ala Ser Val Thr Thr Thr Gly Lys Ala785 790 795 800Ala Lys Leu Lys Ala Asp Ala Asp Arg Lys Thr Ile Thr Ala Asp Gly 805 810 815Lys Asp Leu Ser Tyr Ile Glu Val Asp Val Thr Asp Ala Asn Gly His 820 825 830Ile Val Pro Asp Ala Ala Asn Arg Val Thr Phe Asp Val Lys Gly Ala 835 840 845Gly Lys Leu Val Gly Val Asp Asn Gly Ser Ser Pro Asp His Asp Ser 850 855 860Tyr Gln Ala Asp Asn Arg Lys Ala Phe Ser Gly Lys Val Leu Ala Ile865 870 875 880Val Gln Ser Thr Lys Glu Ala Gly Glu Ile Thr Val Thr Ala Lys Ala 885 890 895Asp Gly Leu Gln Ser Ser Thr Val Lys Ile Ala Thr Thr Ala Val Pro 900 905 910Gly Thr Ser Thr Glu Lys Thr Val Arg Ser Phe Tyr Tyr Ser Arg Asn 915 920 925Tyr Tyr Val Lys Thr Gly Asn Lys Pro Ile Leu Pro Ser Asp Val Glu 930 935 940Val Arg Tyr Ser Asp Gly Thr Ser Asp Arg Gln Asn Val Thr Trp Asp945 950 955 960Ala Val Ser Asp Asp Gln Ile Ala Lys Ala Gly Ser Phe Ser Val Ala 965 970 975Gly Thr Val Ala Gly Gln Lys Ile Ser Val Arg Val Thr Met Ile Asp 980 985 990Glu Ile Gly Ala Leu Leu Asn Tyr Ser Ala Ser Thr Pro Val Gly Thr 995 1000 1005Pro Ala Val Leu Pro Gly Ser Arg Pro Ala Val Leu Pro Asp Gly 1010 1015 1020Thr Val Thr Ser Ala Asn Phe Ala Val Asp Trp Thr Lys Pro Ala 1025 1030 1035Asp Thr Val Tyr Asn Thr Ala Gly Thr Val Lys Val Pro Gly Thr 1040 1045 1050Ala Thr Val Phe Gly Lys Glu Phe Lys Val Thr Ala Thr Ile Arg 1055 1060 1065Val Gln Arg Ser Gln Val Thr Ile Gly Ser Ser Val Ser Gly Asn 1070 1075 1080Ala Leu Arg Leu Thr Gln Asn Ile Pro Ala Asp Lys Gln Ser Asp 1085 1090 1095Thr Leu Asp Ala Ile Lys Asp Gly Ser Thr Thr Val Asp Ala Asn 1100 1105 1110Thr Gly Gly Gly Ala Asn Pro Ser Ala Trp Thr Asn Trp Ala Tyr 1115 1120 1125Ser Lys Ala Gly His Asn Thr Ala Glu Ile Thr Phe Glu Tyr Ala 1130 1135 1140Thr Glu Gln Gln Leu Gly Gln Ile Val Met Tyr Phe Phe Arg Asp 1145 1150 1155Ser Asn Ala Val Arg Phe Pro Asp Ala Gly Lys Thr Lys Ile Gln 1160 1165 1170Ile Ser Ala Asp Gly Lys Asn Trp Thr Asp Leu Ala Ala Thr Glu 1175 1180 1185Thr Ile Ala Ala Gln Glu Ser Ser Asp Arg Val Lys Pro Tyr Thr 1190 1195 1200Tyr Asp Phe Ala Pro Val Gly Ala Thr Phe Val Lys Val Thr Val 1205 1210 1215Thr Asn Ala Asp Thr Thr Thr Pro Ser Gly Val Val Cys Ala Gly 1220 1225 1230Leu Thr Glu Ile Glu Leu Lys Thr Ala Thr Ser Lys Phe Val Thr 1235 1240 1245Asn Thr Ser Ala Ala Leu Ser Ser Leu Thr Val Asn Gly Thr Lys 1250 1255 1260Val Ser Asp Ser Val Leu Ala Ala Gly Ser Tyr Asn Thr Pro Ala 1265 1270 1275Ile Ile Ala Asp Val Lys Ala Glu Gly Glu Gly Asn Ala Ser Val 1280 1285 1290Thr Val Leu Pro Ala His Asp Asn Val Ile Arg Val Ile Thr Glu 1295 1300 1305Ser Glu Asp His Val Thr Arg Lys Thr Phe Thr Ile Asn Leu Gly 1310 1315 1320Thr Glu Gln Glu Phe Pro Ala Asp Ser Asp Glu Arg Asp Tyr Pro 1325 1330 1335Ala Ala Asp Met Thr Val Thr Ala Gly Ser Glu Gln Thr Ser Gly 1340 1345 1350Thr Ala Thr Glu Gly Pro Lys Lys Phe Ala Val Asp Gly Asn Thr 1355 1360 1365Ser Thr Tyr Trp His Ser Asn Trp Thr Pro Thr Thr Val Asn Asp 1370 1375 1380Leu Trp Ile Ala Phe Glu Leu Gln Lys Pro Thr Lys Leu Asp Ala 1385 1390 1395Leu Arg Tyr Leu Pro Arg Pro Ala Gly Ser Lys Asn Gly Ser Val 1400 1405 1410Thr Glu Tyr Lys Val Gln Val Ser Asp Asp Gly Thr Asn Trp Thr 1415 1420 1425Asp Ala Gly Ser Gly Thr Trp Thr Thr Asp Tyr Gly Trp Lys Leu 1430 1435 1440Ala Glu Phe Asn Gln Pro Val Thr Thr Lys His Val Arg Leu Lys 1445 1450 1455Ala Val His Thr Tyr Ala Asp Ser Gly Asn Asp Lys Phe Met Ser 1460 1465 1470Ala Ser Glu Ile Arg Leu Arg Lys Ala Val Asp Thr Thr Asp Ile 1475 1480 1485Ser Gly Ala Thr Val Thr Val Pro Ala Lys Leu Thr Val Asp Arg 1490 1495 1500Val Asp Ala Asp His Pro Ala Thr Phe Ala Thr Lys Asp Val Thr 1505 1510 1515Val Thr Leu Gly Asp Ala Thr Leu Arg Tyr Gly Val Asp Tyr Leu 1520 1525 1530Leu Asp Tyr Ala Gly Asn Thr Ala Val Gly Lys Ala Thr Val Thr 1535 1540 1545Val Arg Gly Ile Asp Lys Tyr Ser Gly Thr Val Ala Lys Thr Phe 1550 1555 1560Thr Ile Glu Leu Lys Asn Ala Pro Ala Pro Glu Pro Thr Leu Thr 1565 1570 1575Ser Val Ser Val Lys Thr Lys Pro Ser Lys Leu Thr Tyr Val Val 1580 1585 1590Gly Asp Ala Phe Asp Pro Ala Gly Leu Val Leu Gln Leu Asn Tyr 1595 1600 1605Asp Asp Asp Ser Thr Gly Thr Val Thr Trp Asn Thr Gln Thr Ala 1610 1615 1620Gly Asp Phe Thr Phe Lys Pro Ala Leu Asp Ala Lys Leu Lys Val 1625 1630 1635Thr Asp Lys Thr Val Thr Val Thr Tyr Gln Gly Lys Ser Ala Val 1640 1645 1650Ile Asp Ile Thr Val Ser Gln Pro Ala Pro Thr Val Ser Lys Thr 1655 1660 1665Asp Leu Asp Lys Ala Ile Lys Ala Ile Glu Ala Lys Asn Pro Asp 1670 1675 1680Ser Ser Lys Tyr Thr Ala Asp Ser Trp Lys Thr Phe Ala Asp Ala 1685 1690 1695Met Ala His Ala Lys Ala Val Ile Ala Asp Asp Ser Ala Thr Gln 1700 1705 1710Gln Asp Val Asp Lys Ala Leu Lys Ala Leu Thr Asp Ala Tyr Ala 1715 1720 1725Gly Leu Thr Glu Lys Thr Pro Glu Pro Ala Pro Val Ser Lys Ser 1730 1735 1740Glu Leu Asp Lys Lys Ile Lys Ala Ile Glu Ala Glu Lys Leu Asp 1745 1750 1755Gly Ser Lys Tyr Thr Ala Glu Ser Trp Lys Ala Phe Glu Thr Ala 1760 1765 1770Leu Ala His Ala Lys Ala Val Ile Ala Ser Asp Ser Ala Thr Gln 1775 1780 1785Gln Asp Val Asp Ala Ala Leu Gly Ala Leu Thr Ser Ala Arg Asp 1790 1795 1800Gly Leu Thr Glu Lys Gly Glu Val Lys Pro Asp Pro Lys Pro Glu 1805 1810 1815Pro Gly Thr Val Asp Lys Ala Ala Leu Asp Lys Ala Val Lys Lys 1820 1825 1830Val Glu Ala Glu Lys Leu Asp Gly Ser Lys Tyr Thr Ala Asp Ser 1835 1840 1845Trp Lys Ala Phe Glu Thr Ala Leu Ala His Ala Lys Ala Val Ile 1850 1855 1860Gly Asn Ala Asn Ser Thr Gln Phe Asp Ile Asp Asn Ala Leu Ser 1865 1870 1875Met Leu Asn Asp Ala Arg Ala Ala Leu Lys Glu Lys Pro Gly Arg 1880 1885 1890Ile Ile Ala Ile Ile Asp Gly Gly Ala Leu Ser Lys Thr Gly Ala 1895 1900 1905Ser Val Ala Ile Ile Ala Ser Val Ala Ala Ala Met Lys Ala Val 1910 1915 1920Gly Ala Gly Val Met Ala Leu Arg Pro Pro Lys Trp 1925 1930 193552021PRTRuminococcus torques 5Met Lys Asn Leu Lys Trp Lys Lys Ala Gly Ser Ala Val Leu Ala Thr1 5 10 15Ala Leu Ala Gly Ser Met Val Leu Pro Ala Thr Ala Tyr Ala Gln Gly 20 25 30Glu Ile Val Gln Leu Glu Gly Gly Thr Ser Thr Gln Thr Asn Thr Ala 35 40 45Pro Glu Gln Val Phe Leu Asn Lys Tyr Ser Gly Thr Val Arg Thr Gln 50 55 60Asn Phe Asn Asp Asn Trp Lys Phe Tyr Leu Gly Asp Ala Ser Gly Ala65 70 75 80Gln Thr Pro Ala Phe Asp Asp Ser Ser Trp Asp Gln Val Asn Leu Pro 85 90 95His Asp Tyr Ser Ile Asp Gln Lys Tyr Ser Gln Lys Met Glu Ala Glu 100 105 110Ser Gly Tyr Leu Pro Gly Gly Thr Gly Trp Tyr Arg Lys Asn Phe Thr 115 120 125Val Asp Glu Ser Leu Lys Gly Lys Arg Ile Ser Ile Asp Phe Gly Gly 130 135 140Val Tyr Met Asn Ala Thr Ile Tyr Val Asn Gly Lys Lys Leu Gly Thr145 150 155 160His Pro Asn Gly Tyr Thr Pro Phe Ser Phe Asp Ile Thr Asp Asn Val 165 170 175Lys Phe Gly Lys Glu Asn Val Ile Ala Val Lys Val Asp His Gln Thr 180 185 190Pro Ser Ser Arg Phe Tyr Ser Gly Ser Gly Ile Tyr Arg Asp Val Asp 195 200 205Phe Val Val Thr Asp Thr Val His Val Asp Lys Asn Gly Thr Lys Ile 210 215 220Glu Thr Pro Asp Leu Lys Asp His Ala Asp Gly Asn Asn Val Ala Val225 230 235 240Lys Val Lys Thr Thr Val Val Asn Glu Ser Glu Asn Asn Ala Ser Val 245 250 255Lys Val Lys His Thr Ile Tyr Pro Lys Asn Gly Thr Ala Glu Gln Ala 260 265 270Val Gly Thr Phe Glu Thr Glu Val Ala Thr Val Asp Lys Gly Lys Ser 275 280 285Lys Asp Val Gln Ala Asp Phe Thr Val Ser Gly Val Lys Leu Trp Ser 290 295 300Thr Thr Thr Pro Asn Leu Tyr Thr Val Lys Thr Glu Val Leu Met Asp305 310 315 320Gly Thr Thr Val Asp Thr Tyr Glu Thr Asp Tyr Gly Phe Arg Tyr Phe 325 330 335Asp Phe Asn Asn Asn Thr Gly Phe Ser Leu Asn Gly Gln Lys Met Lys 340 345 350Leu Gln Gly Val Cys Met His His Asp Gln Gly Ala Leu Gly Ser Val 355 360 365Ala Asn Asp Arg Ser Thr Glu Arg Gln Val Glu Ile Leu Lys Met Met 370 375 380Gly Cys Asn Ser Ile Arg Val Thr His Asn Pro Ala Ser Asp Glu Leu385

390 395 400Ile Asp Ala Cys Asn Lys His Gly Ile Leu Val Ile Asp Glu Ala Phe 405 410 415Asp Gly Trp Val Ala Pro Lys Asn Ser Asn Ser Asn Asp Tyr Ser Lys 420 425 430Trp Phe Asn Lys Lys Ile Glu Asp Gly Asn Glu Ile Met Gly Ala Ala 435 440 445Glu Asn Met Thr Trp Ala Gln Phe Asp Leu Thr Ala Met Ile Glu Arg 450 455 460Gly Gln Asn Asp Pro Ala Ile Ile Met Trp Ser Leu Gly Asn Glu Met465 470 475 480Trp Glu Gly Thr Gly Gly Tyr Ser Asp Asp Tyr Lys Thr Ala Gln Asp 485 490 495Asn Leu Val Lys Trp Ala Lys Ala Ala Asp Thr Thr Arg Pro Val Thr 500 505 510Thr Gly Asp Asn Lys Leu Lys Ser Asn Glu Thr Gly Ala Ile Thr Leu 515 520 525Gly Gln Glu Leu Gln Lys Ala Gly Gly Ile His Gly Met Asn Tyr Ser 530 535 540Gln Glu Trp Lys Asn His Ala Gly Lys Thr His Tyr Asp Met Ile His545 550 555 560Glu Ala Tyr Pro Glu Trp Cys Met Tyr Gly Ser Glu Thr Ala Ser Ala 565 570 575Val Asn Ser Arg Gly Ile Tyr Lys Gly Met Gly Ser Gln Thr Asp Tyr 580 585 590Gly Asp Tyr Asp Leu Thr Ser Tyr Asp Thr Ser Ala Val Gly Trp Gly 595 600 605Ala Thr Ala Ser Ser Ala Trp Tyr Glu Val Ile Lys Arg Asp Phe Ile 610 615 620Ala Gly Glu Tyr Val Trp Thr Gly Phe Asp Tyr Ile Gly Glu Pro Thr625 630 635 640Pro Trp Asn Gly Thr Gly Gln Gly Lys Pro Gly Asn Ala Ser Arg Trp 645 650 655Pro Ala Pro Lys Ser Ser Tyr Phe Gly Ile Val Asp Thr Ala Gly Leu 660 665 670Pro Lys Asp Ser Tyr Tyr Phe Tyr Gln Ser Gln Trp Asn Asp Ser Val 675 680 685Asn Thr Leu His Ile Leu Pro Ala Trp Asn Glu Glu Val Val Tyr Lys 690 695 700Lys Ser Gly Asn Asp Val Pro Val Val Val Tyr Ser Asp Ala Lys Lys705 710 715 720Val Glu Leu Phe Phe Thr Pro Ala Ser Gly Gly Glu Gln Arg Ser Leu 725 730 735Gly Ala Lys Glu Phe Thr Glu Lys Lys Thr Thr Ala Gly Tyr Thr Tyr 740 745 750Gln Met Tyr Glu Gly Thr Gly Lys Ser Asn Thr Glu His Glu Asn Leu 755 760 765Tyr Met Thr Trp Met Val Pro Tyr Glu Ala Gly Thr Ile Thr Ala Lys 770 775 780Ala Trp Asp Lys Asp Gly Lys Glu Ile Thr Glu Asn Leu Gln Gly Arg785 790 795 800Thr Ser Val Thr Thr Ala Gly Glu Ala Lys Lys Leu Lys Val Asp Val 805 810 815Asp Arg Thr Lys Ile Thr Ala Asn Gly Glu Asp Leu Ser Tyr Leu Thr 820 825 830Val Ser Val Thr Asp Asp Lys Gly Asn Leu Val Pro Asn Ala Asp Asn 835 840 845Lys Val Thr Phe Glu Val Ser Gly Asp Gly Val Leu Ala Gly Val Asp 850 855 860Asn Gly Arg Pro Val Asp His Gln Ser Tyr Arg Asp Asp Asn Arg Lys865 870 875 880Ala Phe Ser Gly Gln Leu Val Gly Ile Val Gln Ser Thr Lys Ser Ala 885 890 895Gly Thr Ile Thr Val Lys Val Lys Ala Glu Gly Met Glu Asp Gln Thr 900 905 910Val Thr Ile Thr Thr Thr Pro Ser Ser Asp Ser Ser Glu Ser Lys Lys 915 920 925Ala Ile Ser Ser Val Lys Met Ser Lys Ser Tyr Tyr Val Lys Val Gly 930 935 940Asn Gln Pro Gln Leu Pro Gly Gln Val Glu Val Val Leu Thr Asp Lys945 950 955 960Thr Lys Thr Thr Gly Thr Val Thr Trp Glu Lys Ala Thr Ala Glu Gln 965 970 975Ile Gly Gln Ala Gly Thr Phe Ser Leu Thr Gly Thr Val Ser Val Glu 980 985 990Gly Val Glu Lys Ala Glu Thr Val Ser Val Asn Val Asn Met Ile Asp 995 1000 1005Thr Val Ala Ala Leu Leu Asn Tyr Ser Thr Thr Thr Ser Val Gly 1010 1015 1020Val Ala Pro Ser Leu Pro Thr Ser Arg Pro Ala Val Met Glu Asp 1025 1030 1035Gly Thr Val Leu Thr Ala Ala Phe Pro Val Lys Trp Glu Ala Pro 1040 1045 1050Glu Lys Gly Tyr Asp Ala Glu Gly Ile Val Asn Val Thr Gly Thr 1055 1060 1065Ala Asp Val Phe Gly Glu Ser Met Pro Val Thr Ala Thr Val Arg 1070 1075 1080Val Gln Glu Ala Glu Tyr Thr Val Gly Asn Asn Val Ala Lys Glu 1085 1090 1095Ala Met Thr Leu Ser Gln Asp Ile Pro Gln Glu Met Gln Ser Asp 1100 1105 1110Asp Leu Glu Ala Ile Arg Asp Gly Asn Arg Thr Val Asp Gly Asn 1115 1120 1125Gln Gly Gly Asn Thr Asn Ser Thr Met Trp Ser Asn Tyr Lys Asn 1130 1135 1140Ser Lys Asp Ala Lys Asp Asn Asp Ala Asp Ile Thr Phe Gln Tyr 1145 1150 1155Ala Thr Gln Gln Ile Phe Asn Gln Ile Lys Ile Phe Phe Arg Ser 1160 1165 1170Asp Ser His Ala Ala Ser Tyr Pro Ala Asp Asn Thr Thr Lys Ile 1175 1180 1185Tyr Val Ser Glu Thr Gly Glu Glu Gly Thr Trp Thr Glu Val Thr 1190 1195 1200Ala Thr Glu Ser His Pro Glu Glu Leu Pro Ala Ile Gly Val Val 1205 1210 1215Glu Tyr Thr Tyr Asp Phe Val Pro Thr Lys Ala Val Phe Val Lys 1220 1225 1230Ile His Val Val Asn Asn Pro Asp Ala Ser Gly Lys Gly Gly Gly 1235 1240 1245Phe Thr Cys Thr Gly Ile Val Glu Ala Glu Leu Tyr Leu Ala Asn 1250 1255 1260Gln Ala Asp Phe Thr Thr Asn Thr Thr Ala Lys Leu Glu Ser Leu 1265 1270 1275Lys Ile Asn Glu Thr Ser Ala Pro Ala Glu Val Leu Ala Ala Gly 1280 1285 1290Ala Gly Ser Trp Gly Thr Lys Glu Val Glu Ala Lys Thr Val Glu 1295 1300 1305Ala Val Gly Ala Asp Asn Ala Ala Val Thr Val Leu Pro Thr Tyr 1310 1315 1320Glu Asn Ala Val Arg Ile Ile Ile Glu Ser Glu Asp His Lys Thr 1325 1330 1335Thr Asn Thr Phe Val Val Asn Leu Asp Ala Asp Ala Thr Asp Asp 1340 1345 1350Ser Lys Asp Tyr Asp Lys Ala Lys Ile Thr Ser Thr Val Gly Ser 1355 1360 1365Ala Gln Ser Gly Asn Glu Lys Glu Lys Ala Phe Asp Gly Asp Thr 1370 1375 1380Asn Thr Leu Trp His Thr Gln Trp Asn Asn Thr Asn Pro Ala Glu 1385 1390 1395Arg Trp Ile Glu Met Glu Leu Glu Asp Val Gln Asn Val Ile Gly 1400 1405 1410Leu Arg Tyr Leu Pro Arg Gln Asn Gly Gly Gln Asn Gly Ile Val 1415 1420 1425Lys Thr Tyr Lys Ile Glu Val Lys Ala Ala Glu Gly Asp Glu Trp 1430 1435 1440Lys Glu Val Ala Val Thr Glu Gly Thr Lys Val Trp Ala Val Asp 1445 1450 1455Asn Thr Trp Lys Met Ala Lys Phe Glu Thr Pro Val Gln Ala Lys 1460 1465 1470Tyr Ile Arg Phe Ser Gly Val Glu Thr His Asp Asp Gln Gly Gly 1475 1480 1485Asn Lys Trp Met Ser Ala Ala Glu Ile Arg Val Lys Val Thr Lys 1490 1495 1500Glu Glu Val Val Pro Pro Thr Ala Thr Glu Leu Ser Leu Lys Ala 1505 1510 1515Gln Pro Thr Lys Thr Ala Tyr Ala Val Gly Glu Lys Phe Asp Pro 1520 1525 1530Ala Gly Leu Val Ile Gly Val Lys Tyr Ser Asp Gly Thr Glu Lys 1535 1540 1545Glu Val Ala Tyr Gly Gln Asp Asn Ala Gly Glu Phe Thr Phe Asn 1550 1555 1560Pro Thr Leu Ser Thr Ala Leu Thr Lys Asp Tyr Thr Lys Val Glu 1565 1570 1575Val Gly Tyr Ala Gly Leu Lys Leu Asp Val Asn Ile Thr Val Ser 1580 1585 1590Glu Ser Glu Pro Val Ile Pro Glu Ala Leu Glu Val Val Ser Ala 1595 1600 1605Pro Ala Lys Thr Glu Tyr Glu Glu Gly Glu Met Phe Asn Pro Ala 1610 1615 1620Gly Leu Ser Val Lys Ile Lys Tyr Ser Asp Gly Ser Tyr Gly Asp 1625 1630 1635Glu Val Ala Tyr Gly Thr Ala Asn Ala Asp Gln Phe Thr Phe Asn 1640 1645 1650Pro Thr Leu Asp Thr Ala Leu Lys Thr Ser Asp Glu Lys Val Thr 1655 1660 1665Val Thr Tyr Ala Glu Lys Thr Ala Asp Ile Lys Ile Lys Val Asn 1670 1675 1680Lys Lys Thr Pro Val Val Pro Glu Asn Pro Thr Val Glu Lys Val 1685 1690 1695Glu Ile Lys Ala Asn Pro Ala Lys Thr Glu Tyr Lys Glu Gly Asp 1700 1705 1710Lys Phe Asp Pro Thr Gly Leu Val Leu Thr Val Lys Tyr Asp Lys 1715 1720 1725Gly Glu Asp Lys Glu Val Ala Tyr Gly Asp Ala Thr Lys Ala Asp 1730 1735 1740Phe Thr Phe Ile Pro Ser Leu Asp Thr Ala Leu Lys Thr Ser Asp 1745 1750 1755Glu Lys Val Thr Val Thr Tyr Ala Gly Lys Thr Ala Glu Ile Gly 1760 1765 1770Ile Glu Val Lys Ala Asp Thr Pro Val Glu Pro Glu Lys Pro Thr 1775 1780 1785Val Asp Lys Ile Ala Val Lys Lys Val Pro Ala Lys Thr Thr Tyr 1790 1795 1800Lys Ala Gly Glu Thr Phe Asp Pro Ser Gly Leu Val Leu Thr Val 1805 1810 1815Thr Met Ser Asp Lys Thr Thr Lys Glu Val Ala Tyr Gly Asn Glu 1820 1825 1830Thr Ala Lys Asp Phe Val Phe Asn Pro Thr Leu Asp Thr Ala Leu 1835 1840 1845Thr Glu Gly Met Asn Lys Val Asp Val Thr Tyr Ala Gly Lys Thr 1850 1855 1860Val Asp Ile Gly Ile Glu Val Lys Ala Asp Thr Pro Val Glu Pro 1865 1870 1875Glu Lys Pro Thr Val Glu Lys Val Glu Ile Lys Ala Asn Pro Ala 1880 1885 1890Lys Thr Glu Tyr Lys Ala Gly Glu Thr Phe Asp Pro Thr Gly Met 1895 1900 1905Ser Leu Thr Val Thr Met Ser Asp Gly Thr Thr Lys Val Val Ala 1910 1915 1920Tyr Gly Pro Glu Thr Ala Lys Asp Phe Ser Phe Asn Pro Ser Leu 1925 1930 1935Asn Thr Lys Leu Thr Ala Asp Thr Lys Lys Val Thr Val Thr Tyr 1940 1945 1950Gly Gly Gln Ser Ala Asp Val Ala Val Ser Val Lys Ala Asp Pro 1955 1960 1965Ser Glu Asp Lys Lys Pro Asn Thr Glu Lys Pro Asp Lys Gly Gly 1970 1975 1980Ala Val Gln Thr Gly Asp Asn Phe Asn Val Thr Leu Leu Ile Gly 1985 1990 1995Leu Val Val Leu Ala Gly Ala Val Ala Gly Gly Ala Ala Leu Thr 2000 2005 2010Ile Phe Lys Arg Asn Lys Arg Lys 2015 202061355PRTClostridium perfringens 6Met Gln Ser Phe Asn Lys Arg Gly Thr Ala Leu Gly Ala Ala Ile Ala1 5 10 15Phe Ala Leu Thr Leu Ala Pro Thr Leu Val Met Ala Glu Thr Arg Gln 20 25 30Ile Pro Glu Ser Glu Thr Val Asn Val Gly Phe Ile Lys Asp Gly Glu 35 40 45Arg Ser Thr Ile Phe Asn Gln Asn Trp Lys Phe Phe Lys Gly Asp Pro 50 55 60Ser Gly Ala Glu Gly Val Asp Phe Asp Asp Ser Ser Trp Arg Gly Leu65 70 75 80Asn Leu Pro His Asp Trp Ser Ile Glu Gly Asp Phe Thr Val Glu Gly 85 90 95Glu Ala Glu Ser Gly Phe Leu Leu Gly Gly Thr Gly Trp Tyr Arg Lys 100 105 110Ala Phe Val Val Pro Glu Lys Tyr Asn Ser Lys Asp Phe Thr Leu Asn 115 120 125Phe Asp Gly Val Tyr Met Asn Ala Glu Val Tyr Val Asn Gly Lys Lys 130 135 140Val Gly Glu His Asn Tyr Gly Tyr Thr Ser Phe Ala Phe Asp Ile Thr145 150 155 160Glu Ala Leu Ile Cys Asp Gly Gln Thr Glu Asn Ile Ile Ala Val Lys 165 170 175Val Ser Asn Pro Val Pro Thr Ser Arg Trp Tyr Ser Gly Ser Gly Ile 180 185 190Tyr Arg Asp Val Thr Leu Ser Val Thr Asp Ser Ile His Val Ala His 195 200 205Ser Gly Thr Thr Val Thr Thr Pro Lys Leu Glu Glu Gln Lys Gly Gly 210 215 220Asp Val Asp Val Ala Ile Glu Thr Ile Val Glu Asn Glu Ser Lys Asp225 230 235 240Asn Ser Met Val Thr Val Lys Ser Thr Val Val Asn Ser Lys Gly Glu 245 250 255Glu Val Ser Glu Ala Val Ile Asn Glu Gln Ser Ile Gly Val Asn Glu 260 265 270Ser Tyr Thr Phe Lys Gln Thr Ala Ile Val Asn Asn Pro Asp Leu Trp 275 280 285Ser Val Asp Asn Pro Asn Met Tyr Lys Val Lys Ser Glu Val Leu Leu 290 295 300Asp Gly Lys Val Ile Asp Thr Tyr Phe Thr Asp Phe Gly Phe Arg Tyr305 310 315 320Tyr Asn Phe Asp Lys Asp Thr Gly Phe Ser Leu Asn Gly Glu Asn Met 325 330 335Lys Leu Lys Gly Val Cys Met His His Asp Gln Gly Ala Leu Gly Ala 340 345 350Ala Ser Tyr Tyr Arg Ala Val Glu Arg Gln Met Glu Lys Met Lys Glu 355 360 365Met Gly Val Asn Ala Ile Arg Val Ser His Asn Pro Ala Ser Glu Met 370 375 380Leu Leu Glu Ile Cys Asn Arg Leu Gly Leu Leu Val Ile Asn Glu Ala385 390 395 400Phe Asp Thr Trp Thr Asn Pro Lys Asn Gly Asn Val Asn Asp Phe Ser 405 410 415Lys Tyr Phe Asn Glu Val Ile Gly Glu Asp Asn Glu Ile Leu Asn Gly 420 425 430Ser Pro Glu Met Thr Trp Gly Glu Phe Glu Ala Arg Ser Met Val Lys 435 440 445Asn Ser Lys Asn Asn Pro Ser Ile Ile Met Trp Ser Ile Gly Asn Glu 450 455 460Val Leu Glu Gly Ile Ser Gly Ser Ala Ser Asn Tyr Thr Asn Val Ala465 470 475 480Gln Asn Ile Ile Asp Trp Ile Lys Asp Glu Asp Glu Thr Arg His Val 485 490 495Thr Ile Gly Asp Asn Arg Thr Lys Asn Gly Asp Arg Thr Ala Glu Ala 500 505 510Ile Ser Glu Val Val Asp Asp Asn Gly Gly Leu Val Gly Phe Asn Tyr 515 520 525Ala Asn Glu Thr Gln Val Ala Gln Gln Arg Ala Asn His Pro Asp Trp 530 535 540Thr Leu Tyr Ala Ser Glu Thr Ser Ser Ala Ile His Thr Arg Gly Tyr545 550 555 560Tyr Lys Thr Lys Gly Ile Asp Tyr Gly Asn His Arg Ile Ser Glu Tyr 565 570 575Asp Asn Asn Gln Thr Lys Val Gly Trp Gly His Ser Ala Ser Asp Ala 580 585 590Trp Lys Phe Val Ile Lys Asn Asp Tyr Asn Ala Gly Glu Leu Val Trp 595 600 605Thr Gly Phe Asp Tyr Ile Gly Glu Pro Thr Pro Trp Asn Gly Thr Gly 610 615 620Thr Gly Thr Val Gly Gly Gly Asn Gly Ala Ala Pro Lys Ser Ser Tyr625 630 635 640Phe Gly Ile Val Asp Thr Ala Gly Phe Glu Lys Asp Ile Tyr Tyr Leu 645 650 655Tyr Gln Ser Gln Trp Asn Asp Asp Val Asn Thr Leu His Val Leu Pro 660 665 670Thr Trp Asn Arg Glu Asp Ile Val Ile Glu Asn Gly Asn Val Glu Val 675 680 685Asn Val Phe Thr Asp Ala His Lys Val Glu Leu Tyr Leu Asn Asp Lys 690 695 700Lys Val Gly Glu Gln Thr Ser Thr Glu His Thr Thr Asp Ala Gly Tyr705 710 715 720Lys Tyr Tyr Thr Phe Gly Asn Asp Ser Leu Tyr Pro Val Phe Asn Val 725 730 735Pro Tyr Glu Glu Gly Thr Leu Thr Ala Lys Ala Tyr Asp Lys Glu Gly 740 745 750Asn Glu Ile Thr Asn Thr Glu Gly Arg Asn Thr Val Lys Thr Thr Gly 755 760 765Glu Ala Ser Thr Val Arg Leu Ser Ala Asp Arg Asp Thr Ile Asp Ser 770 775 780Asp Gly Tyr Asp Leu Ser Tyr Ile Thr Val Asp Ile Val Asp Glu Asp785 790 795 800Gly Asn Ile Val Gln Asn Ala Asp Asn Arg Leu Asn Phe Gln Leu Glu 805 810 815Gly Asp Gly Lys Ile Val Gly Val Asp Asn Gly Asp

Gln Thr Asp Thr 820 825 830Asp Ser Tyr Lys Pro Thr Ser Asp Thr Glu Ala Ser Arg Lys Ala Leu 835 840 845Ser Gly Lys Ala Leu Val Ile Val Gln Ser Thr Lys Asp Ala Gly Asn 850 855 860Ile Arg Leu Asn Val Ser Gly Glu Gly Leu Gln Ser Gln Ser Ile Glu865 870 875 880Ile Asn Thr Val Asn Asn Ala Gly Glu Asp Lys Phe Leu Glu Ser Tyr 885 890 895Glu Ile Val Lys Asp Tyr Tyr Val Asn Leu Asn Glu Lys Pro Glu Leu 900 905 910Pro Ser Thr Val Glu Gly Arg Tyr Ser Asp Gly Thr Thr Glu Thr Phe 915 920 925Asn Ile Ser Trp Asn Asp Tyr Asp Glu Ser Gln Leu Asn Thr Pro Gln 930 935 940Val Phe Lys Ile Asn Gly Lys Leu Glu Gly Thr Asp Val Ala Val Asn945 950 955 960Val Asn Val His Val Ile Gly Asp Val Val Ser Met Glu Asn Tyr Ser 965 970 975Thr Phe Thr Tyr Ala Gly Gln Thr Pro Thr Leu Pro Lys Thr Val Lys 980 985 990Gly Tyr Leu Ala Asp Gly Asn Glu Ser Glu Glu Phe Lys Val Asp Trp 995 1000 1005Asn Leu Glu Gly Val Asp Phe Ser Glu Pro Asn Thr Thr Val Glu 1010 1015 1020Val Leu Gly Glu Val Ser Leu Leu Gly Lys Thr Tyr Thr Val Thr 1025 1030 1035Ser Thr Val Arg Val Val Glu Ala Leu Lys Ala Ala Ala Asn Leu 1040 1045 1050Ala Ile Asn Asn Ser Ser Asn Lys Asp Val Pro Ala Leu Ser Gln 1055 1060 1065Ser Cys Val Ser Thr Ala Asp Asn Leu Asn Ser Ile Asn Asn Gly 1070 1075 1080Ile Thr Asn Asn Ser Ser Asn Thr Gly Glu Arg Trp Thr Asn Trp 1085 1090 1095Asn Glu Arg Asn Leu Thr Glu Asn Gly Glu Pro Lys Gly Ala Tyr 1100 1105 1110Val Gln Leu Asp Trp Lys Asn Lys Tyr Asn Ile Asp Arg Leu Asp 1115 1120 1125Leu Trp Leu Phe Thr Asp Asn Ile Tyr Gly Arg Ile Pro Lys Lys 1130 1135 1140Val Glu Ile Ser Tyr Lys Asn Glu Ala Gly Glu Tyr Glu Val Val 1145 1150 1155Thr His Ser Asn Thr Thr Glu Val Ser Tyr Leu Ala Gly Glu Thr 1160 1165 1170Thr Tyr Phe Leu Asp Lys Val Ile Asn Thr Asp Ser Ile Arg Val 1175 1180 1185Tyr Met Gln Gln Pro Glu Val Gly Lys Cys Ile Gly Leu Ser Glu 1190 1195 1200Val Ala Val Tyr Glu Tyr Val Pro Gln Val Ser Ala Asn Glu Gly 1205 1210 1215Asn Lys Leu Ser Glu Ile Lys Leu Asp Gly Glu Ala Leu Glu Gly 1220 1225 1230Phe Asn Pro Asp Thr Asn Glu Tyr Thr Val Asn Leu Lys Glu Leu 1235 1240 1245Pro Lys Thr Val Glu Ala Ser Gly Glu Glu Asn Val Ala Ile Thr 1250 1255 1260Ile Leu Pro Val His Asn Asn Lys Ser Ile Ile Ile Ala Arg Ser 1265 1270 1275Glu Ser Gly Ala Lys Asn Ile Tyr Thr Val Asn Tyr Val Leu Glu 1280 1285 1290Glu Ser Glu Gly Ser Ala Asp Ile Asn Glu Asp Gly Ser Ile Asn 1295 1300 1305Val Gly Asp Leu Ser Ile Val Ser Lys Tyr Gln Gly Glu Val Ile 1310 1315 1320Ser Gly Asn Ala Leu Ser Glu Lys Ser Asp Ile Asn Lys Asp Gly 1325 1330 1335Val Val Asp Lys Ala Asp Ile Gln Ile Val Met Gly Lys Ile Leu 1340 1345 1350Gly Glu 135571355PRTClostridium perfringens 7Met Gln Ser Phe Asn Lys Arg Gly Thr Ala Leu Gly Ala Ala Ile Ala1 5 10 15Phe Ala Leu Thr Leu Ala Pro Thr Leu Val Met Ala Glu Thr Arg Gln 20 25 30Ile Pro Glu Ser Glu Thr Val Asn Val Gly Phe Ile Lys Asp Gly Glu 35 40 45Arg Ser Thr Ile Phe Asn Gln Asn Trp Lys Phe Phe Lys Gly Asp Pro 50 55 60Ser Gly Ala Glu Gly Val Asp Phe Asp Asp Ser Ser Trp Arg Gly Leu65 70 75 80Asn Leu Pro His Asp Trp Ser Ile Glu Gly Asp Phe Thr Val Glu Gly 85 90 95Glu Ala Glu Ser Gly Phe Leu Leu Gly Gly Thr Gly Trp Tyr Arg Lys 100 105 110Ala Phe Val Val Pro Glu Lys Tyr Asn Gly Lys Asp Phe Thr Leu Asn 115 120 125Phe Asp Gly Val Tyr Met Asn Ala Glu Val Tyr Val Asn Gly Lys Lys 130 135 140Val Gly Glu His Asn Tyr Gly Tyr Thr Ser Phe Ala Phe Asp Ile Thr145 150 155 160Glu Ala Leu Ile Cys Asp Gly Gln Thr Glu Asn Ile Ile Ala Val Lys 165 170 175Val Ser Asn Pro Val Pro Thr Ser Arg Trp Tyr Ser Gly Ser Gly Ile 180 185 190Tyr Arg Asp Val Thr Leu Ser Val Thr Asp Ser Ile His Val Ala His 195 200 205Ala Gly Thr Thr Val Thr Thr Pro Lys Leu Glu Glu Gln Lys Asp Gly 210 215 220Asp Val Asp Val Ala Ile Glu Thr Ile Val Glu Asn Glu Ser Lys Asp225 230 235 240Asn Ser Met Val Thr Val Lys Ser Thr Val Val Asn Ser Lys Gly Glu 245 250 255Glu Val Ser Glu Ser Val Ile Asn Glu Lys Ser Ile Gly Ala Asn Glu 260 265 270Ser Tyr Thr Phe Asn Gln Thr Ala Ile Val Asn Asn Pro Gly Leu Trp 275 280 285Ser Val Asp Asn Pro Asn Met Tyr Lys Val Lys Ser Glu Val Leu Val 290 295 300Asp Gly Asn Val Ile Asp Thr Tyr Phe Thr Asp Phe Gly Phe Arg Tyr305 310 315 320Tyr Asn Phe Asp Lys Asp Thr Gly Phe Ser Leu Asn Gly Glu Asn Ile 325 330 335Lys Leu Lys Gly Val Cys Met His His Asp Gln Gly Ala Leu Gly Ala 340 345 350Ala Ser Tyr Tyr Arg Ala Val Glu Arg Gln Met Glu Lys Met Lys Glu 355 360 365Met Gly Val Asn Ala Ile Arg Val Ser His Asn Pro Ala Ser Glu Met 370 375 380Leu Leu Glu Ile Cys Asn Arg Leu Gly Leu Leu Val Ile Asn Glu Ala385 390 395 400Phe Asp Thr Trp Thr Asn Pro Lys Asn Gly Asn Val Asn Asp Phe Ser 405 410 415Lys Tyr Phe Asn Glu Val Ile Gly Glu Asp Asn Glu Ile Leu Asn Gly 420 425 430Ser Pro Glu Met Thr Trp Gly Glu Phe Glu Ala Arg Ser Met Val Lys 435 440 445Asn Ser Lys Asn Asn Pro Ser Ile Ile Met Trp Ser Ile Gly Asn Glu 450 455 460Val Leu Glu Gly Ile Ser Gly Ser Ala Ser Asn Tyr Thr Asn Val Ala465 470 475 480Gln Asn Ile Ile Asp Trp Ile Lys Asp Glu Asp Glu Thr Arg His Val 485 490 495Thr Ile Gly Asp Asn Arg Thr Lys Asn Gly Asp Arg Thr Ala Glu Ala 500 505 510Ile Ser Glu Val Val Asp Asp Asn Asp Gly Leu Val Gly Phe Asn Tyr 515 520 525Ala Asn Glu Ala Gln Val Ala Gln Gln Arg Ala Asn His Pro Asp Trp 530 535 540Thr Leu Tyr Ala Ser Glu Thr Ser Ser Ala Ile His Thr Arg Gly Tyr545 550 555 560Tyr Lys Thr Lys Gly Ile Asp Tyr Ser Asn His Arg Ile Ser Glu Tyr 565 570 575Asp Asn Asn Gln Thr Arg Val Gly Trp Gly His Ser Ala Ser Asp Ala 580 585 590Trp Lys Phe Val Ile Lys Asn Asp Tyr Asn Ala Gly Glu Phe Val Trp 595 600 605Thr Gly Phe Asp Tyr Ile Gly Glu Pro Thr Pro Trp Asn Gly Thr Gly 610 615 620Thr Gly Thr Val Gly Gly Gly Asn Gly Ala Ala Pro Lys Ser Ser Tyr625 630 635 640Phe Gly Ile Val Asp Thr Ala Gly Phe Glu Lys Asp Ile Tyr Tyr Leu 645 650 655Tyr Gln Ser Gln Trp Asn Asp Asp Val Asn Thr Leu His Val Leu Pro 660 665 670Thr Trp Asn Arg Glu Asp Ile Val Ile Glu Asn Gly Asn Val Glu Val 675 680 685Asn Val Phe Thr Asp Ala His Lys Val Glu Leu Tyr Leu Asn Asp Glu 690 695 700Lys Ile Gly Glu Gln Thr Ser Thr Glu His Thr Thr Asp Ala Gly Tyr705 710 715 720Lys Tyr Tyr Thr Phe Gly Asn Asp Ser Leu Tyr Pro Val Phe Asn Val 725 730 735Pro Tyr Lys Glu Gly Thr Leu Thr Ala Arg Ala Tyr Asp Lys Glu Gly 740 745 750Asn Glu Ile Thr Asn Thr Glu Gly Arg Asn Thr Val Lys Thr Thr Gly 755 760 765Glu Ala Ser Thr Val Arg Leu Ser Ala Asp Arg Asp Thr Ile Asp Ser 770 775 780Asp Gly Tyr Asp Leu Ser Tyr Ile Thr Val Asp Ile Val Asp Glu Asn785 790 795 800Gly Asn Ile Val Gln Asn Ala Asp Asn Arg Leu Asn Phe Glu Leu Glu 805 810 815Gly Asn Gly Lys Ile Val Gly Val Asp Asn Gly Asp Gln Thr Asp Thr 820 825 830Asp Ser Tyr Lys Pro Thr Ser Asp Thr Glu Ala Ser Arg Lys Ala Leu 835 840 845Ser Gly Lys Ala Leu Val Ile Val Gln Ser Thr Lys Asp Ala Gly Asn 850 855 860Ile Arg Leu Asn Val Ser Gly Glu Gly Leu Gln Ser Gln Ser Ile Glu865 870 875 880Ile Asn Thr Val Asn Asn Ala Gly Glu Asp Lys Phe Leu Glu Ser Tyr 885 890 895Glu Ile Val Lys Asp Tyr Tyr Val Asn Leu Asn Glu Lys Pro Glu Leu 900 905 910Pro Ser Thr Val Glu Gly Arg Tyr Ser Asp Gly Thr Thr Glu Thr Phe 915 920 925Asn Ile Ser Trp Asn Asp Tyr Asp Glu Ser Gln Leu Asn Thr Pro Gln 930 935 940Val Phe Lys Ile Asn Gly Lys Leu Glu Gly Thr Asp Val Ala Val Asn945 950 955 960Val Asn Val His Val Ile Gly Asp Val Val Ser Met Glu Asn Tyr Ser 965 970 975Thr Phe Thr Tyr Ala Gly Gln Thr Pro Thr Leu Pro Lys Thr Val Lys 980 985 990Gly Tyr Leu Ala Asp Gly Asn Glu Ser Glu Glu Phe Lys Val Asp Trp 995 1000 1005Asn Leu Glu Gly Val Asp Phe Ser Glu Pro Asn Thr Thr Val Glu 1010 1015 1020Val Leu Gly Glu Val Ser Leu Leu Gly Lys Thr Tyr Thr Val Thr 1025 1030 1035Ser Thr Val Arg Val Val Glu Ala Leu Lys Ala Ala Ala Asn Leu 1040 1045 1050Ala Ile Asn Lys Asp Thr Asn Lys Asp Val Pro Ala Leu Ser Gln 1055 1060 1065Ser Cys Val Ser Gln Ala Asp Asn Leu Asn Ser Ile Asn Asn Gly 1070 1075 1080Ile Thr Asn Asn Gly Thr Asp Thr Arg Glu Arg Trp Thr Asn Trp 1085 1090 1095Asn Glu Arg Asp Leu Thr Val Asn Gly Glu Pro Lys Gly Ala Tyr 1100 1105 1110Val Gln Leu Asp Trp Glu Asn Lys Tyr Asn Ile Asp Arg Leu Asp 1115 1120 1125Leu Trp Leu Phe Thr Asp Asn Ile Tyr Gly Arg Ile Pro Lys Lys 1130 1135 1140Val Glu Ile Ser Tyr Lys Asn Glu Ala Gly Glu Tyr Glu Val Val 1145 1150 1155Thr His Ser Asn Thr Thr Glu Val Ser Tyr Leu Ala Gly Glu Thr 1160 1165 1170Thr Tyr Phe Leu Asp Lys Val Ile Asn Thr Asp Ser Ile Arg Val 1175 1180 1185Tyr Met Gln Gln Pro Glu Val Gly Lys Cys Ile Gly Leu Ser Glu 1190 1195 1200Val Ala Val Tyr Glu Tyr Val Pro Gln Val Ser Ala Asn Glu Gly 1205 1210 1215Asn Lys Leu Ser Glu Ile Lys Leu Asp Gly Glu Ala Leu Glu Gly 1220 1225 1230Phe Asn Pro Asp Thr Asn Glu Tyr Thr Val Asn Leu Lys Glu Leu 1235 1240 1245Pro Lys Thr Val Glu Ala Ser Gly Glu Glu Asn Val Ala Ile Thr 1250 1255 1260Ile Leu Pro Val His Asn Asn Lys Ser Ile Ile Ile Ala Arg Ser 1265 1270 1275Glu Ser Gly Ala Lys Asn Ile Tyr Thr Val Asn Tyr Val Leu Glu 1280 1285 1290Glu Ser Glu Gly Ser Ala Asp Ile Asn Glu Asp Gly Ser Ile Asn 1295 1300 1305Val Gly Asp Leu Ser Ile Val Ser Lys Tyr Gln Gly Glu Ile Ile 1310 1315 1320Ser Gly Asn Ala Leu Ser Glu Lys Ser Asp Ile Asn Lys Asp Gly 1325 1330 1335Val Val Asp Lys Ala Asp Ile Gln Ile Val Met Gly Lys Ile Leu 1340 1345 1350Gly Glu 1355

Claims

1-24. (canceled)

25. A method for producing a dairy product comprisinga) providing a milk-based substrate comprising lactose; andb) treating said substrate with an enzyme having lactase activity and selected from the group consisting of: an amino acid sequence which is at least 70% identical to amino acids 28-1331 of SEQ ID NO: 2 or a fragment thereof; an amino acid sequence which is at least 70% identical to amino acids 28-1931 of SEQ ID NO: 1 or a fragment thereof; an amino acid sequence which is at least 70% identical to any of SEQ ID NO's: 3-4 or a fragment thereof; and an amino acid sequence which is at least 70% identical to any of SEQ ID NO's: 5-7 or a fragment thereof.

26. The method of claim 25, wherein the enzyme is derived from a microorganism of the genus Bifidobacterium.

27. The method of claim 25, where the pH optimum of the lactase activity at 37.degree. C. is above pH 5, and where the lactase activity of the enzyme at pH 5 is at least 50% of its lactase activity at pH 6 when measured at 37.degree. C.

28. A method for producing a dairy product comprisinga) providing a milk-based substrate comprising lactose, andb) treating said substrate with an enzyme having lactase activity, where the pH optimum of the lactase activity at 37.degree. C. is above pH 5, and where the lactase activity of the enzyme at pH 5 is at least 50% of its lactase activity at pH 6 when measured at 37.degree. C.

29. The method of claim 28, wherein step b) takes place at a temperature of at least 50.degree. C.

30. A method for producing a dairy product comprisinga) providing a milk-based substrate comprising lactose, andb) treating said substrate with an enzyme having lactase activity,wherein step b) takes place at a temperature of at least 50.degree. C.

31. The method of claim 30, wherein the lactase activity of the enzyme at a temperature of 52.degree. C. is at least 70% of its lactase activity at a temperature of 38.degree. C. when measured at pH 6.5.

32. The method claim 30, wherein the enzyme is added to the milk-based substrate at a concentration of less than 30 LAU per g lactose in the milk-based substrate.

33. The method of claim 30, wherein the enzyme is added to the milk-based substrate at a concentration of less than 1000 LAU per litre milk-based substrate.

34. The method of claim 30, wherein the enzyme when hydrolysing the lactose in the milk-based substrate has a ratio of lactase to transgalactosylase activity of more than 1:1.

35. The method of any of claim 30, further comprising a stepc) fermenting said substrate with a microorganism,wherein the dairy product is a fermented dairy product.

36. The method of claim 35, wherein the fermented dairy product is yoghurt.

37. The method of claim 35, wherein step b) and step c) are performed essentially at the same time.

38. The method of claim 35, wherein the enzyme of step b) and the microorganism of step c) are added to the milk-based substrate at essentially the same time.

39. The method of claim 35, wherein less than 80% of the lactose has been hydrolyzed after two hours of fermentation, and wherein more than 90% of the lactose has been hydrolyzed in the final fermented dairy product.

40. The method of claim 35, wherein less than 80% of the lactose has been hydrolyzed when step c) is completed, and wherein more than 90% of the lactose has been hydrolyzed in the final fermented dairy product.

41. The method of claim 28, wherein the milk-based substrate is raw milk which is not pasteurized before step b).

42. The method of claim 41, wherein step b) is performed for between 10 minutes and 4 hours at a temperature of between 62.degree. C. and 64.degree. C., and wherein step b) is followed by cooling to below 10.degree. C. without further heat treatment.

43. The method of claim 41, wherein less than 80% of the lactose has been hydrolyzed when step b) is completed, and wherein more than 90% of the lactose has been hydrolyzed after one week.

44. The method of claim 41, wherein step b) is performed for between 10 minutes and 4 hours at a temperature of between 62.degree. C. and 64.degree. C., and wherein step b) is followed by UHT treatment.

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