Polypeptides with transgalactosylation activity

Abstract

To provide: polypeptides having transgalactosylation activity; nucleic acids encoding the same; and uses thereof.SOLUTION: There are provided: a polypeptide having transgalactosylation activity, the polypeptide having at least 95% sequence identity to a particular amino acid sequence, consisting of 975 amino acid residues or less, and having the ratio of transgalactosylation activity to β-galactosidase activity of at least 1.5; a nucleic acid encoding the polypeptide; and a cell being able to express the polypeptide; as well as their use, for example, in dairy products.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a polypeptide, particularly a polypeptide having transgalactosylation activity, and their uses, for example in dairy products. [Background technology]

[0002] Galacto-oligosaccharides (GOS) are glycosyl linkers that are usually linked together by glycosidic bonds. Non-specific in humans and animals, containing up to nine, one or more galactose molecules GOS are digestible carbohydrates. They also contain one or more glucose molecules. One of the effects is by selectively stimulating the growth of beneficial bacterial microorganisms such as bacteria. , their ability to act as probiotic compounds and provide physical benefits to the consumer. The established health benefits of glycerol have led to its use as a food additive in various foods. This has resulted in an increase in

[0003] The enzyme β-galactosidase (EC 3.2.1.23) usually converts lactose into a monosaccharide, D-glucose. The normal enzyme reaction of β-galactosidase hydrolyzes glucose and D-galactose. In this reaction, the enzyme hydrolyzes lactose and transiently transports galactose. The galactose monosaccharide of the galactose enzyme complex is bound to the hydroxyl group of water, As a result, D-galactose and D-glucose are produced. However, high lactose At certain concentrations, some β-galactosidases are transgalactosylating. In the process, galactose is transferred to the hydroxyl groups of D-galactose or D-glucose. This allows the production of galacto-oligosaccharides. At low lactose concentrations, some β-galactosidases convert galactose to lactose. The hydroxyl groups of the oligosaccharides or higher can be transferred to the hydroxyl groups of the oligosaccharides.

[0004] Bifidobacteria are a popular bacterial culture medium in the dairy industry for the fermentation of various dairy products. Furthermore, oral intake of bifidobacterium-containing products has been shown to have health-promoting effects. This effect is not only achieved by a lowered pH of the intestinal contents, but also by the anti- Reconstructing the intestinal flora in individuals whose intestinal flora has been disrupted by ingestion of raw materials The ability of the Bifidobacterium genus to They have the ability to compete with and outcompete harmful gut microbes.

[0005] Galactooligosaccharides are known to promote the growth of Bifidobacteria. This effect is due to the fact that bifidobacteria use galacto-oligosaccharides as a carbon source. This appears to be achieved through the unique ability of sodium. Supplements are also believed to have a significant long-term disease prevention effect. For example, galactooligosaccharide intake was found to be highly protective against the progression of colon cancer in rats. It has been shown to be effective and is therefore being used in food supplements and dairy product improvements. To provide a method for the inexpensive and efficient production of galacto-oligosaccharides for use in industry. There is considerable interest in developing methods to

[0006] Bifidobacterium bifidum (Bifidus bifidum) truncated at approximately amino acid 580 obacterium bifidum) DMS20215 (BIF3-d3) in water It has been described as a transgalactosylating enzyme in solutions containing solubilized lactose. (Jorgensen et al. (2001), Appl. Microbio I. Biotechnol., 57:647-652). WO01 / 90317 is a translational WO20 describes a truncation mutant (OLGA347) as a galactosylating enzyme. In 12 / 010597, OLGA347 replaces the galactose moiety with D-fructose and N -acetyl-galactosamine, and xylose.

[0007] In WO2009 / 071539, a compound that is cleaved in a manner different from that of BIF3-d3 was The fragments, when tested in milk, had efficient hydrolytic activity and significantly reduced the production of GOS. The results shown are consistently low.

[0008] The lactase enzyme of Bifidobacterium bifidum is used to produce GOS. High lactose concentrations such as 10% (w / w) are required or heterooligosaccharides are However, the drawback is that a large number of other acceptor molecules are required to produce the desired peptide. Furthermore, this molecule was found to have predominantly beta-galactosidase (hydrolysis) activity. As shown.

[0009] Effective in promoting GOS in products with low lactose substrate levels such as milk There is a need to develop enzymes which Summary of the Invention

[0010] It has a useful ratio of transgalactosylation activity to hydrolysis activity and thus is suitable for use in milk base Even when incubated at low lactose levels, such as in milk products, GOS is efficiently It is an object of this aspect of the invention to provide a polypeptide that produces The aim is to generate galactooligosaccharides (GOS) in situ in dairy products. It is a further object of the present invention to provide a method for producing glass for industrial use. Providing a cheaper and more efficient way to develop tooligosaccharides (GOS) It is a further object of the present invention to provide a method for the preparation of a method for the preparation of a medicament ... s subtilis, e.g. Bacillus subtilis When produced in a suitable microorganism, such as the BG3594 strain of Bacteroides ilis, proteolytic The object of the present invention is to provide a polypeptide that is resistant to further cleavage such as A further object is to develop a polypeptide that is resistant to further cleavage during storage after final formulation. The aim of this study is to provide

[0011] The inventors have demonstrated that the polypeptides disclosed herein are effective in the production of lactobacilli, e.g. milk. The availability of galacto-oligosaccharides in situ when incubated in a galactosidase-containing composition They have made the surprising discovery that they are the most efficient producers of lactose into GOS. This results in a lower concentration of free lactose. - The presence of oligosaccharides is a known In humans or animals consuming these products, the genus Bifidobacterium Development of beneficial microbial strains (probiotics) such as Acterium sp. This provides the advantage of increasing the length.

[0012] In one aspect, disclosed herein is a nucleic acid sequence represented by SEQ ID NO: O:)1, a transgalactivirus containing an amino acid sequence having at least 90% identity to wherein the polypeptide is a polypeptide having silencing activity. The nucleic acid sequence encoding the peptide was identified in Bacillus subtilis When expressed in the BG3594 strain, the product exhibits transgalactosylation activity. The only polypeptide expression product of this nucleic acid sequence is that which has the desired activity.

[0013] In one aspect, disclosed herein is a transgalactosidase inhibitor selected from the group consisting of: It is a polypeptide having ctosylation activity. a. At least 90% sequence identity to SEQ ID NO:1 and up to 980 A polypeptide consisting of amino acid residues, b. At least 97% sequence identity to SEQ ID NO:2 and up to 975 A polypeptide made up of amino acid residues c. At least 96.5% sequence identity to SEQ ID NO:3 and up to 130 A polypeptide consisting of 0 amino acid residues d. under at least low stringency conditions, i) SEQ ID NO:1, 2, 3, SEQ ID NO: 9, 10, 11, 12, or ii) a polynucleotide that hybridizes to the nucleic acid sequence included in i) or the complementary strand of A polypeptide encoded by the nucleotide, e. A nucleic acid sequence encoding a polypeptide of SEQ ID NO:1, 2, 3, 4, or 5. SEQ ID NO: 1. A nucleic acid having at least 70% identity to a sequence or a sequence encoding a mature polypeptide. A polynucleotide comprising a nucleic acid sequence contained in ID NO: 9, 10, 11, 12, or 13. A polypeptide encoded by the leutide, and f. At one or more amino acid residues of SEQ ID NO:1, 2, 3, 4, or 5 A polypeptide containing deletions, insertions, and / or conservative substitutions.

[0014] In another aspect, disclosed herein is a transgalactamylase selected from the group consisting of: It is a polypeptide having ctosylation activity. a. At least 96.5% sequence identity to SEQ ID NO:3 and A polypeptide consisting of 0 amino acid residues, b. At least 90% sequence identity to SEQ ID NO:1 and up to 980 A polypeptide consisting of amino acid residues, c. under at least low stringency conditions, i) SEQ ID NO:1, 2, 3, SEQ ID NO: 9, 10, 11, 12, or 13, or a polynucleotide that hybridizes to the complementary strand of i). A polypeptide encoded by the d. A nucleic acid sequence encoding a polypeptide of SEQ ID NO:1, 2, 3, 4, or 5. SEQ ID NO: 1. A nucleic acid having at least 70% identity to a sequence or a sequence encoding a mature polypeptide. A polynucleotide comprising a nucleic acid sequence contained in ID NO: 9, 10, 11, 12, or 13. A polypeptide encoded by the leutide, and e. At one or more amino acid residues of SEQ ID NO:1, 2, 3, 4, or 5 A polypeptide containing deletions, insertions, and / or conservative substitutions.

[0015] In one aspect, disclosed herein is a method for the preparation of a galactosylating agent having transgalactosylation activity. A C-terminal truncated fragment of SEQ ID NO:22, which is a C-terminal truncated fragment of Bacillus subtilis (Bacillus subtilis), e.g., Bacillus subtilis (Bacil When produced in useful microorganisms such as BG3594 strain of Bacillus subtilis, Resistant to further cleavage, such as proteolysis, and / or storage after final formulation It is a polypeptide that is resistant to further cleavage between

[0016] In one aspect, disclosed herein is a method for detecting a mutated nucleotide sequence comprising at least 9 amino acids in SEQ ID NO:1. A polypeptide comprising an amino acid sequence having a sequence identity of up to 980 amino acids. It is a polypeptide made up of amino acid residues.

[0017] In one aspect, disclosed herein is a method for detecting a mutated nucleotide sequence comprising at least 9 amino acids in SEQ ID NO:2. A polypeptide comprising an amino acid sequence having a sequence identity of 7% and a maximum of 975 amino acids. It is a polypeptide made up of amino acid residues. In one aspect, disclosed herein is a nucleic acid sequence comprising at least 9 amino acids in SEQ ID NO:3. A polypeptide comprising an amino acid sequence having a sequence identity of 6.5% and up to 130 It is a polypeptide consisting of 0 amino acid residues.

[0018] In one aspect, disclosed herein is a polypeptide as disclosed herein. It is a nucleic acid capable of encoding the

[0019] In one aspect, the present invention relates to a nucleic acid comprising the nucleic acid disclosed herein or Expression vectors and / or plasmids capable of expressing the disclosed polypeptides. It is.

[0020] In one aspect, disclosed herein is a polypeptide of the present invention. The present invention relates to a cell capable of expressing the

[0021] In one aspect, disclosed herein is a method of expressing a polypeptide. The method includes obtaining a cell as described herein and causing the cell to express a polypeptide. and optionally a step of purifying the polypeptide.

[0022] In one aspect, disclosed herein is a method for the preparation of a polypeptide comprising the polypeptide described herein. The composition is preferably a food composition, more preferably a dairy product.

[0023] In one aspect, disclosed herein is a lactase comprising the polypeptides described herein. The present invention relates to a method for treating a milk-based substrate containing tocopherol to produce a food product such as a dairy product.

[0024] In one aspect, disclosed herein is a polypeptide as described herein. Galacto-oligosaccharides or their derivatives obtained by treating a substrate containing lactose It is a composition. [Brief description of the drawings]

[0025] [Figure 1] Figure 1 shows the plasmid map of the BIF_1326 mutant for recombinant expression in Bacillus subtilis. [Diagram 2] FIG. 2 shows an SDS-PAGE showing the truncation mutants purified using a HyperQ column eluted with a NaCl gradient. [Diagram 3] Figure 3 shows the ratio of transgalactosylation activity, which was calculated by dividing Abs420 with acceptor by Abs420 without acceptor and multiplying by 100. [Figure 4] FIG. 4 shows the galacto-oligosaccharide (GOS) purification efficiency of selected mutants in yogurt matrix at 30° C. for 3 hours. [Diagram 5] Figure 5 is an SDS-PAGE showing the expression of different mutants from Table 2 and the degradation of the detected fragments. The lower panel shows the extent and identity of the degradation bands. Array List

[0026] SEQ ID NO:1 (also referred to herein as (BIF_917)) is SEQ ID It is a truncated 887 amino acid fragment of NO:22. SEQ ID NO:2 (also referred to herein as (BIF_995)) is SEQ ID It is a truncated 965 amino acid fragment of NO:22. SEQ ID NO:3 (also referred to herein as (BIF_1068)) is It is a 1038 amino acid truncation fragment of NO:22. SEQ ID NO:4 (also referred to herein as (BIF_1172)) is It is a truncated 1142 amino acid fragment of NO:22. SEQ ID NO:5 (also referred to herein as (BIF_1241)) is It is a truncated 1211 amino acid fragment of NO:22. SEQ ID NO:6 (also referred to herein as (BIF_1326)) is It is a truncated 1296 amino acid fragment of NO:22. SEQ ID NO:7 is a sequence diagram of Bifidobacterium bifidum. The glycoside hydrolase catalytic core of Pseudomonas aeruginosa (Actium bifidum). SEQ ID NO:8 is a sequence diagram of Bifidobacterium bifidum. Encoding extracellular lactase from Bacillus subtilis bifidum DSM20215 It is a base sequence. SEQ ID NO:9 is the nucleotide sequence encoding BIF_917. SEQ ID NO:10 is the nucleotide sequence encoding BIF_995. SEQ ID NO:11 is the nucleotide sequence encoding BIF 1068. SEQ ID NO:12 is the nucleotide sequence encoding BIF_1172. SEQ ID NO:13 is the nucleotide sequence encoding BIF_1241. SEQ ID NO:14 is the nucleotide sequence encoding BIF_1326. SEQ ID NO:15 is the forward primer for generating the BIF mutants described above. be. SEQ ID NO:16 is the reverse primer for BIF917. SEQ ID NO:17 is the reverse primer of BIF995. SEQ ID NO:18 is the reverse primer of BIF1068. SEQ ID NO:19 is the reverse primer for BIF1241. SEQ ID NO:20 is the reverse primer for BIF1326. SEQ ID NO:21 is the reverse primer for BIF1478. SEQ ID NO:22 is a sequence identified as Bifidobacterium bifidum. It is an extracellular lactase derived from Lactobacillus subtilis (Ac. erium bifidum) DSM 20215. SEQ ID NO:23 is a sequence identified as Bifidobacterium bifidum. The signal sequence of extracellular lactase from Erythrium bifidum DSM 20215. Detailed Description of the Invention

[0027] definition In accordance with this detailed description, the following abbreviations and definitions are used. As used herein, the singular forms "a," "an," and "the" are used unless expressly defined otherwise. Thus, for example, "(a) polypeptide" refers to all such polypeptides. "The formulation" also includes reference to one or more formulations, and This includes equivalents known to those skilled in the art.

[0028] Unless otherwise defined, all technical and scientific terms used herein are understood to be of ordinary skill in the art. The terms are used in the same sense as they are commonly understood by those skilled in the art. The definitions of the terms are provided below.

[0029] "Transgalactosylase" is, among other things, the enzyme that converts galactose into D-galactose. or to the hydroxyl group of D-glucose to produce galacto-oligosaccharides. In one aspect, transgalactosylase refers to an enzyme that can produce The amount of galactose produced in a given time is less than the amount of glucose produced. It is defined by the reaction of an enzyme on a tocopherol.

[0030] In this context, the term "transgalactosyl activity" refers to the conversion of galactose moieties to molecules other than water. This activity is the transport of [glucose] produced at a given time during the reaction to the target. The amount of galactose produced at a given time during the reaction is measured as [glucose]-[galactose] or This can be measured by quantifying the amount of GOS. This can be done by several methods, such as the PLC method. For example, 3, 4, 5, 6, 7, 8, 9 or 10% (W / W) The initial lactose concentration was given.

[0031] In the present context, the term "β-galactosidase activity" refers to the ability of the enzyme to hydrolyze β-galactosides. For example, the enzyme that breaks down lactose into monosaccharides such as glucose and galactose. It means ability.

[0032] Transgalactosylation activity: in the context of the calculation of .BETA.-galactosidase activity β-galactosidase activity is the ratio of [galactose This measurement can be performed using several methods, such as the HPLC method, as shown in the examples. This can be carried out by the method described below.

[0033] In this context, ortho-nitrophenol-β-D-galactosidase (ONPG) The term "ratio of transgalactosylation activity" using the above formula was calculated as follows: Abs420 with receptor present divided by Abs420 without receptor present and multiplied by 100 Mutants with an index below 100 are pure hydrolysis mutants, while those with an index above 100 are pure hydrolysis mutants. The lower index indicates the comparative transgalactosylation activity.

[0034] Transgalactosylation activity ratio = (Abs420 +セロビオース / Abs420 -セ ロビオース )*100 In the formula, Abs420 +セロビオース The reaction involves cellobiose. is the absorbance at 420 nm, Abs420 -セロビオース During the reaction, cellobiolate The absorbance at 420 nm was measured using Method 3 described below, without any added fluoride. It is effective when diluted to a luminosity between 0.5 and 1.0.

[0035] In one aspect, the activity is measured at 15 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, Thus, in one aspect, for example, the relative transgalaxy The ctosyl activity was measured 15 minutes after the addition of the enzyme, 30 minutes after the addition of the enzyme, and 6 minutes after the addition of the enzyme. 0 min, 90 min after enzyme addition, 120 min after enzyme addition, 180 min after enzyme addition After that, it is measured.

[0036] In this context, the "ratio of galactosylation activity:β-galactosidase activity" refers to ([Glu [galactose]-[galactose] / [galactose]). In this context, the expression "glucose" is determined by HPLC method and is expressed as a percentage by weight. This refers to the concentration of glucose measured.

[0037] In this context, the expression "galactose" is given as a percentage by weight, as determined by HPLC. This refers to the concentration of galactose present in the culture.

[0038] In the present context, the statement "lactose is galactosylated" refers to the galactose molecule is covalently bonded to the lactose molecule, e.g., Either covalently linked to the α-galactosidase or internal transgalactosidase, e.g., arabinose formation It is covalently attached to a lactose molecule, produced by tosylation.

[0039] In this context, the present invention relates to the production of galactooligosaccharides (GOS) The performance of the polypeptides was evaluated using a "milk-based assay (fake yogurt product)" Batch testing in a volume of 100 μl was performed with fresh 98.60% (W / v) Fresh pasteurized low-fat milk (Arla Mini-maelk) and 1.4% ( Yogurt made from whey ingredient (Arla) (w / v) Nutrilac YQ-5075 The experiment was carried out in 96-well MTP plates using Tomix. Nutrilac YQ-50 75 The mixture is left to mix for 20 hours to completely hydrolyze the whey components. Then, 20 mM Na phosphatase pH 6.5 was added to adjust the pH to 6.5. The yogurt base is available plain or with added lactose, fucose, maltose, It is used either with xylose or with various supplements such as salt. Mix 90 μl with 10 μl of purified enzyme or crude fermentation product, seal with tape, and The reaction was incubated for 3 hours at 3°C. The reaction was stopped with 100 μl of 10% NaCO3. The samples were stored at -20°C. Galactooligosaccharides (GOS), lactose, The quantitative determination of galactose was performed by HPLC. Samples were analyzed by DinonexICS. The IC parameters were as follows: Mobile phase: 150 mM N aOH, Flow: Isochratic, 0.25ml / min, Column: Carbop ac PA1, Column temperature: RT, Note: 10 μl, Detector: PAD, Injector: Manual ,Sample preparation: 100-fold dilution with Milli-Q water (0.1 ml sample + 9.9 ml water) and Filter through a 0.45um syringe filter. Quantitative: Percentage of peak area of ​​standard substance. Peak area of ​​GOS syrup (VianalGOS, Friesland Campi The results of such an evaluation are shown in Figure 4. This is further described in Example 2.

[0040] In this context, the term "polypeptide lyophilized (lyophilized polypeptide)" refers to The term "lyophilized" refers to the process in which a solution of this polypeptide is subjected to a freeze-drying process under a suitable pressure for a suitable period of time to remove the water. This means that it is obtained by drying.

[0041] In the present context, the term "polypeptide in solution" refers to a solvent that is not precipitated in the solution. Solvents for this purpose include aqueous buffers or cyclohexane. Any environment in which the polypeptide occurs, such as a solution, a fermentation broth, or the cytoplasm of an expression host cell ( millieu).

[0042] In the present context, the term "stabilizer" refers to any agent that stabilizes a polypeptide. For example, glycerol or propylene glycol, sugar or sugar alcohol, butyric acid, Boric acid or a boric acid derivative (e.g., an aromatic boric acid ester). In this case the stabilizer is glycerol.

[0043] In the present context, the term "carbohydrate substrate" has the general formula C(HO), i.e., At least two hydrogen to oxygen pairs, consisting only of carbon, hydrogen, and oxygen, such as saccharides This refers to an organic compound having an atomic ratio of 1:2.

[0044] In this context, the term "disaccharide" refers to a disaccharide consisting of a hydrogen atom from one monosaccharide and a disaccharide from another monosaccharide. The formation of conjugated bonds known as glycosidic bonds through dehydration reactions, in which a hydroxyl group is lost from the glycosyl group. The formula for the unmodified disaccharide is C. 12 H 22 O 11 In one aspect, the disaccharide is lactulose, trehalose, , rhamnose, maltose, sucrose, lactose, fucose, and cellobiose. In a further aspect, the disaccharide is lactose.

[0045] The term "isolated" refers to a substance that is naturally associated with and has its natural sequence as found in nature. This means that the sequence is at least substantially free from at least one other component. In one aspect, the "isolated polypeptide" as used herein is SDS- At least 30% pure, at least 40% pure, at least 60% pure, at least 80% pure, at least 90% pure, at least 95% pure It means a polypeptide comprising:

[0046] The term "substantially pure polypeptide" as used herein refers to a polypeptide that is at most 10%, more preferably At most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most is at most 4%, at most 3%, even more preferably at most 2%, and most preferably at most 1 %, and most preferably up to 0.5% by weight of other naturally occurring polypeptide material. It therefore refers to a polypeptide preparation containing, by amount, a substantially pure polypeptide. The peptide is at least 92% pure, preferably 100% pure, by weight of the total polypeptide material present in the preparation. Preferably at least 94% pure, more preferably at least 95% pure, more preferably "At least 96% pure, more preferably at least 97% pure, more preferably at least More preferably, both are 98% pure, even more preferably at least 99% pure, and most preferably 99.5% pure. % pure, and most preferably 100% pure. The polypeptide is preferably in a substantially pure form. It is preferably in a pure form, i.e., free from other polypeptides with which it is naturally associated. Preferably, the polypeptide preparation is essentially free of substances. By preparing the polypeptide by known recombinant methods or by conventional purification methods. The term "substantially pure polypeptide" means "isolated polypeptide." The term "polypeptide" is synonymous with "polypeptide in isolated form."

[0047] The terms "purified" or "purified" refer to a substance in which a given component is present in a high level, e.g., at least about 51% pure, such as at least 51% pure, or at least 75% pure; At least 75% pure, such as at least 80% pure, at least 80% pure At least about 95% pure, at least about 95% pure, Such an ingredient is the major component present in the composition, such as 98% pure or at least 98% pure. It is preferably an active ingredient.

[0048] The term "microorganism" in the context of the present invention may include or may not include the nucleic acid sequence of the present invention. A nucleic acid sequence encoding a polypeptide having specific properties as defined in the specification, and / or In this context, the term includes any "microorganism" that can contain the compounds or products obtained therefrom. In this context, the term "microorganism" refers to any bacteria or cell capable of fermenting a milk substrate. Contains bacteria.

[0049] The term "host cell" as used in the present invention refers to a host cell that is capable of expressing a polypeptide having specific properties as defined herein. A nucleic acid sequence encoding a polypeptide as defined above and as defined herein. Any cell containing any of the expression vectors used to produce a polypeptide having the property In one aspect, the product is a recombinant product.

[0050] The term "milk" in the context of the present invention refers to milk from cows, sheep, goats, buffalo, camels, etc. In this context, "milk" is understood to be the milk secretion obtained from any mammal. The term "base substrate" refers to any raw and / or processed dairy substance or milk component derived therefrom. Useful milk-based substrates are any milk or milk-based substance that contains lactose. including but not limited to solutions / suspensions of milk-like products, whole or low fat milk, skim milk Buttermilk, Buttermilk, Reconstituted Milk Powder, Condensed Milk, Dry Milk Solution, UHT Milk, White Milk whey, whey permeate, acid whey, or cream. The milk-based substrate is an aqueous solution of milk or skim milk powder. It is more concentrated than raw milk. In one embodiment, the milk-based substrate is lactose-free. The ratio of protein to protein is at least 0.2, preferably at least 0.3, At least 0.4, at least 0.5, at least 0.6, and more preferably at least 0.7. The milk-based substrate may be homogenized and / or pasteurized by known methods. stomach.

[0051] As used herein, the term "homogenize" refers to the thorough mixing of a mixture to obtain a soluble suspension or emulsion. Homogenization means mixing the milk fat into smaller pieces so that it does not separate from the milk. Homogenization is done by forcing milk through small openings at high pressure. .

[0052] As used herein, "pasteurization" refers to the process of reducing the presence of viable microorganisms in a milk-based matrix. Preferably, pasteurization is performed by maintaining a given temperature for a given time. This particular temperature is usually maintained by heating. To kill or reduce certain bacteria, such as harmful bacteria, and / or This is followed by a rapid refrigeration step. The term "food" or "food composition" means any foodstuff or feed suitable for animal or human consumption. It is fine to use a food product.

[0053] In the context of this invention, the term "dairy product" refers to any food product in which one of the main ingredients is milk-based. Preferably, the main ingredient is milk-based. More preferably, the main ingredient is milk-based. The key component is a milk-based substrate that has been treated with an enzyme that has transgalactosylation activity. .

[0054] In this context, "one of the major components" means at least 20% of the total dry weight of the dairy product, preferably or more than 30% or more than 40% of the dry matter. The term "major ingredient" means any ingredient that constitutes not less than 50%, preferably not less than 60%, of the total dry weight of the dairy product; More preferably, it means an ingredient having a dry matter content of 70% or more.

[0055] The term "fermented dairy product" in this context refers to any type of fermentation that produces this product. It is understood that any dairy product in which fermentation is part of the process is a fermented dairy product. - Gurt, buttermilk, crème fraîche, quark and fromage fraîche Another example of a fermented milk product is cheese. The fermented milk product is produced by known methods.

[0056] The term "fermentation" refers to the process by which carbohydrates, such as a starting medium, are converted into alcohol or other alcoholic beverages through the action of microorganisms. In one aspect, fermentation converts lactose to lactic acid. Includes.

[0057] In this context, the term "microorganism" refers to any bacteria capable of fermenting a milk substrate. Contains terrier or germa.

[0058] In this context, the term "Pfam domain" refers to a multi-sequence alignment. and Hidden Markov Motifs (" The Pfam pr otein families database ”:RDFinn,J.Mis try, J. Tate, P. Coggill, A. Heger, JEPolling ton, OL Gavin, P. Gunesekaran, G. Ceric, K. Fo rslund, L. Holm, EL Sonnhammer, SREddy, A. Bateman Nucleic Acids Research(2010)Data Based on the presence of the base Issue 38:D211-222), Pfam-A or refers to a region in a protein sequence that is identified as either Pfam-B or Pfam-B. Examples of am domains are Glyco_hydro2N (PF02837), Glyc o_hydro(PF00703), Glyco_hydro 2C(PF02836) and bacterial Ig-like domain (group 4) (PF07532).

[0059] As used herein, the term "position corresponding to a position" refers to a position corresponding to a particular polypeptide of interest. It means an alignment as disclosed herein between a reference polypeptide. Alignment of positions that correspond to specific positions in a reference polypeptide with high sequence identity. The amino acids are identified as the corresponding amino acids in the

[0060] The term "variant" or "variants" refers to either a polypeptide or a nucleic acid. The term "variant" is used interchangeably with the term "mutant." Insertions, substitutions, transversions, truncations, or deletions at one or more positions in the amino acid or base sequence, respectively. "Mutant polypeptide," "polypeptide variant," "polypeptide The terms "polypeptide," "mutant," and "mutant enzyme" refer to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 2 , 4, or 5, or or a polypeptide having an amino acid sequence modified compared to a selected amino acid sequence. It means protein.

[0061] As used herein, a "reference enzyme," "reference sequence," "reference polypeptide" The term "variant polypeptide" refers to any of the variant polypeptides, e.g., SEQ ID NO:1, 2, 3, 4, or or 5. "Reference nucleic acid" refers to a nucleic acid sequence that encodes a reference polypeptide.

[0062] As used herein, the terms "reference sequence" and "subject sequence" are used interchangeably. will be done.

[0063] As used herein, the term "question sequence" refers to a sequence that is not within the scope of the present invention. It means a foreign sequence aligned with a reference sequence to check for the presence of such a The sequence in question may be, for example, a conventional sequence or a third-party sequence.

[0064] As used herein, the term "sequence" refers to either a polypeptide sequence or a nucleic acid sequence, depending on the context. It can mean either:

[0065] As used herein, the terms "polypeptide sequence" and "amino acid sequence" refer to Used interchangeably.

[0066] The signal sequence of the "variant" may be the signal sequence of the wild-type Bacillus signal peptide or The signal sequence may be the same as or different from any signal sequence that provides for secretion of the polypeptide.

[0067] To describe the various variants contemplated by this specification, the following terminology is used: is used for ease of reference. When a substitution includes a number and a position, for example, 592P, {position based on the numbering system / substituted amino acid}. The amino acid substitution at position 592 to proline is designated as 592P. When a position is described by a letter, e.g., D592P, this description is {original amino acid / number The term "substituted amino acid" refers to the position based on the barring system.

[0068] Thus, for example, a substitution of alanine with proline at position 592 would be represented as A592P. do.

[0069] When more than one substitution is possible at a particular position, such substitutions are It may be written as a sequence of letters or, optionally, separated by a slash mark " / ". For example, G303ED or G303E / D.

[0070] The positions and substitutions may be, for example, any of SEQ ID NOs: 1, 2, 3, 4, or 5. For example, equivalent positions in other sequences are listed in relation to this sequence and SEQ ID High sequence identity with either NO:1, 2, 3, 4, or 5 followed by any of SEQ ID NOs: 1, 2, 3, 4, or 5 This is done by determining the amino acid sequence corresponding to the amino acid at a particular position. Such an alignment and using one sequence as the first sequence is well within the skill of the art. It's just something that normally happens.

[0071] As used herein, the term "expression" refers to the expression of a polypeptide based on the nucleic acid sequence of a gene. This refers to the process by which a gene is produced. This process includes transcription and translation.

[0072] As used herein, the term "polypeptide" refers to an "amino acid sequence," "enzyme," "protein" is used interchangeably with "peptide" and / or "protein." As used herein, a "nucleotide sequence" or "nucleic acid sequence" refers to an oligonucleotide sequence or polynucleotide sequence. The term "nucleotide sequence" refers to a nucleotide sequence, its variants, homologues, fragments, and derivatives thereof. The octide sequence may be genomic, synthetic, or recombinant and may be single-stranded or double-stranded. The present invention may be directed to a single strand, in which sense or antisense sequences may be present. As used herein, the term "nucleotide sequence" refers to DNA, cDNA, synthetic DNA, and RNA. Contains A.

[0073] A "homolog" is a molecule that has a particular degree of sequence identity or similarity to the subject amino acid sequence and the subject nucleic acid sequence. means having "homology." In one aspect, the subject amino acid sequence is SQE ID NO: 1, 2, 3, 4, or 5, and the subject sequence is preferably Q ID NO:9, 10, 11, 12, or 13.

[0074] A "homologous sequence" is one that is identical to another sequence by a certain percentage, e.g., 80%, 85%, 90%, Contains polynucleotides or polypeptides with 95% or 99% sequence identity The percentage of identity is the number of bases or amino acid residues that match when aligned. indicates the degree to which two sequences are the same when compared. If an amino acid sequence is deleted or added, the amino acid sequence will not be identical. The page usually represents the mature sequence of the subject protein, i.e., the sequence after, for example, the signal sequence has been removed. Homologues are measured by reference to the amino acid sequence of the subject. Typically, a homolog contains the same active site residues as the subject amino acid sequence. Although the homologues have different enzymatic properties than the wild type, they also retain enzymatic activity.

[0075] As used herein, "hybridization" refers to the polymerase chain reaction (PCR). In addition to the amplification process that occurs in the R technology, the strands of nucleic acid undergo base pairing. The mutant nucleic acid may be a single-stranded or double-stranded DNA or RNA, It may exist as an RNA / DNA heteroduplex or an RNA / DNA copolymer. As used herein, the term "copolymer" refers to a copolymer of ribonucleotides and deoxyribonucleotides. This variant is codon-optimized for further expression. It can enhance the reality.

[0076] As used herein, a "synthetic" compound is one that is synthesized by in vitro chemical or enzymatic means. It is produced by selecting the codons appropriate for the host microorganism, such as yeast or other selected expression host. These include, but are not limited to, optimized mutant nucleic acids.

[0077] As used herein, a "transformed cell" refers to a cell that has been transformed using recombinant DNA techniques. This includes transformed bacterial cells and cells that contain bacterial cells. Transformation is usually performed by This occurs by inserting one or more nucleic acid sequences into a heterologous nucleic acid sequence. That is, sequences that are not in their native form in the cell being transformed, such as fusion proteins.

[0078] As used herein, the term "operably linked" refers to a combination of the components described that is intended to For example, operably linked to a coding sequence means that the coding sequence is capable of functioning in a specific manner. The regulatory sequence linked to the coding sequence is capable of achieving expression under conditions that are competitive with the control sequence. They are combined so that they can be

[0079] As used herein, the term "fragment" refers to a fragment that has activity and is and / or have one or more amino acids removed from the carboxy terminus It is defined as a polypeptide.

[0080] In one aspect, the term "fragment" refers to a fragment having transgalactosylation activity, The amino terminus and / or amino acid sequence of the polypeptide of SEQ ID NO:1, 2, 3, 4, or 5 or a polypeptide having one or more amino acids removed from the carboxy terminus. Defined as petide.

[0081] The term "galactose-binding domain-like" is abbreviated herein as "GBD" and is intended to mean any It is used in the same sense.

[0082] Degree of identity The relationship between two amino acid sequences or two base sequences is measured by a parameter called "identity." Explained.

[0083]

[0084] In one embodiment, the degree of sequence identity between a query sequence and a reference sequence is determined by: 1) differential Any combination of the default scoring matrix and default gap penalties. 2) aligning the two sequences using a suitable alignment program; identifying the number of matches, where an exact match is any match found in the alignment; Identical amino acids or bases in two aligned sequences at a given position are and 3) the length of the two sequences. Divide the number of exact matches by the length of the other sequence.

[0085] In other embodiments, the degree of sequence identity between a query sequence and a reference sequence is determined by: 1) a default Any suitable scoring matrix and default gap penalties are used. 2) aligning the two sequences using a suitable alignment program; identifying the number of matches found in the alignment, where an exact match is a given match in the alignment; Identical amino acids or bases in two aligned sequences at positions and 3) "the length of the aligned sequences." Divide the number of exact matches by the number of aligned sequences. The length of the alignment is the length of the entire alignment, including any overhanging sequences.

[0086] Sequence identity comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commonly available computer programs use multiple comparison algorithms. Use it to best identify the evolutionary events that may have led to differences between two or more sequences. These algorithms align two or more sequences that closely reflect each other. or similar amino acids are rewarded, and gap insertion, gap extension, and similar are not allowed. The alignment is driven by a scoring system that penalizes alignments of missing amino acids. Scoring systems for comparative alignments include: i) Assign a penalty for each time a gap is inserted (Gap Penalty (Score) ii) the penalty score for each time a gap is extended at an additional position Allocation (Delay Penalty Score) iii) assigning a high score to the alignment of identical amino acids; and iv) Assignment of variation scores in alignments of non-identical amino acids. Most alignment programs are modified by gap penalties. However, However, it is recommended to use the default values ​​when using such software for sequence comparisons. It is preferable that The score given for the alignment of non-identical amino acids is also called the substitution matrix. Such a permutation matrix is ​​assigned according to the scoring matrix. The scores provided in are based on the degree to which one amino acid should be replaced by another during evolution. These reflect the possibility of modifying and depending on the physical / chemical properties of amino acids. For example, , the probability of one polar amino acid being substituted with another polar amino acid is higher than the probability of it being substituted with a hydrophobic amino acid. Therefore, the scoring matrix is The highest score is assigned to identical but similar amino acids, and a lower score is assigned to non-identical but similar amino acids. The nucleotide sequence will be assigned a score equal to the amino acid sequence of the matched sequence, and non-identical and dissimilar amino acids will be assigned a lower score. The most frequently used scoring matrix is ​​the PAM matrix (Dayho ff et al. (1978), Jones et al. (1992)), the BLOSUM matrices(Henikoff and Henikoff(19 92)) and the Gonnet matrix (Gonnet et al. (1992)).

[0087] A suitable computer program for carrying out such an alignment is Vector or NTI (Invitrogen Corp.) and ClustalV, Clu stalW and ClustalW2 programs (Higgins DG & Sh arp PM (1988), Higgins et al. (1992), Thompsons On et al. (1994), Larkin et al. (2007) The selection of alternative alignment tools is not limited to these. www.expasy.or g This is made possible by the ExPASy proteomics server. Other examples of software that can perform intervention include the National Center for Available from the Biotechnology Information webpage http: / / www.ncbi.nlm.nih.gov / , Altschul et al. 1990) J. Mol. Biol. 215; 403-410, (BLAST)(Basic Local Alignment Search Too l).

[0088] In a preferred embodiment of the present invention, the alignment program aligns the entire full-length sequence. A global alignment program is being implemented to optimize alignment. In a preferred embodiment, the global alignment program is Needleman -Wunsch algorithm (Needleman, Saul B.; and Wu nsch, Christian D. (1970), “A general meth od applicable to the search for similari ties in the amino acid sequence of two p Journal of Molecular Biology 48 (3):443-53). An example of a recent program that uses the EMB There are OSS Needle and EMBOSS Stretcher programs, both of which teeth http: / / www.ebi.ac.uk / Tools / psa / Available in It is.

[0089] The EMBOSS Needle is a Needleman-Wunsch alignment It uses a genomic algorithm to find the optimal alignment of the entire length of two sequences (gap Perform an optimal global sequence alignment (including all sequences).

[0090] EMBOSS Stretcher uses a larger globally aligned sequence to A modification of the Eedleman-Wunsch alignment is used.

[0091] In one embodiment, the sequences are aligned using a global alignment to obtain the sequence Identity is calculated by dividing the number of exact matches identified by the program by the "alignment length". In this case, the alignment length is calculated by taking into account sequence gaps and overhangs. This is the length of the entire alignment, including any gaps.

[0092] In a further preferred embodiment, the global alignment is performed using the Needleman-Wuns method. Using the ch algorithm, sequence identity is calculated by the number of exact matches identified by the program. The alignment length is calculated by dividing the length of the alignment by the number of sequences. This is the length of the entire alignment, including gaps and overhanging gaps.

[0093] In a further embodiment, the global alignment program is EMBOSS Need le and EMBOSS Stretcher, and the sequence identity is It is calculated by dividing the number of exact matches identified by the program by the "alignment length". In this context, the alignment length includes gaps and overhanging gaps in the sequences. This is the length of the entire alignment, including gaps.

[0094] Once the software has produced the alignment, it calculates the percentage similarity and sequence identity. This software can calculate the identity percentage of normal sequences. This is done as a part to generate a numerical result.

[0095] In one embodiment, ClustalW software is used to perform sequence alignment. Preferably, alignments in ClustalW are pairwise. The alignment is performed with the following parameters: Table 1 TIFF0007612445000001.tif27153

[0096] ClustalW2 is available on the EMBL-EBI web page. www.ebi.ac.uk European Bioinformatics Institute Bioinformatics Institute's tools (under too ls) - sequence analysis - ClustalW2 The exact address of the recent ClustalW2 tool is ww w.ebi.ac.uk / Tools / clustalw2 It is.

[0097] In another embodiment, the sequence alignment is performed using Vector NTI The program Align X (Invitrogen) was used. In one embodiment, Exp10 is used with the following default settings: Did: Gap Open Penalty: 10 Gap extension penalty: 0.05 Gap Separation Penalty Range: 8 In other embodiments, one amino acid sequence is fused to or with another amino acid sequence. The alignment to columns is done using a score matrix set as follows: Determined using sum62mt2 and VectorNTI for pairwise alignments To be: TIFF0007612445000002.tif34141

[0098] In one embodiment, one amino acid sequence is combined with or with another amino acid sequence. The percentage of identical sequences is calculated using the BLOS This can be determined by using Blast with UM62. Polypeptides In one aspect, disclosed herein is a galactosylation inhibitor having transgalactosylation activity: - The ratio of galactosidase activity at a starting lactose concentration of 3% w / w or more is at least at least 0.5, at least 1, at least 2, at least 2.5, at least 3, at least At least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least In some embodiments, the polypeptide has at least 10, at least 11, or at least 12 amino acids.

[0099] In one aspect, disclosed herein is an amino acid sequence of SEQ ID NO:7. It is a polypeptide having a glycoside hydrolysis catalytic core having an acid sequence.

[0100] In one aspect, disclosed herein is a method for producing Glyco_hydro2N(P F02837), Glyco_hydro (PF00703) and / or Glyco A polypeptide containing the _hydro 2C (PF02836) domain.

[0101] In one aspect, disclosed herein are bacterial Ig-like domains (group Ig-like domains). It is a polypeptide containing peptide 4) (PF07532).

[0102] In one aspect, disclosed herein is a method for treating a pulmonary circulation comprising administering to a patient a therapeutically effective amount of a pulmonary circulation comprising administering to said patient a therapeutically effective amount of a pulmonary circulation ... is a polypeptide having transgalactosylation activity: a. Contains an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1 A polypeptide comprising 980 or less amino acid residues. Lipeptide b. Contains an amino acid sequence having at least 97% sequence identity to SEQ ID NO:2 A polypeptide comprising 975 or fewer amino acid residues. Lipeptide c. An amino acid sequence having at least 96.5% sequence identity to SEQ ID NO:3 a polypeptide comprising 1,300 or less amino acid residues; Polypeptide di) encoding the polypeptide of SEQ ID NO: 1, 2, 3, 4, or 5 or a nucleic acid sequence contained in SEQ ID NO:9, 10, 11, 12, or 13, ii) hybridizes under at least low stringency conditions to a sequence complementary to i); Polypeptides encoded by polynucleotides e. A nucleic acid sequence encoding the polypeptide of SEQ ID NO:1, 2, 3, 4, or 5. A polynucleotide or mature polypeptide containing a base sequence that is at least 70% identical to the sequence A salt contained in SEQ ID NO: 9, 10, 11, 12, or 13 encoding A polypeptide encoded by a polynucleotide comprising the base sequence, and f. a deletion of one or more amino acid residues in SEQ ID NO:1, 2, 3, 4, or 5; Polypeptides containing insertions and / or conservative substitutions.

[0103] In one aspect, disclosed herein is a method for treating a pulmonary circulation comprising administering to a patient a therapeutically effective amount of a pulmonary circulation comprising administering to said patient a therapeutically effective amount of a pulmonary circulation ... is a polypeptide having transgalactosylation activity: a. An amino acid sequence having at least 96.5% sequence identity to SEQ ID NO:3 a polypeptide comprising 1,300 or less amino acid residues; Polypeptide b. Contains an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1 A polypeptide comprising 980 or less amino acid residues. Lipeptides c) encoding a polypeptide of SEQ ID NO: 1, 2, 3, 4, or 5; or a nucleic acid sequence contained in SEQ ID NO:9, 10, 11, 12, or 13, ii) hybridizes under at least low stringency conditions to a sequence complementary to i); a polypeptide encoded by a polynucleotide d. A nucleic acid sequence encoding the polypeptide of SEQ ID NO:1, 2, 3, 4, or 5. A polynucleotide or mature polypeptide containing a base sequence that is at least 70% identical to the sequence A salt contained in SEQ ID NO: 9, 10, 11, 12, or 13 encoding A polypeptide encoded by a polynucleotide comprising the base sequence, and e. A deletion of one or more amino acid residues in SEQ ID NO:1, 2, 3, 4, or 5; Polypeptides containing insertions and / or conservative substitutions.

[0104] In one aspect, disclosed herein is a nucleic acid sequence comprising SEQ ID NO:1, 2, 3, 4, or 5 of the mature amino acid sequence have at least 68%, 70%, 72%, 74%, or 7 6%, 78%, 80%%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, Polypeptides consisting of amino acids with 96%, 97%, 98%, or 99% sequence identity Chid.

[0105] In one aspect, disclosed herein is a mature version of SEQ ID NO:1. It is a polypeptide having 90% sequence identity to the amino acid sequence of

[0106] In one aspect, disclosed herein is a nucleic acid sequence similar to that of SEQ ID NO:2. % sequence identity.

[0107] In one aspect, disclosed herein is a nucleotide sequence similar to that of SEQ ID NO:3, but different from that of SEQ ID NO:4. It is a polypeptide having 0.5% sequence identity.

[0108] In one aspect, disclosed herein is a nucleotide sequence similar to that of SEQ ID NO:4, but different from that of SEQ ID NO:5. It is a polypeptide having 0.5% sequence identity.

[0109] In one aspect, disclosed herein is a nucleotide sequence similar to that of SEQ ID NO:5, but different from that of SEQ ID NO:96. It is a polypeptide having 0.5% sequence identity.

[0110] In one aspect, disclosed herein is a nucleic acid sequence comprising SEQ ID NO:1, 2, It is a polypeptide comprising or consisting of a sequence of 3, 4, or 5 amino acids.

[0111] In one aspect, disclosed herein is a Bifidobacterium bifidum strain. It is a polypeptide derived from Bifidobacterium bifidum.

[0112] In one aspect, disclosed herein is a strain having an optimum pH of 6.5 to 7.5. It is a polypeptide.

[0113] In one aspect, disclosed herein is a method for cultivating a yeast strain having an optimum temperature of 30 to 60 degrees Celsius; For example, a polypeptide between 42 and 60 degrees Celsius.

[0114] Carbohydrate-active polypeptides are classified by IUBMB based on their substrate specificity. In one or more embodiments of the system or the recent 125 glycoside hydrolase family The CaZy database is used to classify the genus CaZy. In this case, assignments are made based on sequence and product information combined with knowledge of the stereochemistry of the substrate and product. It is based on both structural and nonstructural information.

[0115] Disclosed herein is a Bacillus subtilis strain having a nucleic acid sequence encoding this polypeptide. Expression in Bacillus subtilis BG3594 It is a polypeptide which is a product of a nucleic acid sequence having transgalactosylation activity. The polypeptide expression product is merely a polypeptide that can be expressed using known techniques known to those skilled in the art. The samples to be evaluated were subjected to SDS-PAGE and analyzed using a Bio-Rad clonidine gel electrophoresis system. The Criterion System is a suitable system for protein quantification. The gels were visualized using a suitable dye. Using a suitable densitometric scanner, such as the Terion System The dynamic range of the photograph obtained by scanning is ensured. SEQ ID NO:8 The bands corresponding to the derived mutants / fragments were quantified to determine the percentage of polypeptides The percentage of polypeptides tested is calculated as follows: Percentage of polypeptides tested = Polypeptides tested polypeptide / (sum of all polypeptides with transgalactosyl activity)*100 . The total number of polypeptide variants / fragments derived from SEQ ID NO:8 in the composition Western blot using polyclonal antibodies by methods known to those skilled in the art The method is to detect a fragment derived from SEQ ID NO:8. can be done.

[0116] The polypeptides disclosed herein contain at least two separate functional domains within one enzyme. First, the polypeptide contains a glycosidic hydrolase as described below. This catalytic core is a member of the GH family of related glycoside hydrolases. The GH-A clan belongs to the -A clan. The GH-A clan maintains the glycosidic bond by cleaving the glycosidic bond. It is characterized by its ability to bind to and has a catalytic domain based on the TIM barrel fold (W ierenga,2001,FEBS Letters,492(3),p193-8) The catalytic domain acts as a proton donor and nucleophile, and binds the strands of the barrel domain. It contains two glutamic acid residues released from 4 and 7 (Jenkins, 1995, FEBS Letters, 362(3), p281-5).

[0117] The overall structure of the TIM barrel is composed of eight β strands and eight α helices (β / α In one aspect, the glycoside hydrolases disclosed herein are The catalytic core of the glycoside hydrolase belongs to either the glycoside hydrolase family GH-2 or GH-35. All these TIM barrel enzymes belong to the GH-A clan. The galactosyl hydrolase catalytic core is a member of the GH-2 or GH-35 family. In a further aspect, the glycoside hydrolase catalytic core belongs to the GH-2 These enzymes belong to the same family. The common factor is that these enzymes are called retention enzymes and The structure of the substance is transformed into a product (Henrissat, 1997, Curr Opin Struct Biol,7(5),637-44).

[0118] In one aspect, the polypeptides described herein have a β(1→4) structure. This actually makes the enzyme the I type of β-galactosidase. It is classified in the UBMB EC 3.2.1.23 class. This activity is probably due to para-nitro Phenol-β-D Galactopyranoside (PNPG), ortho-nitrophenol-β- D-galactopyranoside (ONPG), β-D-galactopyranoside with a pigment-forming aglycone The enzyme activity is determined using synthetic substrates such as, but not limited to, xenoside (XGal). A proxy for determining whether an enzyme belongs to the EC 3.2.1.23 class of β-galactosidases. An alternative method is incubation with a substrate such as lactose and subsequent analysis by HPLC, TLC, or measure glucose release by other methods known to those skilled in the art.

[0119] Using the accumulation of several known facts to predict the functional properties of a polypeptide For example, Pfam (Nucl. Acids Res. (2010) 38 (sup pl 1):D211-D222.doi:10.1093 / nar / gkp985) and Interpro (Nucl. Acids Res. (2009) 37 (suppl 1):D211-D215. doi:10.1093 / nar / gkn785) When such an analysis is carried out, it should be made available in publicly available databases. It should be specified that the technique should be performed on the full-length sequence of a polypeptide capable of being purified.

[0120] In a further aspect, Glyco_hydro2N (PF02837), Glyco_ hydro(PF00703), Glyco_hydro 2C(PF02836) and and Bacterial Ig-like domains (group 4) (PF07532). In a further aspect, a polypeptide is provided comprising one or more of the Pfam domains comprising: The Pfam domains Glyco_hydro2N (PF02837), Glyco_ hydro(PF00703), Glyco_hydro 2C(PF02836) and and bacterial Ig-like domain (group 4) (PF07532 In a further aspect, a polypeptide is provided comprising the catalytic domain of the polypeptide. In is Glyco_hydro2N (PF02837), Glyco_hydro (PF 00703), and Glyco_hydro 2C (PF02836). A peptide is provided.

[0121] In a further aspect, the present invention relates to a method for the preparation of a lactic acid bacteria comprising the steps of: Measured after 15, 30 or 180 minutes at 100 ppm in a milk-based matrix The ratio of transgalactosylation activity to β-galactosidase activity obtained is at least 1. At least 2.5, at least 3, at least 4, at least 5, at least 6, at least At least 7, at least 8, at least 9, at least 10, at least 11, or at least In a further aspect, the polypeptide is a bifurcation. From Bifidobacterium bifidum It is coming.

[0122] In one aspect, the polypeptides disclosed herein are capable of being cultured at 37° C. at 5% w / w lactate. In a milk-based substrate, the concentration of 100 ppm was measured for 15, 30, or 18 minutes. Measured at 0 minutes and found to be 20%, 30%, or 40% higher than the initial lactose level , up to 50% is galactosylated.

[0123] In a further aspect, the polypeptides disclosed herein are capable of being incubated at 37° C. for 5% w / w lactate. In a milk-based substrate, the concentration of 100 ppm was measured for 15, 30, or 18 minutes. After 0 minutes, the lactose content was measured as below 80%, below 70%, below 60%, below 50%, β-galactosylation activity was determined so that less than 40%, less than 305, and less than 20% of the Yes.

[0124] In one aspect, the β-galactosidase activity and / or transgalactosidase activity The silylating activity was determined by Method 4 at 100 ppm, which corresponds to 2.13 LAU. It is measured in concentration.

[0125] In further aspects, the polypeptides described herein have one or more of the following properties: : i) At 37°C, 5% w / w lactose, at a concentration of 100 ppm in a milk-based matrix Transgalactosylation activity versus β-galactosylation activity measured after 15, 30, or 180 min. The lactosidase activity ratio is at least 1, at least 2.5, at least 3, at least At least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least at least 10, at least 11, or at least 12 and / or ii) 100 ppm in a milk-based matrix at 37°C and 5% w / w lactose and >20% of the initial lactose measured after 15, 30, or 180 minutes. Greater than 30%, greater than 40%, up to 50% are galactosylated.

[0126] In one aspect, a nucleic acid sequence having at least 96.5% sequence identity to SEQ ID NO:3. The present invention provides a polypeptide having up to 1300 amino acid residues, comprising an amino acid sequence having the above structure. In a further aspect, the present invention relates to a method for the preparation of a nucleic acid sequence having at least 90% identity with SEQ ID NO:1. At least 95%, at least 96%, at least 97%, at least 98%, at least 99% or a sequence having at least 100% sequence identity, up to 980 amino acids In a further aspect, a polypeptide is provided comprising the amino acid residues of SEQ ID NO: A sequence of up to 980 amino acids having at least 90% sequence identity to NO:1 In a further aspect, there is provided a polypeptide consisting of the amino acid sequence of SEQ ID NO: At least 90% sequence identity to NO:1, e.g., at least 91%, at least 9 2%, at least 93%, at least 94%, at least 95%, at least 96%, Amino acids having at least 97%, at least 98%, or at least 99% sequence identity. In another aspect, a polypeptide is provided comprising the amino acid sequence of SEQ ID NO:2. at least 96.5% sequence identity to, for example, at least 98%, or At least 97% sequence identity, such as at least 99%, is provided. In the above, up to 975 amino acid residues, for example up to 970 amino acid residues , up to 950 amino acid residues, up to 940 amino acid residues, up to 93 0 amino acid residues, up to 920 amino acid residues, up to 910 amino acid residues amino acid residues, up to 900 amino acid residues, up to 895 amino acid residues, Alternatively, a polypeptide of up to 890 amino acid residues is provided. In one aspect, the present invention relates to a nucleic acid sequence that is at least 97%, e.g., at least 99%, similar to SEQ ID NO:2. or a sequence with 100% sequence identity, up to a maximum of 975 amino acids. up to 970 amino acid residues, or at least 965 amino acid residues. In one aspect, a polypeptide is provided that comprises the amino acid residues of SEQ ID NO. :2, with at least 97% sequence identity and up to 975 amino acids. A peptide is provided.

[0127] In a further preferred aspect, the polypeptide comprises SEQ ID NO: 1, 2, 3, 4, or 5. In a further aspect, a polypeptide is provided comprising SEQ ID NO: 1, 2, 3, 4, or 5, particularly a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2. The present invention provides a polypeptide comprising:

[0128] In a further aspect, a polypeptide having at least 96.5% sequence identity to SEQ ID NO:3, At least 97% sequence identity, e.g., at least 98%, at least 99%, or at least Contains an amino acid sequence with at least 100% sequence identity and up to 1300 amino acids. Polypeptides consisting of acid residues are provided.

[0129] In a further aspect, the nucleic acid sequence of the present invention is at least 98.5%, at least 9 Polypeptides having sequence identity of 9%, or at least 99.5%, etc. are provided. In one aspect, up to 1290 amino acid residues, e.g., up to 1280 Up to 1270, Up to 1260, Up to 1250, Up to Large up to 1240, Maximum up to 1230, Maximum up to 1220, or Maximum In one preferred aspect, a polypeptide is provided that is a polypeptide of up to 1215 amino acid residues. A polypeptide consisting of 1211 amino acid residues is provided.

[0130] In a further aspect, the sequence of the nucleic acid is at least 96%, e.g., at least 96% relative to SEQ ID NO:4. Polypeptides having at least 97%, such as at least 98%, or 99% sequence identity. In one aspect, peptides are provided that are up to 1210 amino acid residues long, e.g. For example, up to 1200, up to 1190, up to 1180, Large: up to 1170, Maximum: 1160, Maximum: 1150, Maximum: 11 Polypeptides of up to 45 and up to a maximum of 1142 amino acid residues are provided.

[0131] In a further aspect, the sequence of the amino acid sequence of the present invention is at least 96.5% relative to SEQ ID NO:3, e.g. , at least 97%, such as at least 98% or 99% sequence identity. In one aspect, up to 1130 polypeptides, e.g., up to 1120, up to 1110, up to 1100, up to 1090, up to 1080 pcs, up to 1070 pcs, up to 1060 pcs, up to 1050 pcs, up to 1055 pcs or Polypeptides of up to 1040 amino acid residues are provided. In the present invention, a polypeptide consisting of 1038 amino acid residues is provided.

[0132] In a further aspect, the polypeptides disclosed herein have a nucleotide sequence greater than 100%, e.g., 150%. %, 175% or 200% higher transgalactosylation activity.

[0133] Proteins usually consist of one or more functional regions called domains. The occurrence of separate domains in various combinations in the structure of naturally occurring proteins They form a diverse repertoire. One way to describe the domains is as follows: The Pf amprotein families database ”:RDFinn,J .Mistry, J. Tate, P. Coggill, A. Heger, JEPo. llington, OL Gavin, P. Gunesekaran, G. Ceri c, K. Forslund, L. Holm, EL Sonnhammer, SRE ddy, A. Bateman Nucleic Acids Research(201 0)Tags such as those described in Database Issue 38:D211-222 Based on the Pfam database, a large collection of protein domain families Each family is represented by a multiple sequence alignment and is modeled using Markov mod els (HMMs) are hidden. In a further aspect, the inventors have , Glyco_hydro2N(PF02837), Glyco_hydro(PF00 703), Glyco_hydro 2C(PF02836) and Bacterial Ig-like domain (group 4) (PF07532) In one aspect, the polypeptides provided herein are The tides are Glyco_hydro2N (PF02837) and Glyco_hydro (PF 00703), Glyco_hydro 2C(PF02836) and Bacteri al Ig-like domain (group 4) (PF07532).

[0134] In one aspect, the pH range is 4-9, such as 5-8, such as 5.5-7.5. It has useful transgalactosylation activity throughout the range, such as 6.5-7.5.

[0135] The present invention relates to a method for the preparation of a compound having an amino acid sequence as defined herein or a specific sex as defined herein. A polypeptide having a particular degree of sequence identity or sequence homology to a polypeptide having the same quality. The present invention particularly relates to SEQ ID NOs: 1, 2, 3, 4, or 5, or a homologue thereof. Includes chid.

[0136] In one aspect, the homologous amino acid / nucleotide sequence is a functional transgalactosidase. and / or a polypeptide of SEQ ID NO:1, 2, 3, 4, or 5. and / or to provide a polypeptide having a higher transgalactosylation activity compared to a peptide. is coded.

[0137] In this context, a homologous sequence is one that is at least 65%, 70%, 75%, 78% , at least 8015, at least 85%, at least 90%, at least 95%, at least and the amino acid sequence is at least 97%, at least 98%, or at least 99% identical to the Typically, the homolog will contain the same active site as the subject amino acid sequence. The nature of the similarity is considered in terms of similarity (i.e., amino acid residues with similar chemical properties / functions). However, in the context of the present invention, it expresses homology in terms of sequence identity. It is preferred to do so.

[0138] Thus, the present invention relates to any amino acid of the proteins or polypeptides described herein. Variants of sequences, in particular SEQ ID NOs: 1, 2, 3, 4 or 5 as described below, Includes homologues and derivatives.

[0139] A variant of the sequence, in particular SEQ ID NO: 1, 2, 3, 4 or 5, as defined below. , homologs, and derivatives have sequences that contain minor variations in amino acids that are functionally equivalent. This also includes the deletion, insertion, or substitution of residues. Deliberate amino acid substitutions can improve secondary binding activity to substrates. As long as the molecular properties are maintained, the molecular weight can be determined by the polarity, charge, solubility, hydrophilicity, hydrophobicity, and / or amphiphilicity. For example, the negatively charged amino acids are aspartic acid and The positively charged amino acids, including glutamine, have similar hydrophilic groups including lysine and arginine. Amino acids with uncharged polar head groups that have a value of 1:1 are isoleucine, valine, glycine, Alanine, Asparagine, Glutamine, Serine, Threonine, Phenylalanine, and tyrosine.

[0140] The present invention is a method for converting basic to basic, acid to acid, polar to polar, etc. Conservative substitutions include substitutions in which an amino acid is replaced with a similar amino acid (substitutions and replacements are not considered to be (This means that the amino acid residue in the sequence is replaced with an alternative residue.) Non-conservative substitutions are The residues of the ras are ornithine (hereinafter referred to as Z) and ornithine diaminobutyrate (hereinafter referred to as B). (hereinafter referred to as O), norleucine ornithine, pyrilalanine, thienyl Others include unnatural amino acids such as alanine, naphthylalanine and phenylglycine. This means that substitution of the same or an alternative amino acid occurs.

[0141] Conservative substitutions include, for example, basic amino acids (arginine, lysine, and histidine), acids Amino acids (glutamic acid and aspartic acid), aliphatic amino acids (alanine, Amino acids (glutamine, asparagine, serine, sucrose, isoleucine), polar amino acids (glutamine, asparagine, serine, leonine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), Amino acids containing hydroxyl groups (serine, threonine), large amino acids (phenylalanine, and tryptophan) and within the group of small amino acids (glycine, alanine) This is carried out by.

[0142] In one aspect, the polypeptide sequences used in the present invention are in a purified form. In this aspect, the polypeptide or protein used in the present invention is in an isolated state. be.

[0143] In one aspect, the polypeptides of the invention are produced using recombinant techniques.

[0144] The variant polypeptides may, for example, have at least one sequence similar to that of SEQ ID NO:1 or 2. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or It has 99% sequence identity.

[0145] The variant polypeptide may, for example, have at least the sequence of SEQ ID NO:3, 4, or 5. 96%, 97%, 98%, or 99% sequence identity to

[0146] In one aspect, the polypeptide disclosed herein has the structure of SEQ ID NO:2. Bifidobacterium bifidum (shown in Fig. 2) ifidum DSM 20215 encoding a transgalactosylase at least 90% to the amino acid sequence of the mature polypeptide encoded by the sequence, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% distribution SEQ ID NO: 1, 2, 3, 4, or 5. All considerations and limitations relating to sequence identity and functionality discussed in this specification are intended to be understood as including all of the above. The definitions apply mutatis mutandis to the sequence identity and functionality of these polypeptides and bases.

[0147] In one aspect, the subject amino acid sequence is SEQ ID NO:1, 2, 3, 4, or or 5, and the subject nucleotide sequences are preferably SEQ ID NOs: 9, 10, 11, 12 , or 13.

[0148] In one aspect, the polypeptide has the sequence set forth in SEQ ID NO: 1, 2, 3, 4, or 5 deletions from the amino and / or carboxy terminus of the polypeptide It is a fragment with one or more (several) amino acids missing.

[0149] In one aspect, at least 500, 550, 600, 650, 700, 750, Contains 800, 850, 900, 950, or 1000 amino acid residues.

[0150] In a further aspect, the length of the polypeptide variant is between 500 and 1300. In an aspect, the length of the polypeptide variant is between 600 and 1300. In a further aspect, the polypeptide variant has a length of 700 to 1300. The length of the polypeptide variant is between 800 and 1300. The length of the mutants ranges from 800 to 1300 amino acids.

[0151] Polypeptide variants of SEQ ID NO:1, 2, 3, 4, or 5 In one aspect, an improved version of SEQ ID NO:1, 2, 3, 4, or 5. At one or more positions, which affect modified properties such as transgalactosylation Variants of SEQ ID NOs: 1, 2, 3, 4, or 5 having substitutions in Such mutant polypeptides are referred to herein for convenience as "mutant polypeptides." In one aspect, the polypeptides described herein are referred to as "polypeptide variants" or "variants." This polypeptide as defined in claim 1 is a polypeptide of SEQ ID NO: 1, 2, 3, 4, or 5. In another aspect, the peptide has improved transgalactosylation activity. The polypeptides defined herein are selected from SEQ ID NOs: 1, 2, 3, 4, or 5, showing improved reaction kinetics.

[0152] In one aspect, the polypeptides and variants defined herein have enzymatic activity. In one aspect, the polypeptides and variants defined herein are It has glycylating activity.

[0153] In one aspect, the ratio of transcosylating activity:β-galactosidase activity is 3%w / w initial lactose concentration, and 30 minutes after reaction, at least 0.5, at least Also be at least 1, at least 1.5 or at least 2.

[0154] In one aspect, the ratio of transcosylating activity:β-galactosidase activity is 3%w / w initial lactose concentration, and 30 minutes after reaction, at least 2.5, at least At least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least It will be at least 9, at least 10, at least 11, or at least 12.

[0155] In one aspect, the polypeptides and variants defined herein are derived from microbial sources, particularly The enzymes are typically derived from fungi or yeasts, or from bacteria. us), e.g., Agricus bisporus; Ascovaginosporum Ascovaginospora; Aspergillus, For example, Aspergillus niger (A. niger), Aspergillus awamori (A. awamori), Aspergillus foetidus (A. foetidus), Aspergillus Gillus japonicus (A. japonicus), Aspergillus oryzae (Ao ryzae);Candida;Chaetomium;Ke Chaetotomastia; Dictyosterium ostelium, e.g. D. discoi deum); Kluveromyces, e.g. Kluveromyces K. fragilis, K. lactis tis);Mucor, e.g. Mucor javanicus (M. javanicus) icus), Mucor mucedo (M.mucedo), Mucor subtilisium (M. subtilissimus; Neurospora, e.g., N.crassa; Rhizomucor For example, Rhizomucor pusillus; Rhizopus s), e.g. Rhizopus arrhizus, Rhizopus japinicus (R.japonicus), Rhizopus stolonifer (R.stolonifer) Sclerotinia, e.g. Sclerotinia liberti S.libertiana; Torula; Torulopsis lopsis); Trichophyton, e.g. Trichophyton T.rubrum; Whetzelinia, e.g. For example, W. sclerotiorum; Bacillus Bacillus, e.g., B. coagulans ), Bacillus circulans (B. circulans), Bacillus megaterium ( B. megaterium), Bacillus novalis, Bacillus subtilis, Bacillus pumilus, B. stearothermophilus, Bacillus thuringiensis (B. thuringiensis); Bifidobacterium (Bifidobacterium), e.g. Bifidobacterium longum (BI ongum), Bifidobacterium bifidum (B. bifidum), Bifidoba B.animalis; Chrysobacterium eobacterium; Citrobacter, e.g. Robacter freundii (C.freundii); Clostridium um), e.g. Clostridium perfringens (C. perfringens); Diplodia, e.g. Diplodia pocipina (D.go ssypina); Enterobacter, e.g. E. aerogenes, Enterobacter cloacae (E.cloacae) Edwardsiella, Edwardsiella E. tarda; Erwinia, e.g. Erwinia erwinia Rubicola (E. herbicola); Escherichia (E. E. coli; Klebsiella, e.g. For example, Klebsiella pneumoniae; iococcum;Myrothesium;Mucor ; Neurospora, e.g., Neurospora crassa (N .crassa); Proteus, e.g., Proteus vulgaris ( P. vulgaris); Providencia, e.g. Densia stuartii (P.stuartii); Pycnoporus For example, Pycnoporus cinnabarinus s), Pycnoporus sanguineus; Ruminococcus, e.g., Ruminococcus turkes (R .torques);Salmonella, e.g. Salmonella typhimurium S. typhimurium; Serratia, e.g. , Serratia liquefaciens (S. liquefasciens), Serratia marcescens S. marcescens; Shigella, e.g., Shigella La flexneri (S. flexneri); Streptomyces (Streptomyces ces), e.g., Streptomyces antibioticus (S.antibiotics icus), Streptomyces castaneoglobisporus (S.castaneoglobisporus) lobisporus, Streptomyces violeceorba (S. violece oruber;Trametes;Trichoderma ma), e.g., Trichoderma reesei, Trichoderma viride T.viride); Yersinia, e.g. Yersinia enterica There is Y. enterocolitica.

[0156] An isolated polypeptide comprising a polypeptide or variant polypeptide as defined herein. and / or the polypeptide produced. In one embodiment, the mutant polypeptide A peptide is a mature form of a polypeptide (SEQ ID NO: 1, 2, 3, 4, or 5). In one aspect, the variant comprises the C-terminal domain.

[0157] In one aspect, the polypeptide defined herein has the sequence of SEQ ID NO:1, Between one and about 25 amino acid residues are substituted for 2, 3, 4, or 5 sequences. In one aspect, the present invention includes variants in which a sequence has been added or deleted, as defined herein. The mutant polypeptides as described above have the sequences of SEQ ID NOs. 1, 2, 3, 4, or 5. Between 1 and 25 amino acid residues per sequence are substituted, added, or deleted. In one aspect, the variant is a variant of SEQ ID NO: 1, 2, 3 , 4, or 5 amino acid sequences, with any number of amino acids between 1 and about 25 substituted. In a further aspect, the variant is a variant of SEQ ID NO: 1, 2, 3, 4 or 5 amino acid sequence with any number of amino acids between 3 and 12 substituted. In a further aspect, the variant has SEQ ID NO:1, 2, 3, 4, or has a 5 amino acid sequence in which any number between 5 and 9 amino acids are substituted.

[0158] In one aspect, at least two of SEQ ID NOs: 1, 2, 3, 4, or 5 in another aspect, at least three, and in a further aspect, at least five amino acids. Has been replaced. In one aspect, the polypeptide disclosed herein is selected from SEQ ID NOs: 1, 2, In one aspect, the polysaccharides disclosed herein comprise 1, 2, 3, 4, or 5 . The peptide has the sequence of SEQ ID NO: 1, 2, 3, 4, or 5, and is , 9, 8, 7, 6, 5, 4, 3, 2, 1 amino acid substitution and / or is missing.

[0159] The enzymes and their variants are characterized by their nucleic acid and primary polypeptide sequences, and their three-dimensional folding. As defined herein, the polypeptides are characterized by their folding structure, their specific activity, and / or their specific amino acid sequence. Thus, additional characteristics of a polypeptide or polypeptide variant may be, for example, stability, The level of expression and enzyme activity can be determined by those skilled in the art, including pH range, oxidative stability, and thermal stability. In another aspect, the variant can be evaluated using known assays. For example, for the polypeptide of QID NO: 1, 2, 3, 4, or 5, It exhibits improved performance characteristics, such as improved stability at high temperatures, such as 85°C.

[0160] A polypeptide variant, as defined herein, is a polypeptide having a sequence similar to that of SEQ ID NO:1, 2, , 3, 4, or 5 sequences had at least 66%, 68%, 70%, 72%, 74%, 7 8%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 9 7%, 98%, 99%, and 100% sequence identity.

[0161] nucleotide In one aspect, the present invention provides a method for the preparation of ... With respect to isolated polypeptides, each polypeptide is purified under very low stringency conditions. Preferably, the reaction is carried out under low stringency conditions, more preferably under moderate stringency conditions. More preferably, the conditions are medium to high stringency, and even more preferably, high stringency. i) under stringent conditions, most preferably under very stringent conditions, SEQ ID NO: 1, 2, 3, 4, or 5 encoding the mature sequence O: 9, 10, 11, 12, or 13 nucleic acid sequence, and ii) the cDNA sequence of i), or or iii) by a polynucleotide that hybridizes to the complementary sequence of i) or ii). Encoded (J. Sambrook, E. F. Fritsch, and T. M aniatis,1989,Molecular Cloning,A Laborat ory Manual,2d edition,Cold Spring Harbor , New York). SEQ ID NO: 9, 10, 11, 12, or 13 A subsequence should consist of at least 100 contiguous nucleotides, preferably at least 200 nucleotides. Furthermore, this subsequence produces a polypeptide having lactase activity. It encodes a fragment.

[0162] The amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5, or a flag thereof ment and nucleotides of SEQ ID NO:9, 10, 11, 12, or 13 The sequences, or subsequences thereof, may be isolated from other genuses or species according to methods well known to those of skill in the art. A DNA encoding a polypeptide having transgalactosidase activity was isolated from the strain. It is used to design nucleic acid probes for identifying and cloning such The probes are hybridized to genomic or cDNA of the desired genus or species to perform Southern The corresponding gene is identified and isolated by blotting. The fragment is much shorter than the entire sequence, at least 14 bases, preferably at least 25 bases, and more preferably More preferably at least 35, and most preferably at least 70 nucleotides. However, the nucleotide probe length must be at least 100 nucleotides long. For example, the nucleic acid probes are preferably at least 200, more preferably at least At least 300, more preferably at least 400, or most preferably at least At least 500 nucleic acids in length. Longer probes may also be used, e.g., at least 600 nucleic acids in length. nucleotides, preferably at least 700 nucleotides, more preferably at least A probe of at least 800 nucleotides in length, or most preferably at least 900 nucleotides in length. Both DNA and RNA can be used. The probe is usually To detect genes that 32 P, 3 H, 35 S, proton, or avid Such probes are encompassed by the present invention.

[0163] Genomic DNA libraries prepared from such organisms can therefore be used in accordance with the abovementioned protocols. DNA that hybridizes to the polypeptide and encodes a polypeptide having lactase activity. Genomic or other DNA from such microorganisms is screened for agarologs. The proteins are separated by gel electrophoresis or polyacrylamide gel electrophoresis, or other separation techniques. The DNA from the library or isolated DNA is then transferred to nitrocellulose or other suitable support. SEQ ID NOs: 9, 10, 11, 12, and To identify clones or DNA homologous to 13 or their subsequences, The carrier material is used in Southern blotting.

[0164] For purposes of the present invention, hybridization refers to SEQ ID NOs: 9, 10, 1 1, 12, or 13, or a complementary sequence thereof, or any subsequence thereof A labeled nucleic acid probe corresponding to the sequence is subjected to nucleotide synthesis under highly stringent conditions. These conditions indicate that the nucleic acid probe hybridizes to the nucleotide sequence of the target nucleic acid. Hybridizing molecules can be detected using X-ray film.

[0165] In other preferred aspects, the nucleic acid probe is SEQ ID NO:9, 10, 11, 12, or 13, mature polypeptide coding regions.

[0166] Very low stringency for long probes of at least 100 nucleotides in length The lowest to highest stringency conditions were 42°C, 5x SSPE, 0.3% SD S, 200 g / ml shear-denatured salmon sperm DNA, and very low and low stringency 25% formamide for high stringency, 35% formamide for medium and medium to high stringency. 50% formamide for high and very high stringency This is followed by Southern blotting for 21 to 24 hours, optionally followed by prehybridization. The term is defined as fusion and hybridization.

[0167] For long probes of at least 100 nucleotides in length, the carrier material is finally diluted twice 0.5% SSC, 0.2% SDS, preferably at 45°C (very low stringency) -), more preferably 50°C (low stringency), more preferably at least 5 5°C (medium stringency), more preferably 60°C (medium-high stringency) 0°C (high stringency), more preferably at least 65°C (high stringency), and most preferably Most of the washes are performed at at least 70° C. (very high stringency).

[0168] In a particular embodiment, this washing is preferably performed with 0.2x SSC, 0.2% SDS. at least 45°C (very low stringency), more preferably at least 5 0°C (low stringency), more preferably at least 55°C (medium stringency) 50° C. (medium-high stringency), more preferably 60° C. (medium-high stringency), and even more preferably at least 65°C (high stringency), and most preferably at least 70°C ( In another particular embodiment, the washing is performed at 0 1x SSC, 0.2% SDS, preferably at least 45°C (very low storage temperature) more preferably at least 50° C. (low stringency), more preferably at least 50° C. (low stringency), Preferably at least 55°C (medium stringency), more preferably at least 60°C ( medium-high stringency), and more preferably at least 65°C (high stringency) -20°C (high stringency), and most preferably at least 70°C (very high stringency). can be.

[0169] For short probes that are about 15 to about 70 nucleotides in length, The concurrency conditions were: prehybridization, hybridization, and R Per milliliter of NA, 0.9 M NaCl, 0.09 M Tris-HCl, pH 7.6, 6mM EDTA, 0.5% NP-40, 1x Denhardt's solution, 1mM Sodium phosphate, 1 mM sodium monophosphate, 0.1 mM ATP, and 0.2 mM g yeast, Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences) edings of the National Academy of Sciences es USA 48:1390) This is defined as post-hybridization washes at 20°C, followed by standard Southern blotting. The law is carried out.

[0170] For short probes, which are between about 15 and about 70 nucleotides in length, the carrier The quality was calculated by performing one run of 15 min in 6x SSC plus 0.1% SDS and two runs of 15 min each. Wash with 6x SSC at 5-10°C below the Tm.

[0171] Under salt-containing hybridization conditions, the effective Tm indicates successful hybridization. The degree of identity required between the probe and the filter-bound DNA for the The following formula is used to control the hybridization rate under each stringency condition. Determine the degree of identity required for the two DNA fragments to be resized.

[0172] Effective T m =81.5+16.6(log M[Na + ])+0.41(%G+C)-0 .72 (% formamide) ( www.ndsu.nodak.edu / instruct / mcclean / pls See c731 / dna / dna6.htm ) The G+C content of SEQ ID NO:1 is 42% and the G+C content of SEQ ID NO:11 is 42%. At medium styrancy, the +C content is 44%. At medium styrancy, the formamide is 35%. The Na+ concentration in 5x SSPE is 0.75M.

[0173] Another relevant correlation is that a 1% mismatch between two DNAs reduces the Tm by 1.4°C. Two DNAs that hybridize under moderately stringent conditions at 42°C To determine the degree of identity required, the following formula is used: % identity = 100 - [(effective Tm - hybridization temperature / 1.4)] (www.ndsu.nodak.edu / instruct / mcclean / pls See c731 / dna / dna6.htm. ) The variant nucleic acid is a nucleic acid encoding SEQ ID NO: 1, 2, 3, 4, or 5; For example, 60%, 65%, 75%, 805, 85%, 90%, 95%, or 99% of the sequence In one aspect, the present invention includes polynucleotides having a specific percentage of identity, such as Nucleic acids capable of encoding the polypeptides defined herein are provided. In a further aspect, the nucleic acid disclosed herein is selected from SEQ ID NOs: 9, 10, 11, 12, or 13 at least 60%, at least 65%, at least 70%, or at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical nucleic acid sequence.

[0174] In one aspect, there is provided a plasmid comprising a nucleic acid as described herein. In one aspect, an expression vector comprising the nucleic acid disclosed herein is provided. In one aspect, a nucleic acid as defined herein or a nucleic acid as defined herein is An expression vector capable of expressing a polypeptide comprising the polypeptide is provided.

[0175] Complementary to a nucleic acid encoding any polypeptide variant as defined herein. A nucleic acid is provided. That is, a nucleic acid capable of hybridizing to a complement is provided. Therefore, the sequences used in the methods and compositions disclosed herein are synthetic sequences. These include, but are not limited to, sequences that are codon-optimized for expression in suitable host microorganisms. The polypeptide variants provided herein may be produced synthetically, as known to those of skill in the art. In one aspect, the present invention is produced intracellularly or by recombinant expression in a host cell. The polypeptides disclosed herein are recombinant polypeptides. The expressed polypeptide variants are optionally isolated prior to use. Polypeptide variants as defined herein are produced after expression. Methods for genetic modification and recombinant production of variants are described, for example, in U.S. Pat. Nos. 7,371,525, 7,372,525, and 7,373,525. 52; 7,166,453; 6,890,572; and 6,667,065; and U.S. Patent Application Publication Nos. 2007 / 0141693;2007 / 0072270;20 07 / 0020731;2007 / 0020727;2006 / 0073583;200 6 / 0019347;2006 / 0018997;2006 / 0008890;2006 2005 / 0137111; and 2005 / 0008888; Encoding polynucleotide sequences, primers, vectors, selection methods, host cells, expression Useful buffers for purification and reconstitution of expressed polypeptide variants and for enzyme assays. , pH range, Ca2+ concentration, substrate concentration, and enzyme concentration of the polypeptides of the present invention. The relevant disclosures contained in these documents, including characterizations, are incorporated herein by reference. .

[0176] or encoding a protein of SEQ ID NO: 1, 2, 3, 4, or 5; A nucleic acid encoding a protein of SEQ ID NO:1, 2, 3, 4, or 5 At least 60%, 68%, 70%, 72%, 74%, 78%, 80%, 85%, 90%, Nucleic acid sequences with 95%, 96%, 97%, 98%, 99% or 100% sequence identity is provided.

[0177] vector In one aspect, the invention relates to a vector comprising the polynucleotide. In some embodiments, the bacterial cell contains the vector. A DNA construct containing an encoding nucleic acid may include a regulatory sequence operably linked to the coding sequence. The vector is delivered to the host cell in an expression vector containing the gene. Any vector that can be integrated into the genome of a filamentous fungal host cell and replicate therein can be used. stomach. The FGSC Catalog of Strains,Universi The University of Missouri lists suitable vectors. Additional examples of integrating vectors are described in Sambrook et al., Mol. ecular Cloning: A Laboratory Manual, 3 rd e d.,Cold Spring Harbor Laboratory Press,C old Spring Harbor,New York(2001);Bennett et al.,More Gene Manipulations in Fungi ,Academic Press,San Diego(1991),pp.396-4 28; and U.S. Pat. No. 5,874,276. Exemplary vectors include pFB6, pBR322, PUC18, pUC100, pENTR / D, and pDON TM 201 , pDONR TM 221, pENTR TM , pGEM R 3Z and pGEM R Recorded in 4Z Examples for use in bacterial cells include pBR322 and pBR322. UC29, which replicates in Escherichia coli (E. coli) and Additionally, pE194 replicates in, for example, Bacillus.

[0178] In some embodiments, the nucleic acid encoding the variant is operably linked to a suitable promoter. The promoter is homologous to the host cell and is transcribed in the host cell. The proteins are derived from genes encoding either native or heterologous proteins. Suitable, non-limiting examples of promoters include cbh1, cbh2, egl1, and egl2 promoters. In one embodiment, the promoter is native to the host cell. For example, Pseudomonas saccharophila (P. saccharophila a) is the host cell, this promoter is expressed in the native Pseudomonas saccharophila (P. saccharophila promoter. An "inducible promoter" is an environmentally or is a promoter that is active in developmental regulation. The gene is one that is heterologous to the host cell.

[0179] In some embodiments, the coding sequence is engineered to a DNA sequence that encodes a signal sequence. In another embodiment, the representative signal peptide is represented by SEQ ID NO: The representative signal peptide is Bacillus subtilis. SEQ ID NO: 1 is the native signal sequence of the aprE precursor of S. subtilis. In another embodiment, the DNA encoding the signal sequence is Nucleotides encoding a signal sequence from an exogenous Bacillus subtilis precursor In one embodiment, the DNA encoding the signal sequence is directly This is then upregulated, leading to inflation of the polynucleotide encoding this polypeptide. The signal sequence is selected from the same species as the host cell. The signal sequence and promoter comprising the DNA construct or vector to be introduced into a filamentous fungal host cell. The promoter sequences are from the same source. In some embodiments, the expression vector contains a termination sequence. In one embodiment, the termination sequence and the promoter sequence are of the same origin. In embodiments, the terminator sequence is homologous to the host cell.

[0180] In some embodiments, the expression vector comprises a selectable marker. Examples include those that confer resistance to antibiotics such as hygromycin and phleomycin. Nutritional selection markers are also suitable and include amdS, argB, and pyr4. In one embodiment, the selectable marker is the amdS gene, which encodes the enzyme acetamidase. The selection vector encodes a nucleotide sequence that allows the transformed cells to grow on acetamide as a nutrient source. Aspergillus nidulans amdS gene as a marker The article is based on Kelley et al., EMBO J. 4:475-479 (1985) and and Penttila et al., Gene 61:155-164 (1987). The present invention relates to a method for the preparation of a nucleic acid sequence for a human ovarian cancer cell line comprising the steps of: A suitable expression vector is capable of autonomous replication in a given host cell and of integrating into the DNA of the host cell. In some embodiments, the expression vector is a plasmid. In some embodiments, two types of expression vectors are used to obtain expression of genes: The first expression vector includes a promoter, a coding region, and a and a DNA sequence including a terminator. The NA sequence was deleted to create a domain expressed under the control of its own transcriptional and translational regulatory sequences. The second type of expression vector is a vector that induces high levels of transcription. It is an expression vector that has been pre-constructed to contain sequences required for transcription and selection markers. In some embodiments, the coding region of a gene or a portion thereof is a gene for general purpose expression. Inserted into the vector, the expression construct is under the control of a promoter and terminator sequence. In some embodiments, the gene or portion thereof is It is inserted downstream of the

[0181] Expression host / host cell In a further aspect, a plasmid as described herein or a gene encoding a gene as described herein. Host cells are provided which contain, or preferably are transformed with, the present vectors. In a further aspect, a cell capable of expressing a polypeptide as described herein is Provided. In one aspect, a host cell as described herein, or a method for the production of a Suitable cells are bacteria, fungi, or yeast cells. In a further aspect, the host cell is a Ruminococcus. Bifidobacterium, Lactococcus ococcus, Lactobacillus, Streptococcus Streptococcus, Leuconostoc, Escherichia coli Escherichia, Bacillus, Streptoma Streptomyces, Saccharomyces ), Kluyveromyces, Candida, Torula, Torulopsis, and Aspergillus The fungus is selected from the group consisting of Aspergillus. In a further aspect, the host cell is Ruminococcus hansenii. ccus hansenii, Bifidobacterium breve terium breve, Bifidobacterium longum erium longum, Bifidobacterium infantis bacterium infantis, Bifidobacterium bifidum (Bif idobacterium bifidum and Lactococcus lactis (Lac tococcus lactis).

[0182] In another embodiment, a suitable host cell is Bacillus subtilis. ubtilis), Bacillus licheniformis, Bacillus lentus (B. lentus), Bacillus brevis (B. brevis), Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefa amyloliquefaciens, Bacillus coagulans B. coagulans), Bacillus circulans, Bacillus lautus (B.lautus), Bacillus thuringiensis (B.thuri ngiensis), Streptomyces lividans (Streptomyces l ividans, or Streptomyces murinus or Escherichia coli. ichia coli) or Pseudomonas speci es), including gram-negative bacteria.

[0183] In some embodiments, the host cell comprises SEQ ID NO:1, 2, 3, 4, or 5. at least about 66%, 68%, 70%, 72%, 74%, 78%, 80%, %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% %, 99%, 100% identity to the amino acid sequence of the polypeptide as defined herein. In some embodiments, the polypeptide is modified to express a peptide variant as defined herein. The polynucleotide encoding the polypeptide variant to be treated is SEQ ID NO:1, Nucleic acid sequences encoding proteins 2, 3, 4, or 5 or SEQ ID N O: At least about 6 to the nucleic acid sequence encoding protein 1, 2, 3, 4, or 5 6%, 68%, 70%, 72%, 74%, 78%, 80%, 85%, 90%, 95%, 9 6%, 97%, 98%, 99%, or 100% sequence identity. In one embodiment, the nucleic acid sequence is SEQ ID NO:9, 10, 11, 12, or Thirteen nucleic acids have at least about 60%, 66%, 68%, 70%, 72%, 74%, 78%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity has.

[0184] Methods for Producing Polypeptides In a further aspect, a method of expressing a polypeptide disclosed herein comprises the steps of: Obtaining the indicated host cell or cells, and extracting the polypeptide from the host cell or cells. The method comprises expressing a polypeptide, and optionally purifying the polypeptide.

[0185] Expression properties refer to the altered expression level of a variant when the variant is produced in a particular host cell. Expression refers to the expression of a gene for a given time using standard methods known to those of skill in the art. Expression is related to the amount of active mutant that can be recovered from the fermentation broth produced by the host cell. It also refers to the amount or proportion of mutant polypeptides produced in a cell or cells. It also affects the rate of translation of the mRNA encoding the peptide.

[0186] Host cell transformation, expression, and culture Introduction of a DNA construct or vector into a host cell is known as transformation, eletrophoresis, or transfection. Microporation, nuclear microinjection, transduction, transfection, For example, lipofection-mediated and DEAE-dextrin-mediated transfection mediated by calcium phosphate DNA precipitates, incubation with DNA-coated microplates These include high-velocity bombardment with a projector and protoplast fusion. Conversion techniques are known. See, e.g., Ausubel et al. (1987), sup ra,chapter 9;Sambrook et al. (2001), supr a; and Campbell et al., Curr. Genet. 16:53-56 (1989). Expression of heterologous proteins in Trichoderma has been disclosed, e.g. For example, U.S. Patent No. 6,022,725; U.S. Patent No. 6,268,328; Hark ki et al., Enzyme Microb.Technol.13: 227 -233(1991);Harkki et al.,BioTechnol.7:59 6-603 (1989); EP 244,234; and EP 215,594. In one embodiment, a generally stable transformant contains a nucleus encoding the mutant. The gene is constructed in a vector system that allows the gene to be stably integrated into the host cell chromosome. The transformant is then refined by local techniques.

[0187] In one non-limiting example, stable transformants containing the amdS marker exhibit a reduced growth rate. and a smooth rather than jagged outline on solid media containing acetamide. They are distinguished from unstable transformants by the formation of small round colonies. In cases where further testing for stability is required, a solid non-selective medium such as acetamide-free medium may be used. Growth of transformants on acetamide, harvesting of spores from the culture medium, and incubation of these spores in acetamide. This is done by determining the percentage of budding and growth on a selective medium containing Other methods known to those skilled in the art can also be used to select transformants.

[0188] Identification of activity To assess expression of the variant in a host cell, the assay comprises measuring the expressed protein, The corresponding mRNA or galactosidase activity can be measured. For example, Preferred assays are Northern and Southern blotting using suitably labeled probes. method, RT-PCR (reverse transcription polymerase chain reaction), and in situ hybridization Suitable assays include measuring activity in a sample. Suitable assays for the activity of Such methods include ONPG-based assays or methods for measuring glucose in a reaction mixture. Not limited to these.

[0189] Methods for purifying the polypeptides disclosed herein Generally, variants produced in cell culture are secreted into the medium, e.g., The medium is purified or isolated by removing undesired components. In such cases, the mutants are recovered from the cell lysate. These are purified from cells produced by techniques that are routinely used in the art. Examples include: ion exchange chromatography methods, including affinity chromatography and high resolution ion exchange; Hydrophobic interaction chromatography, biphasic partitioning, ethanol precipitation, reversed-phase HPLC, DEAE, etc. Chromatography, chromatofocusing, etc. on silica or cation exchange resins. SDS-PAGE, ammonium sulfate precipitation, and separation using e.g. Sephadex G-75 Depending on the mode of use, the methods disclosed herein may include, but are not limited to, gel filtration. The polypeptides are, for example, lyophilized or prepared in solution. In other embodiments, the polypeptides disclosed herein are in a lyophilized form. The polypeptides disclosed herein are in solution.

[0190] Compositions, Products and Uses Methods for immobilizing and formulating the polypeptides disclosed herein The polypeptide compositions may be prepared by methods known to those skilled in the art and may be in the form of a liquid or dry composition. For example, the polypeptide composition is in the form of granules or microgranules. The polypeptides of the invention may be stabilized according to methods known to those skilled in the art. Examples of suitable uses of the peptide or polypeptide compositions are given below.

[0191] In one aspect, disclosed herein is a method for producing a polypeptide as disclosed herein. A method for treating a substrate containing lactose with a peptide to produce a food product.

[0192] In one aspect, disclosed herein is a polypeptide as disclosed herein. and a process for treating a milk-based substrate containing lactose to produce a dairy product.

[0193] In one aspect, the lactose-containing substrate is further treated with a hydrolyzing beta-galactosidase. To be processed.

[0194] The enzyme preparations, such as in the form of food ingredients prepared according to the present invention, can be used and / or may be in the form of a solution or a solid, depending on the mode of use and / or the method of administration. The product form can be either a dried enzyme powder or granular enzyme. Examples of formulations include spray-dried products, mixed granule products, granules produced in a fluidized bed, and extrusion. or layered products such as granules produced by pelletization, small spherical products, freeze-dried products, etc. be.

[0195] The enzyme preparations, such as in the form of food ingredients prepared according to the present invention, can be used and / or may be in the form of a solution or a solid, depending on the mode of use and / or the method of administration. The product form can be either a dried enzyme powder or a granular enzyme.

[0196] In one aspect, preferably the food composition, more preferably the food composition disclosed herein. In accordance with another aspect of the present invention, there is provided a composition that is a dairy product comprising the cells or polypeptides.

[0197] Further disclosed herein are 72%, 74%, 74% and 75% homologues of SEQ ID NO:22. %, 78%, 80%, 82%, 84%, 86%, 88%, 90% 0% sequence identity to the polypeptides disclosed herein based on the total amount of the composition. One or more polypeptides containing 10%, 15%, 20%, 25%, 30%, 35%, 40% This is a composition containing at least 5%, such as 45%, 50% w / w. The samples to be evaluated are subjected to SDS-PAGE. The dyes are suitable for protein quantification, such as the Bio-Rad Criterion system. The gel is then visualized using a Bio-Rad Criterion system. The resulting photograph is scanned using a suitable densitometric scanner. Any variant / s derived from SEQ ID NO:8 are guaranteed to be in the genetic range. The bands corresponding to the fragments were quantified and the percentages of polypeptides were as follows: It is calculated as follows: Percentage of intended polypeptide = Intended polypeptide / (T The sum of all polypeptides with transgalactosylation activity) * 100. The total number of mutants / fragments derived from SEQ ID NO:8 in the composition is known to those skilled in the art. According to the method, Western blotting using polyclonal antibodies was performed to detect S This can be determined by detecting a fragment derived from EQ ID NO:8.

[0198] In one aspect, the composition of the invention comprises SEQ ID NO: 1, 2, 3, 4, or 5, comprising one or more polypeptides selected from the group consisting of: In a further aspect, the composition comprises a polypeptide consisting of SEQ ID NO:1 or 2. The polypeptides include one or more polypeptides selected from the group consisting of:

[0199] In one aspect, the present invention relates to an enzyme conjugate of the present invention, an enzyme carrier, and optionally a stabilizer. and / or a preservative.

[0200] In a further aspect of the invention, the enzyme carrier is selected from the group consisting of glycerol or water. can be.

[0201] In a further aspect, the preparation / composition comprises a stabilizing agent. The agent is selected from the group consisting of inorganic salts, polyols, sugars, and combinations thereof. In one aspect, the polyol is glycerol, propylene glycol, or sorbitol. In yet another aspect, the sugar is a small carbohydrate, particularly glucose, galactose, or the like. Sweet substances such as sugar, fructose, and sucrose.

[0202] In a further aspect, the preparation comprises a preservative. In one aspect, the preservative is methylparaben. Laben, propylparaben, benzoic acid, sorbic acid, or other food preservatives or It is a mixture of.

[0203] The method of the present invention involves the use of immobilized enzymes, such as immobilized lactase or other galactase. This is done using a tooligosaccharide-forming enzyme, which can be used on any organic or inorganic support. Exemplary inorganic supports include alumina, celite, Dowex, etc. Exemplary organic supports include DEAE-1-chloride, glass beads, and silica gel. The present invention includes cellulose, alginate hydrogel, or alginate beads or equivalents. In various aspects, the immobilization of lactase is achieved by physical adsorption onto inorganic supports. The enzymes used in the practice of the invention are stored in water, Tris-HCl buffer, and Enzymes can be immobilized in other media, including phosphate buffer and cellulose. Any type of substrate, such as a filter, fiber, column, bead, colloid, gel, hydrogel, or mesh The antibody can be immobilized on any type of substrate.

[0204] In one aspect, a milk-based substrate containing lactose is treated with a polypeptide of the invention. In a further aspect, a method is provided for producing a dairy product by processing 15 After 1 min reaction, relative transgalactosylation of 60%, 70%, and 75% or more was observed. US201002326333A1 - Treating milk-based substrates containing lactose with polypeptides having the activity to produce dairy products - Google Patents In one aspect, a method is provided for determining whether the relative transgalactosylation activity is greater than or equal to 30. After 1 min of reaction, the relative transgalactosylation activity is 3 or more. is greater than or equal to 6 after 30 minutes of reaction. The activity is greater than or equal to 12 after 30 minutes of reaction. Here, treatment with the polypeptides disclosed herein provides optimal temperatures for the activity of the enzyme. In a further aspect, the polypeptide is at a concentration of 0.01-1000 ppm. In a further aspect, the polypeptide is added to a milk-based matrix at a concentration of 0.1-100 pM. In a further aspect, the polypeptide is added to the milk-based matrix at a concentration of 1- pm. In one aspect, the microbial dairy product is added to a milk-based substrate at a concentration of 10 ppm. In a further aspect, the method further comprises the step of fermenting a substrate such as a food product. The dairy product is yogurt. In a further aspect, the treatment with the polypeptide and the microorganism In one aspect, the polypeptide and the microorganism are simultaneously In one aspect, the cells or Dairy products comprising the polypeptide are provided. The polypeptide is added at a concentration of 0.01-1000 ppm. Dairy products are provided that include an inactivated polypeptide as described herein. In this respect, the inactivated polypeptides described herein are used at a concentration of 0.01-1000 ppm. In one aspect, a dairy product is provided comprising a polypeptide as defined herein. A dairy product is provided that includes a GOS formed in situ by a peptide. In accordance with the present invention, there is provided a dairy product comprising the cells described herein. For example, skim milk, low-fat milk, whole milk, cream, UHT milk, long-life milk, fermented milk Milk products, cheese, yogurt, butter, dairy spreads, buttermilk, acidulants Milk drinks, sour cream, whey-based drinks, ice cream, condensed It can be milk, such as dulce de leche or a flavored milk drink. The product may be manufactured by any method known to those skilled in the art.

[0205] Dairy products may additionally contain non-dairy ingredients, e.g. vegetable oils, vegetable proteins, and / or vegetable ingredients such as vegetable carbohydrates. In addition, enzymes, flavors, microbial culture media such as probiotic culture media, salts, sweeteners, sugars, acids, fruits, Root juice, or any ingredient in or added to dairy products known to those skilled in the art. It may also contain further additives, such as any other ingredients.

[0206] In one embodiment of the invention, the one or more milk components and / or milk fractions are dairy products. At least 70%, for example at least 80%, preferably at least 90%, of the product. , accounting for at least 50% (w / w).

[0207] In one embodiment of the present invention, a galactosylating agent as defined herein having transgalactosylation activity is provided. One or more milk-based substrates treated with the enzymes described herein are at least 70% dairy, e.g. At least 50% (weight / weight), for example at least 80%, preferably at least 90%. (amount)

[0208] In one embodiment of the present invention, the dairy product is a pre-produced galacto-oligosaccharide. This is a dairy product that does not become thicker due to the addition of amides.

[0209] In one embodiment of the invention, the polypeptide-treated milk-based substrate is It is not dried before being used as an ingredient.

[0210] In one embodiment of the invention, the dairy product is ice cream. Ice cream can be full fat ice cream, low fat ice cream, or yogurt or It can be any type of ice cream, such as ice cream based on other fermented milk products. The ice cream is prepared by methods known to those skilled in the art.

[0211] In one aspect of the invention, the dairy product is milk or condensed milk. In one embodiment of the present invention, the dairy product is UHT milk. Milk is pasteurized to kill all microorganisms, including bacterial spores. The UHT (ultra-high temperature) treatment temperature is 130℃ for 30 seconds or 145℃ for 1 second. The processing temperature.

[0212] In one preferred embodiment of the present invention, the dairy product is ESL milk. The ESL milk used is filtered and / or heat treated to extend its shelf life and can be stored at 2-5°C. Remains fresh for at least 15 days, preferably at least 20 days, when stored at room temperature. It is a milk that can be used.

[0213] In another preferred embodiment of the present invention, the dairy product is a fermented dairy product, such as yogurt. It is a product.

[0214] The microorganisms used for most fermented dairy products are usually referred to as lactic acid bacteria. As used herein, "lactic acid bacteria" refers to a group of bacteria that are The term "lactic acid" refers to a strain that produces lactic acid as the primary acid produced, with a range of acids including acetic and propionic. In the production, it is intended to use gram-positive, microanaerobic or aerobic bacteria that ferment sugars. The most useful lactic acid bacteria in the world are the Lactococcus spp. .), Streptococcus spp., Lactobacillus Lactobacillus spp., Leuconostoc spp. stoc spp., Pseudoleuconost oc spp.), Pediococcus spp., Brevi Bacterium spp., Enterococcus spp. Interococcus spp. and Propiobacterium spp. It is found in the order "Lactobacillus", which includes Lactobacillus spp. Generally, lactic acid-producing bacteria are classified as anaerobic bacteria, bifidobacteria, i.e. Bifidobacteria are frequently used as food cultures either alone or in combination with lactic acid bacteria. Bifidobacterium spp. is a group of lactic acid bacteria. Lactic acid bacteria are usually found in frozen or freeze-dried media for bulk starter propagation or in "Die" for direct inoculation into fermentation tubes or vats for the production of fermented dairy products It is called the “Direct Vat Set” (DVS). Such media are commonly referred to as "starting media." These are called "culture medium" or "starters."

[0215] Commonly used starting culture strains of lactic acid bacteria usually have an optimum growth temperature of about 30°C. and a thermophilic microorganism having an optimum growth temperature in the range of about 40-45° C. A typical microorganism belonging to the mesophilic group is Lactococcus lactis ( Lactococcus lactis, Lactococcus lactis subsp. cremoris (Lactococcus lactis subsp.cremoris), Leucono Stock Mesenteroides subsp. cremoris (Leuconostoc mesenter oides subsp. cremoris), Preseudoleuconococcus mesentero Ides subsp. cremoris (Pseudoleuconostoc mesenteroides cremoris), Pediococcus persaceus (Pedi ococcus pentosaceus), Lactococcus lactis subsp. lactis Biovariety Diacetylactis (Lactococcus lactis lactis biovar. diacetylactis), lactis Lactobacillus casei subsp. casei and Lactobacillus paracasei subsp. paracasei (Lactobaci paracasei subsp. paracasei). Thermophilic lactic acid bacteria species include, for example, Streptococcus thermophilus. tococcus thermophilus), Enterococcus faecium (E interococcus faecium, Lactobacillus delbrueckii subsp. lactobacillus Lactobacillus delbrueckii subsp. lact is), Lactobacillus helveticus icus, Lactobacillus delbrueckii subsp. bulgaris delbrueckii subsp. bulgaricus), and Lactobacillus Lactobacillus acidophilus is one example. Bifidobacterium bifidum ), Bifidobacterium anima lis) and Bifidobacterium longum (Bifidobacterium l ongum) belongs to the Bifidobacterium Anaerobic bacteria are also commonly used in dairy starter cultures and belong to the group of lactic acid bacteria. Additionally, Propionibacteria species include It is used as a dairy starter medium, especially in cheese production. Brevibacterium is commonly used as a starting medium for food. Other groups of microbial starting media include yeast media and fungal media, which include media for filamentous fungi. , which are particularly used in the production of certain types of cheese and beverages. Exemplary filamentous fungi include , Penicillium roqueforti, Penicillium candidum, Geotrichum Geotrichum candidum, Torula kefir la kefir, Saccharomyces kefi r) and Saccharomyces cerevisiae siae).

[0216] In one embodiment of the invention, the microorganism used for fermentation of the milk-based substrate is Lactobacillus subtilis. Lactobacillus casei or Streptococcus casei Streptococcus thermophilus and Lactococcus Bacillus delbrueckii subspecies bulgari (Lactobacillus delbruec kii subsp. bulgaricus).

[0217] The fermentation processes used in the method of the present invention are well known and will be understood by those skilled in the art to , oxygen, the amount and nature of microorganisms, additives such as hydrocarbons, flavors, minerals, enzymes, and processing They know how to select suitable process conditions, such as the fermentation time, etc. The conditions are selected to support the accomplishment of the present invention. The fermentation results in the production of a milk-based substrate. The pH of the fermented dairy product of the present invention is preferably in the range of 3.5-5. Most often it will be in the range of 3.5-6, such as 3.8-4.8.

[0218] In one aspect, a polypeptide or one or more oligosaccharides are used to Methods are provided for using cell types to produce the oligosaccharides. galacto-oligosaccharides, isomalto-oligosaccharides lysides, malto-oligosaccharides, lactosucrose, and xyloligosaccharides These include, but are not limited to, ido.

[0219] In one embodiment of the invention, the oligosaccharides are lactose, trehalose, rum In a medium containing disaccharides such as glucose, maltose, sucrose, lactose or cellobiose, The antibody is produced by incubating cells expressing the polypeptide in a . The incubation is carried out under conditions that allow the production of oligosaccharides. , cheese, fermented milk products, food supplements, and probiotic foods. Alternatively, the oligosaccharides are recovered and subsequently It is added to the desired product before or after its preparation.

[0220] In one aspect, yogurt, cheese, fermented milk products, food supplements, and and a probiotic food product, Uses of the cells are provided.

[0221] In one aspect, the polypeptides disclosed herein are used in the preparation of cheese products. and a method for producing said cheese product. The cheese is selected from the group consisting of cheese, cottage cheese, and processed cheese. By adding peptides, cheeses are enriched with galacto-oligosaccharides. In one aspect, the final cheese product has a higher lactose content and a lower lactose level. The lactose level of the lactose is at least 25 percent, preferably at least 50 percent. and more preferably at least 75 percent lower. This amount has been reduced to less than 1 gram per serving, an amount that is tolerated by most lactose intolerant individuals. The amount of lactose in a cheese product that can be produced is called lactose.

[0222] The cheese products provided herein are high in soluble fiber, low in calories, and have excellent Nutritionally improved cheese with improved organoleptic properties, improved texture and aroma Further, the polypeptides disclosed herein are capable of binding to lactose or other sugars. It is absorbed more slowly than hydrolysates, thus reducing the glycemic index of this cheese product. Finally, this polypeptide, GOS, surprisingly provided improved technology for cream cheese. Provides texture to reduce stabilizer use or to increase moisture content without syneresis This reduces the production costs of cheese products, especially cream cheese.

[0223] In a further aspect, a composition comprising a polypeptide as disclosed herein and a carbohydrate substrate is provided. In a further aspect, the carbohydrate substrate is a disaccharide. Disaccharides include, for example, lactose, trehalose, rhamnose, maltose, sucrose, In a further aspect, the carbohydrate substrate is lactose or cellobiose. The composition is prepared to produce oligosaccharides. The disclosed polypeptides may be used in products such as yogurt, cheese, fermented milk products, and food supplements. , and probiotic foods. In one aspect, a composition is provided that includes a polypeptide disclosed herein and a stabilizer. Exemplary stabilizers include, for example, polyols, such as glycerol or propylene glycol; Sugar or sugar alcohol, lactic acid, boric acid, or boric acid derivatives (e.g. aromatic boric acid esters) (L).

[0224] In one aspect, the transgalactosylated polypeptides disclosed herein or Use of the cells disclosed herein for the production of galacto-oligosaccharides is provided. In one aspect, the transgalactosylated polypeptides disclosed herein are or the cells disclosed herein. and probiotic foods. In one aspect, the product is a method for producing a saccharide comprising the steps of: In one aspect, the present invention relates to a yogurt, cheese, or fermented milk product. The transgalactosylated polypeptide or the cell disclosed herein, The use of galacto-oligosaccharides to enhance the growth of bacteria is provided. In one aspect, the transgalactosylated polypeptides disclosed herein are or the cells disclosed herein in mixed medium fermentation of Bifidobacterium ( Galacto-oligosaccharides for enhancing the growth of Bifidobacterium The use of

[0225] In one aspect, the present invention is characterized in that the polypeptide is expressed in a suitable medium under suitable conditions. The method includes culturing the polypeptide disclosed herein and recovering the polypeptide from the culture medium. For the production of the transgalactosylated polypeptides disclosed herein, comprising a harvesting step. A method is provided for producing the polypeptide of the present invention by culturing the polypeptide of the present invention in a suitable medium under suitable conditions for expression of the polypeptide. The present invention relates to a method for culturing a polypeptide and a method for recovering the polypeptide from a culture medium. A method for producing galacto-oligosaccharides is provided, comprising the steps of:

[0226] The addition of oligosaccharides increased the activity of Bifidobacterium Bifidobacterium, either alone or in a mixture Increase the growth of either.

[0227] Treating milk with enzymes that convert lactose to monosaccharides or GOS has several advantages. First, this dairy product may be harmful to people with lactose intolerance, who may experience symptoms such as bloating and diarrhea. Second, lactase-treated dairy products contain less lactose than lactose. The sweetness of the glucose and galactose was higher than that of the similar untreated product due to the higher sweetness perception of glucose and galactose. This effect is particularly evident in yogurt, ice cream and other products where a high degree of sweetness is desired in the final product. Of particular interest in applications such as creams, where this can reduce the net sugar content in the product. Third, in ice cream production, lactose molecules crystallize, decreasing the relative solubility of milk sugar. The phenomenon known as "gritty" is often observed due to the low alcohol content. When converted to simple sugars or GOS, the mouthfeel of the ice cream is significantly improved compared to the untreated counterpart. The presence of lactose crystallization can be eliminated, and the whey powder is skimmed off the fat. By replacing milk powder, raw material costs can be reduced. Main effects of enzyme treatment is an increase in sweetness.

[0228] In one aspect, the transgalactosylated polypeptides disclosed herein are Proteases such as sarcolemma or renin, lipases such as phospholipase, amylase, It can be used together with other enzymes such as transferase and lactase. In an aspect, the transgalactosylating polypeptides disclosed herein are capable of binding to lactase. The transgalactosylated polypeptides disclosed herein are used together. If subsequent lactose reduction is desired, sometimes lower lactose levels are preferred. Lactase in the context of the present invention is useful for converting the disaccharide lactose to galactose. and any glycosylation hydrolase having the ability to hydrolyze glucose into its monosaccharide components. The lactose group is assigned to the subclass EC 3.2.1.108. Other sub-categories assigned to EC3.2.1.23, etc. The class is also lactase in the context of the present invention. The transgalactosylation active saccharide may have an activity other than the lactose hydrolysis activity of the transgalactosylation active saccharide. In the context of the present invention, the lactose hydrolyzing activity of a lactase is also referred to as its lactase activity or The galactosidase activity of the lactose used in the method of the present invention is Enzymes with tase activity may be of animal, vegetable or microbial origin. They are derived from biological sources, in particular from filamentous fungi or yeasts, or from bacteria. For example, Agaricus, such as Agaricus porus (A. bispo rus); Ascovaginospora; Aspergillus (Aspergillus), e.g. Aspergillus niger (A. niger), Aspergillus Aspergillus awamori (A. awamori), Aspergillus foetidus (Af oetidus), Aspergillus japonicus (A. japonicus), Aspergillus A.oryzae; Candida; Chaetomium (Chaetomium); Chaetotomastia; Dictyostelium, e.g. Dictyostelium di D.discoideum; Kluveromyces es) for example, K. fragilis, K. K.lactis; Mucor, e.g. Mucor japonica M.javanicus, M. mucedo, Muco M. subtilissimus; Neurospora rospora), e.g. Neurospora crassa; Rhizomucor Rhizomucor, for example, R. pusillus Rhizopus, for example (Rhizopus arrhizus) s), Rhizopus japonicus (R. japonicus), Rhizopus stolonifer (R. stolonifer); Sclerotinia, e.g. For example, Sclerotinifa libertiana (S. libertiana); Torula ula);Torulopsis;Trichophyton yton), such as Trichophyton rubrum (T. rubrum); Wetzeria ( Whetzelinia, e.g. Wetzelia sclerotium (W. sclerotium) iorum; Bacillus, e.g., Bacillus coagulans (B .coagulans), Bacillus circulans (B.circulans), Bacillus megaterium (B. megaterium), Bacillus novalis (B. nova lis), Bacillus subtilis (B. subtilis), Bacillus pumilus (B. pumilus), Bacillus stearothermo philus), Bacillus thuringiensis (B. thuringiensis); Bifidobacterium, e.g. Bifidobacterium longum (B. longum), Bifidobacterium bifidum (B. bifid um), Bifidobacterium animalis (B. animalis); Chryseo- bacterium Chryseobacterium; Citrobacterium r), e.g. Citrobacter freundii; Clostridium Clostridium, e.g. Clostridium perfringens (C. perfringens) ingens);Diplodia, e.g. Diplodia gossypina ( D. gossypina); Enterobacter, e.g. Enterobacter aerogenes (E. aerogenes), Enterobacter coloar E. cloacae; Edwardsiella, e.g. Dowageria tarda (E. tarda); Erwinia, e.g. Luwinia herbicola (E. herbicola); Escherichia hia, e.g. Escherichia coli (E. coli); Klebsiella lla), e.g. Klebsiella pneumoniae; Miriococcum; Myrothesium; Muco Mucor; Neurospora, e.g. Neurospora ·N.crassa; Proteus, e.g. Proteas · vulgaris (P. vulgaris); Providencia (Providencia), e.g. For example, Providencia stuartii (P. stuartii); noporus, e.g. Pycnoporus cinnabarinus abarinus, Pycnoporus sanguyens ineus); Ruminococcus, e.g. Ruminococcus R. torques; Salmonella, e.g., Sal Monera typhium (S.typhimurium); Serratia, For example, Serratina liquefaciens, Serratina ma S. marcescens; Shigella, e.g. Shigella flexneri; Streptomyces es), e.g. Streptomyces antibioticus (S. antibiotic us), Streptomyces castaneoglobisporus (S. castaneoglo bisporus), Streptomyces violeceorba (S. violeceor uber; Trametes; Trichoderma ), e.g. Trichoderma reesei (T. reesei), Trichoderma viride (Tv iride); Yersinia, e.g. Yersinia enterocolitica It is derived from (Y.enterocolitica).

[0229] In one embodiment, the lactase is from Kluyveromyces and Bacillus subtilis. Kluyveromyces, especially Kluyveromyces fragilis (K.fragilis) gilis and K. lactis, as well as Candida Candida, Torula and Torulops Other filamentous fungi such as Bacillus subtilis are common sources of fungal lactase, while Bacillus coagulans B. coagulans and B. circulans culans) is a well-known source of bacterial laccases. Several commercially available lactase preparations are available, for example, Lactozym.RTM (Novoz ymes, Denmark), HA-Lactase (Chr. Hans en, Denmark) and Maxilact.RTM (available ble from DSM, the Netherlands) (all All of these laccases are pH-dependent. These laccases are called neutral laccases, having an optimum pH of 6 to 8. For example, when used to produce low-lactose yogurt, the enzyme treatment is a separate step before fermentation. This must be done either at low temperatures or with a higher enzyme dosage. Because their activity decreases as the pH decreases during fermentation. Cutase reduces the number of microorganisms in some cases and ensures good milk quality. For the hydrolysis of lactose in milk at high temperatures, which may be useful for In one embodiment, the enzyme is derived from bacteria, e.g. Bifidobacterium 71539, such as lactase Bifidobacteria, such as those from the genus Bifidobacteria ceae) family of lactase.

[0230] Further aspects of the invention Side 1 Contains an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1, A polypeptide having transgalactosylation activity, said polypeptide comprising: Peptide-encoding Bacillus subtilis If the polypeptide is an expression product of a nucleic acid sequence of the BG3594 strain, the polypeptide is a trans The polypeptide expression product of said nucleic acid sequence having galactosylation activity is a polypeptide. Lipeptide. Side 2 A polypeptide having transgalactosylation activity selected from the group consisting of: a. Contains an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1 A polypeptide comprising a maximum of 980 amino acid residues, Polypeptides, b. Contains an amino acid sequence having at least 97% sequence identity to SEQ ID NO:2 A polypeptide comprising a maximum of 975 amino acid residues, Polypeptides, c. An amino acid sequence having at least 96.5% sequence identity to SEQ ID NO:3 A polypeptide comprising at most 1300 amino acid residues. A polypeptide comprising: di) encoding the polypeptide of SEQ ID NO: 1, 2, 3, 4 or 5 or ii) a nucleic acid consisting of SEQ ID NO: 9, 10, 11, 12, or 13; A polynucleotide that hybridizes to the complementary strand of at least one of the A polypeptide encoded by the e. A nucleic acid encoding a polypeptide of SEQ ID NO:1, 2, 3, 4 or 5. SEQ ID NOs: 9, 10, 11, 1 encoding the sequence or mature polypeptide 2, or 13 nucleotides having at least 70% sequence identity to a nucleic acid sequence A polypeptide encoded by a polynucleotide comprising the sequence, and f. Deletion or insertion of one or more amino acid residues in SEQ ID NO:1, 2, 3, 4 or 5 Polypeptides containing insertions and / or substitutions. Aspect 3 A polypeptide having transgalactosylation activity selected from the group consisting of: a. Contains an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1 A polypeptide comprising a maximum of 980 amino acid residues, Polypeptides, b. An amino acid sequence having at least 96.5% sequence identity to SEQ ID NO:3 A polypeptide comprising at most 1300 amino acid residues. A polypeptide comprising: c) encoding the polypeptide of SEQ ID NO: 1, 2, 3, 4 or 5 or ii) a nucleic acid consisting of SEQ ID NO: 9, 10, 11, 12, or 13; A polynucleotide that hybridizes to the complementary strand of at least one of the A polypeptide encoded by the d. A nucleic acid encoding a polypeptide of SEQ ID NO:1, 2, 3, 4 or 5. SEQ ID NOs: 9, 10, 11, 1 encoding the sequence or mature polypeptide 2, or 13 nucleotides having at least 70% sequence identity to a nucleic acid sequence A polypeptide encoded by a polynucleotide comprising the sequence, and e. Deletion or insertion of one or more amino acid residues of SEQ ID NO:1, 2, 3, 4 or 5 Polypeptides containing insertions and / or substitutions. Side 4 The polypeptide of any of the preceding aspects, wherein the polypeptide is Bacillus subtilis BG, which encodes When the polypeptide is an expression product of a nucleic acid sequence of the 3594 strain, the polypeptide is transgalactam A polypeptide that is simply a polypeptide expression product of said nucleic acid sequence having ctoxylation activity. Chid. Side 5 Contains an amino acid sequence having at least 96.5% sequence identity to SEQ ID NO:3. A polypeptide comprising at most 1300 amino acid residues. a polypeptide according to any of the preceding aspects. Side 6 A polypeptide having an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1. The polypeptide comprises up to 980 amino acid residues. 2. A polypeptide according to any one of the preceding aspects. Side 7 A polypeptide having an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1. A polypeptide comprising up to 980 amino acid residues. A polypeptide according to any one of 1, 2 and 4. Side 8 A polypeptide having an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1. The polypeptide comprises up to 980 amino acid residues. 2. A polypeptide according to any one of the preceding aspects. Side 9 The polypeptide of aspect 8, wherein the polypeptide has transgalactosylation activity. Side 10 The polypeptide is a Bacillus subtilis strain encoding the polypeptide. In the case of an expression product of the nucleic acid sequence of the BG3594 strain of B. lus subtilis, The polypeptide is merely a polypeptide of the nucleic acid sequence that has transgalactosylation activity. 10. The polypeptide of any one of aspects 8 and 9, which is a tide expression product. Side 11 The polypeptide of any one of the preceding aspects, wherein the sequence identity is at least at least 96%, at least 97%, at least 98%, at least 99%, or At least 95%, such as at least 100%, of the polypeptide. Side 12 A polypeptide having at least 90% sequence identity to SEQ ID NO:1. Side 13 13. The polypeptide of aspect 12, having transgalactosylation activity. Side 14 The polypeptide of any one of the preceding aspects, wherein the sequence identity is S At least 91%, at least 92%, at least 93%, at least at least 94%, at least 95%, at least 96%, at least 97%, at least 9 Polypeptides having at least 90% sequence identity, such as at least 8%, at least 99%, Chid. Side 16 16. The polypeptide of any one of aspects 1 to 15, comprising a sequence of interest and a reference The degree of sequence identity between sequences is calculated using 1) a default scoring matrix and a default The alignment of two sequences using any suitable alignment program with default gap penalties is performed. 2) identifying the number of exact matches, A match is a match between two aligned sequences at a given position in the alignment. This means that the alignment program has identified the amino acids or bases as identical in and 3) divide the number of exact matches by the length of the longer of the two sequences. A polypeptide determined by the steps of: Side 17 17. The polypeptide of any one of aspects 1 to 16, comprising: 1) a default sequence Any suitable algorithm using a coring matrix and default gap penalties. The process of aligning two sequences using an alignment program; 2) the number of exact matches. In the process of identifying a sequence, an exact match is a match at a given position in the alignment. If the amino acids or bases in two aligned sequences are identical, and 3) the length of the aligned sequences is exactly the same. Here, the "length of the aligned sequence" is defined as the length of the aligned sequence including gaps and alignment errors. A polypeptide, the length of the entire alignment including the hanging bar portion. Side 18 Aspect 1, wherein the preferred alignment program is a global alignment program. 18. The polypeptide of any one of aspects 5, 16 and 17. Side 19 The global alignment program uses the Needleman-Wunsch algorithm. The polypeptide of aspect 18, using a method of Side 20 The global alignment program is EMBOSS Needle and EMBO SS stretcher, any one of sides 18 and 19 selected from the group consisting of A polypeptide as described in aspect. Side 21 A polypeptide according to any one of the preceding aspects, comprising up to 970 amino acids. 950 amino acid residues, 940 amino acid residues, 930 amino acid residues amino acid residues, up to 920 amino acid residues, up to 910 amino acid residues, up to 900 5 amino acid residues, up to 895 amino acid residues, up to 890 amino acid residues, etc. A polypeptide consisting of 975 amino acid residues. Side 22 A polypeptide according to any one of the preceding aspects, comprising 887 amino acid residues A polypeptide consisting of a base. Side 23 The polypeptide of any one of the preceding aspects, consisting of SEQ ID NO:1. Do. Side 24 The method according to any one of the preceding aspects, comprising the amino acid sequence of SEQ ID NO:1. The polypeptide described above. Side 25 A polypeptide according to any one of the preceding aspects, comprising 965 amino acid residues A polypeptide consisting of a base. Side 26 26. The polypeptide according to any one of aspects 1 to 23 and 25, A polypeptide having at least 96.5% sequence identity to SEQ ID NO:2. Chid. Side 27 27. The polypeptide according to any one of aspects 1 to 23, 25 and 26, A peptide that is at least 98%, at least 99%, etc., similar to SEQ ID NO:2. A polypeptide having at least 97% sequence identity. Side 28 28. A polypeptide according to any one of aspects 1 to 23, 25, 26 and 27, The polypeptide comprises SEQ ID NO:2. Side 29 29. The polypeptide according to any one of aspects 1 to 23 and 25 to 28, A polypeptide, wherein the peptide consists of the amino acid sequence of SEQ ID NO:2. Side 30 From an amino acid sequence having at least 96.5% sequence identity to SEQ ID NO:3 A polypeptide comprising at most 1300 amino acid residues. A polypeptide. Side 31 31. The polypeptide of aspect 30, wherein the polypeptide has transgalactosylation activity. Side 32 32. The polypeptide according to any one of aspects 30 and 31, wherein the polypeptide Bacillus subtilis encoding the polypeptide lis) if the polypeptide is an expression product of the nucleic acid sequence of the BG3594 strain, It is merely the polypeptide expression product of said nucleic acid sequence that has transgalactosylation activity. A polypeptide. Side 33 The polypeptide according to any one of aspects 1 to 15, 15 to 20, and 30 to 32. wherein the sequence identity is at least 98%, at least 99% or at A polypeptide that has at least 97% sequence identity, such as 100%. Side 34 Sides 1 to 15, 15 to 20, and 30 to 33 of SEQ ID NO:3 2. A polypeptide according to any one of claims 1 to 11. Side 35 Flanks 1 to 15, 15 to 20, and 35. A polypeptide according to any one of claims 30 to 34. Side 36 The polypeptide according to any one of aspects 1 to 15, 15 to 20, and 30 to 35. Maximum of 1280, maximum of 1270, maximum of 1260, maximum of 1250 1240 pieces, 1230 pieces, 1220 pieces, 1215 pieces A polypeptide consisting of up to 1290 amino acid residues, such as cyclic carboxylic acid residues. Side 37 Flanks 1 to 15, 15 to 20, 30 to 34, and 37. The polypeptide according to any one of claims 36 to 37. Side 38 Any one of aspects 1 to 15, 15 to 20, 30 to 34, 36, and 37. A polypeptide of the present invention having at least 98.5% sequence identity to SEQ ID NO:5. A polypeptide having the formula: Side 39 Any one of sides 1-15, 15-20, 30-34, 36, 37, and 38 5. A polypeptide as set forth in claim 1, which has a sequence identity of at least 99% or less with SEQ ID NO:5. A polypeptide having at least 99.5% sequence identity. Side 40 Any one of sides 1 to 15, 15 to 20, 30 to 34, and 36 to 39 is described. The polypeptide as described above, which consists of SEQ ID NO:5. Side 41 Any one of sides 1 to 15, 15 to 20, 30 to 34, and 36 to 39 is described. The polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:5. Do. Side 42 Any one of sides 1 to 15, 15 to 20, 30 to 34, and 36 to 40 is described. The polypeptide of claim 1, wherein the polypeptide comprises at most 1200, at most 1190, at most 1180, Up to 1170, up to 1160, up to 1150, up to 1145 amino acids A polypeptide consisting of up to 1210 amino acid residues, such as 1,210 amino acid residues. Side 43 Any one of sides 1 to 15, 15 to 20, 30 to 34, 36 to 40, and 42 2. The polypeptide according to claim 1, which is a polypeptide consisting of 1,142 amino acid residues. Side 44 Any of sides 1 to 15, 15 to 20, 30 to 34, 36 to 40, 42, and 43 a polypeptide according to any one of the preceding claims, which has at least 9 amino acid sequence similar to that of SEQ ID NO:4; A polypeptide with 6% sequence identity. Side 45 Sides 1 to 15, 15 to 20, 30 to 34, 36 to 40, 42, 43, and 44 5. A polypeptide according to any one of claims 1 to 4, wherein the polypeptide has at least one sequence similar to that of SEQ ID NO:4. At least 97% sequence identity, such as 98% or at least 99% sequence identity. A polypeptide having the following properties: Side 46 Sides 1 to 15, 15 to 20, 30 to 34, 36 to 40, and 42 to 45 The polypeptide according to any one of the preceding claims, comprising SEQ ID NO:4. Do. Side 47 Sides 1 to 15, 15 to 20, 30 to 34, 36 to 40, and 42 to 46 The polypeptide according to any one of claims 1 to 5, comprising the amino acid sequence of SEQ ID NO:4. A polypeptide. Side 48 Sides 1 to 15, 15 to 20, 30 to 34, 36 to 40, and 42 to 46 The polypeptide according to any one of claims 1 to 5, wherein the polypeptide has a maximum of 1120, a maximum of 1110, or a maximum of 1120, 1100, up to 1090, up to 1080, up to 1070, up to 106 0, up to 1050, up to 1055, up to 1040 amino acid residues, etc. A large polypeptide consisting of 1,130 amino acid residues. Side 49 Sides 1 to 15, 15 to 20, 30 to 34, 36 to 40, 42 to 46, and 4 8. The polypeptide according to any one of claims 1 to 8, which is a polypeptide consisting of 1038 amino acid residues. Lipeptide. Side 50 Sides 1 to 15, 15 to 20, 30 to 34, 36 to 40, 42 to 46, 48, and 49. A polypeptide according to any one of claims 1 to 49, comprising at least one amino acid sequence similar to that of SEQ ID NO:3. A polypeptide having at least 96.5% sequence identity. Side 51 Sides 1 to 15, 15 to 20, 30 to 34, 36 to 40, 42 to 46, 48, 4 9 and 50, comprising the polypeptide of SEQ ID NO:3 At least 9% sequence identity, such as at least 98% or at least 99% sequence identity to A polypeptide with 7% sequence identity. Side 52 Sides 1 to 15, 15 to 20, 30 to 34, 36 to 40, 42 to 46, and 4 8 to 51, comprising a polypeptide of any one of SEQ ID NOs: 3 to 51. A polypeptide. Side 53 Sides 1 to 15, 15 to 20, 30 to 34, 36 to 40, 42 to 46, and 4 8 to 51, comprising the polypeptide of SEQ ID NO:3 A polypeptide consisting of a sequence of amino acids. Side 54 The polypeptide according to any one of the preceding aspects, wherein the initial lactose concentration is 3% ( Transgalactosylation activity after 30 minutes of reaction at 0.1% or more (w / w): β-galactosidase activity such as a ratio of enzyme activity of at least 1, at least 1.5, at least 2, at least 2, A polypeptide that is at least 0.5. Side 55 The polypeptide according to any one of the preceding aspects, wherein the initial lactose concentration is 3% ( Transgalactosylation activity after 30 minutes of reaction at 0.1% or more (w / w): β-galactosidase activity The ratio of enzyme activities is at least 3, at least 4, at least 5, at least 6, at least 7 , at least 8, at least 9, at least 10, at least 11, at least 12, etc. For example, a polypeptide having a β-amino acid sequence of at least 2.5. Side 56 56. The polypeptide according to any one of aspects 54 and 55, wherein the initial lactose The polypeptide has a concentration of 3% w / w. Side 57 A polypeptide according to any one of the preceding claims, wherein the polypeptide is isolated and / or purified. , polypeptides. Side 58 The polypeptide according to any one of the preceding claims, wherein the polypeptide is recombinantly produced. Do. Side 59 The polypeptide according to any one of the preceding claims, wherein the polypeptide is 100% or more A polypeptide having a ratio of transgalactosylation activity. Side 60 The polypeptide according to any one of the preceding claims, wherein the polypeptide is 150%, 17 A polypeptide having a transgalactosylation activity ratio of 5%, or 200% or more. Side 61 The polypeptide according to any one of the preceding claims, which comprises the group consisting of SEQ ID NO:7. A polypeptide having a glycoside hydrolase catalytic core with an amino acid sequence selected from . Side 62 The polypeptide according to any one of the preceding claims, F02837), Glyco_hydro (PF00703) and / or Glyco A polypeptide containing the _hydro 2C (PF02836) domain. Side 63 The polypeptide according to any one of the preceding claims, comprising a bacterial Ig-like domain. Polypeptides containing the nucleotide sequence (group 4) (PF07532). Side 64 The polypeptide according to any one of the preceding claims, A polypeptide derived from Bifidobacterium bifidum. Side 65 Any one of the preceding polypeptides, having an optimum pH of 6.7-7.5. Polypeptides. Side 66 The polypeptide according to any one of the preceding claims, A polypeptide having an optimum temperature of .degree. Side 67 A polypeptide according to any one of the preceding claims, wherein the polypeptide further comprises a single amino acid sequence. The sequence identity of amino acid sequences was calculated using BLOSUM62 with a word size of 3 as the substitution matrix. Polypeptides as determined by using Blast. Side 68 The polypeptide of any one of the preceding claims, which is a recombinant polypeptide. Side 69 A lyophilized polypeptide according to any one of the preceding claims. Side 70 A polypeptide according to any preceding claim, in solution. Side 71 The polypeptide of any one of the preceding claims, which is an isolated polypeptide. Side 72 The polypeptide of any one of the preceding claims, which is a purified polypeptide. Side 73 A nucleic acid capable of encoding a polypeptide according to any one of the preceding. Side 74 Expressing the nucleic acid of aspect 73 or the polypeptide of any one of aspects 1 to 72. An expression vector capable of: Side 75 A cell capable of expressing a polypeptide of any one of aspects 1 to 72. Side 76 A method for expressing a polypeptide, the method comprising obtaining a cell of the invention; expressing said polypeptide from said cell, and optionally purifying said polypeptide. The method comprising the steps of: Side 77 A composition comprising a polypeptide according to any one of aspects 1 to 72, preferably The composition is preferably a food composition, more preferably a dairy product. Side 78 The composition of aspect 77, comprising one or more polypeptides according to any one of aspects 1 to 72. At least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% w / w, etc., at least 5%, For EQ ID NO:2, 72%, 74%, 74%, 78%, 80%, 82%, 8 4%, 86%, 88%, 90% or more of sequence identity. Composition. Side 79 79. The composition of any one of aspects 77 and 78, wherein the one or more polypeptides A composition selected from the group consisting of SEQ ID NO:1, 2, 3, 4, and 5. Side 80 The composition of any one of aspects 77, 78, and 79, comprising one or more polypeptides. The peptide is selected from the group consisting of SEQ ID NOs: 1, 2, and 3. Side 81 81. The composition of any one of aspects 77 to 80, wherein the one or more polypeptides are S A composition selected from EQ ID NO:1 or 2. Side 82 Treating a substrate containing lactose with a polypeptide as defined in any one of aspects 1 to 72. A method for producing food by using this method. Side 83 A milk-based substrate containing lactose is coated with a polypeptide as defined in any one of aspects 1 to 72. 2. The method of claim 1, wherein the dairy product is treated with lactose. Side 84 The method according to any one of aspects 82 and 83, further comprising hydrolyzing the substrate to form a β-galactosidase. A method comprising a step of treating with exosidase. Side 85 Treating a substrate containing lactose with a polypeptide as defined in any one of aspects 1 to 72. 2. A galacto-oligosaccharide or a composition thereof obtained by the process of: Side 86 73. A nucleic acid capable of encoding a polypeptide according to any one of aspects 1 to 72. Side 87 Nucleic acids at least 60% identical to SEQ ID NO:9, 10, 11, 12, or 13 Flanking 86 a nucleic acid having a sequence. Side 88 88. A plasmid comprising the nucleic acid of any one of aspects 86 and 87. Side 89 87. The nucleic acid according to any one of aspects 86 and 87, or according to any one of aspects 1 to 72. An expression vector capable of expressing the polypeptide according to any one of the above. Side 90 A host cell preferably transformed with the plasmid of aspect 88 or the expression vector of aspect 89. cell. Side 91 A cell capable of expressing a polypeptide according to any one of aspects 1 to 72. Side 92 A host cell on side 90, which may be a bacterial, fungal, or yeast cell, or a cell on side 91. Cell. Side 93 93. The cell of aspect 92, wherein the cell is Ruminococcus ), Bifidobacterium, Lactococcus (La ctococcus, Lactobacillus, Streptococcus Streptococcus, Leuconostoc , Escherichia, Bacillus, Strep Streptomyces, Saccharomyces es), Kluyveromyces, Candida ), Torula, Torulopsis and Aspergillus A cell selected from the group consisting of Aspergillus. Side 94 The cell of aspect 93, wherein the cell is Ruminococcus hansenii. cus hansenii, Ruminococcus lactalis lactaris, Bifidobacterium breve um breve, Bifidobacterium longum m longum), Bifidobacterium infantis erium infantis, Bifidobacterium bifidum acterium bifidum and Lactococcus lactis lactis). Side 95 A method for expressing a polypeptide, the method being described in any one of aspects 91 to 94. obtaining said host cell or cells, and extracting the polypeptide from said host cell or cells. A method comprising expressing and optionally purifying the polypeptide. Side 96 73. A composition comprising a polypeptide as defined in any of aspects 1 to 72 and a stabiliser. Side 97 73. A composition comprising a polypeptide as defined in any of aspects 1 to 72 and a carbohydrate substrate. Side 98 98. The method of aspect 97, wherein the carbohydrate substrate is a disaccharide. Side 99 99. The method of aspect 98, wherein the disaccharide is lactose. Side 100 73. The polypeptide according to any one of aspects 1 to 72, further comprising a milk-based base comprising lactose. A method for producing a dairy product comprising treating milk quality. Side 101 The method of aspect 90, wherein the polypeptide has the transgalactosylation activity ratio as described above. How to do it. Side 102 A method according to any one of aspects 100 and 101, wherein the milk-based substrate comprises yogurt, cheese, or a fermented dairy product. Side 103 The method of any one of aspects 100, 101, and 102, further comprising: The method further comprises fermenting the substrate with a microorganism capable of fermenting. Side 104 104. The method of aspect 103, wherein the milk-based substrate is yogurt. Side 105 The method according to any one of aspects 100 to 104, comprising binding the polypeptide to a microorganism. A method in which the processing is performed simultaneously. Side 106 106. The method of any one of aspects 100 to 105, comprising: and are always added simultaneously to the milk-based substrate. Side 107 From yogurt, cheese, fermented dairy products, food supplements and probiotic foods 20. Use of a cell of any of the above aspects for the manufacture of a product selected from the group consisting of: Side 108 A dairy product comprising the cells of any of the above aspects. Side 109 73. A dairy product comprising a polypeptide as defined in any one of aspects 1 to 72. Side 110 Polypeptides defined in any one of aspects 1 to 72 at a concentration of 0.01-1000 ppm Dairy products containing chitosan. Side 111 73. A dairy product comprising an inactivated polypeptide as defined in any one of aspects 1 to 72. Side 112 Inactivation defined as any one of sides 1 to 72 at concentrations between 0.01 and 1000 ppm A dairy product comprising the selected polypeptide. Side 113 GO formed in situ by a polypeptide as defined in any one of aspects 1 to 72. Dairy products containing S. Side 114 The method of any one of aspects 1 to 72 for producing galacto-oligosaccharides. Use of an ingalactosylated polypeptide or a cell of any one of the above aspects. Side 115 Yogurt, cheese, fermented dairy products, food supplements, and probiotic foods To produce a galacto-oligosaccharide that is part of a product selected from the group consisting of A transgalactosylated polypeptide according to any one of aspects 1 to 72, or Use of any one cell on the face. Side 116 Galactam to enhance the growth of Bifidobacterium The transglycosylation of any one of aspects 1 to 72 for producing a tri-oligosaccharide. Use of a lactosylated polypeptide or a cell of any one of the above aspects. Side 117 Growth of Bifidobacterium in mixed culture fermentations Any of aspects 1 to 72 for producing galacto-oligosaccharides for enhancing or one of the transgalactosylated polypeptides or the use of any one of the above aspects in a cell. For. Side 118 A method for producing a transgalactosylated polypeptide according to any one of aspects 1 to 72. In a suitable culture medium under conditions allowing expression of said polypeptide, and recovering the polypeptide obtained from the culture medium. Includes, a method. Side 119 A method for producing galacto-oligosaccharides, comprising the steps of: The polypeptide or any of the above-mentioned aspects is then soaked in a milk-based solution containing lactose. The method comprises the step of contacting the Side 120 C-terminal truncation fragments of SEQ ID NO:22 with transgalactosylation activity Bacillus subtilis BG3594 In microorganisms such as Bacillus subtilis strains Resistant to further proteolytic cleavage and / or optimally A polypeptide that is stable to further cleavage during storage after final formulation. Side 121 The polypeptide of aspect 120, further defined in any one of aspects 1 to 72. Lipeptide.

[0231] Materials and Methods Method 1 Polypeptide production For expression in Bacillus subtilis Don Optimized Bifidobacterium bifidum A synthetic sequence was designed to encode the full-length (1752 residues) gene of L. bifidum. The gene (SEQ ID No. 8) was submitted to GeneART (Regensburg, Germany). I purchased it from many.

[0232] A reverse primer capable of specifically amplifying a selected region of the synthetic gene is provided. Using the polymerase chain reaction, Bifidobacterium bifidum (Bifid A truncation mutant of the genus taurate (Proc. obacterium bifidum) was constructed. Forward primer: GGGGTA ACTAGT GGAAGATGCAACAAGAA G (SpeI is underlined) (SEQ ID NO:15) Reverse Plamer: TIFF0007612445000003.tif98148

[0233] The synthetic gene was cloned into pBNspeI using the unique restriction sites SpeI and PacI (Figure 1). Cloning into Bacillus subtilis expression vector The isolated plasmid was then transformed into Bacillus subtilis strain BG3594. Transformants were cultured in LB medium containing 10 μg / mL neomycin as a selection agent. The streaking was repeated on the plate. P64 Prepare a spare medium in LB medium containing 10 μg / mL neomycin and shake at 180 rpm. The cells were harvested at 37°C for 7 hours while keeping the medium in a cool place. The mixture was used to inoculate 50 mL of Grant medium containing 100 μg / L of lysine, and incubated for 3 h at 180 rpm. The cells were grown at 3°C ​​for 68 hours.

[0234] Lysozyme (Sigma-Aldrich) was added directly to the medium at a final concentration of 1 mg / ml. h) and 10 U / ml Benzonase (Merck) were added to lyse the cells. The lysate was thawed and incubated at 33°C for 1 hour with shaking at 180rpm. The mixture was centrifuged at 0xg for 20 minutes, then filtered and washed.

[0235] Grant's modified medium was prepared as follows: Part I (Autoclave) Soytone 10g 500 mg per litre Part II 1M K2HPO43mL Glucose 75g Urea 3.6g Grant's 10X MOPS 100mL Make it 400ml per litre Part I (2 w / w % Soytone) was prepared and autoclaved at 121°C for 25 minutes. I put it on. Part II was prepared and mixed with Part I, and the pH was adjusted to 7.3 with HCl / NaOH. The volume was brought to full volume and sterilized through a 0.22-μm PES filter. A 10x MOPS buffer solution was prepared as follows. 83.72g Tricin 7.17g KOH pellets 12g NaCl 29.22g 0.276M K2SO4 10 mL 0.528M MgCl2 10 mL 100x Grant Micronutrients Make up to 900ml with water, dissolve, adjust pH to 7.4 with KOH, and make up to 1 liter. The solution was then sterilized through a 0.22-μm PES filter. The 100x Grant micronutrients were adjusted as follows: Sodium citrate.2H2O 1.47g CaCl2.2H2O 1.47g FeSO4.7H2O 0.4g MnSO4.H2O 0.1g ZnSO4.H2O 0.1g CuCl2.2H2O 0.05g CoCl2.6H2O 0.1g Na2MoO4.2H2O 0.1g Dissolve and make up to 1 liter. Sterilized through a 0.2-μm PES filter. It was stored in the dark at 4°C.

[0236] Method 2 Purification and enzyme preparation The filtered enzyme isolate was filtered using a VivaSpin ultrafiltration device with a 10 kDa cutoff. The solution was concentrated using Vivaspin 20 (Sartorius, Lot#12V S2004), and the concentrate was introduced into a PD10 desalting column (GE Healthcare, Lot# 6284601) and eluted with pH 8.6 Tris-HCL. The data was collected according to the Akta FPLC system (GE Healthcare) manual. A 4 ml sample containing approximately 20 mg of protein was desalted in 2 ml of HyperQ The column (HyperCel™, Q Solvent) was loaded with 20 mM Tris-HCl. The column was eluted at 1 ml / min with 30 column volumes of washing buffer. After washing, the bound β-galactosidase was removed using 20 mM Tris-HCl pH 8.6. The column was eluted with a 100-column-volume-long gradient to 250 mM NaCl. The remaining impurities were removed using 20 mM Tris-HCl pH 8.6, 500 mM NaCl. The β-galactosidase activity of the proteins eluted in the flow-through was measured. The sex was analyzed and SDS-PAGE was performed.

[0237] SDS-PAGE gel method was performed using Invitrogen NuPageR Novex 4- 12% Bis-Tris gel 1.0 mm, 10 well (Cat#NP 0321box), See-BlueR Plus2 Pre-stained Standard (Cat # LC5925) and NuPAGER MES SDS running buffer (Ca The procedure was performed using a 350-mL ELISA kit (Model No. NP0002) according to the manufacturer's specifications.

[0238] Method 3 Measurement of β-galactosidase activity Enzyme activity was assayed using the commercially available substrate, 2-nitrophenyl-β-D-galactopyranoside (O Measurements were performed using NPG (Sigma N1127). No ONPG acceptor 100mM KPO4 pH 6.0 12.3mM ONPG Acceptor-supplemented ONPG 100mM KPO4 pH 6.0 20mM cellobiose 12.3mM ONPG Stop solution 10% Na2CO3 10 μl serial dilutions of purified enzyme were added to 90 μl with or without acceptor. The samples were added to the wells of a microtiter plate containing ONPG buffer. Incubate at 37°C for 10 minutes, then add 100 μl of stop solution to each well. The absorbance measurements were controlled by the Softmax software package. The plate was read using a Molecular Devices SpectraMax plate reader. The absorbance was measured at 420 nm.

[0239] Dilutions with an absorbance between 0.5 and 1.0 indicate transgalactosylation activity. The sex ratio calculation is as follows: Transgalactosylation activity ratio = (Abs420 +セロビオース / Abs420 -セロ ビオース )*100 (Figure 3).

[0240] Method 4 Determination of LAU activity principle: The principle of this assay is that lactase is activated by 2-o-nitrophenyl-β-D- Galactopyranoside (ONPG) was synthesized by the reaction of 2-o-nitrophenol (ONP) and galactose This reaction is stopped with sodium carbonate, and the released ONP is It is measured at 420 nm by a spectrophotometer or colonimeter. reagent: MES buffer pH6.4 (100mM MES pH6.4, 10mM CaCl2) :19.52g MES hydrate (Mw:195.2 g / mol, Sigma-al drich #M8250-250G) and 1.470g CaCl2 dihydrate (Mw : 147.01g / mol, Sigma-Aldrich) in 1000 ml of ddHO Dissolve in 20 mL of 10 M NaOH and adjust the pH to 6.4 with 10 M NaOH. Filter through a 0.2 μm filter. Filter. Store at 4°C for up to one month (storable).

[0241] ONPG substrate pH 6.4 (12.28mM ONPG, 100mM MES pH 6 .4, 10 mM CaCl2): 0.370 g of 2-o-nitrophenyl-β-D-galactosidase Topyranoside (ONPG, Mw: 301.55g / mol, Sigma-Aldrich #N1127) was dissolved in 100 ml of MES buffer, pH 6.4, and incubated at 4°C for 7 days. Store for a while. Stopping reagent (10% Na2CO3): 20.0 g of Na2CO3 in 200 ml of ddH2 Dissolve in HO, filter through a 0.2 μm filter, and store at room temperature for up to 1 month. procedure: Serial dilutions of the enzyme samples were performed in MES buffer pH 6.4, with 10 μL of each sample diluted. The dilutions were placed in a 96-well microtiter plate containing 90 μl of ONPG substrate pH 6.4. The samples were mixed and transferred to a Comfort Thermomix Incubate at 37°C for 5 minutes using a 100 μl The reaction was stopped by adding a stop reagent to each well. The increase in absorbance at 420 nm relative to the blank was measured using EL S buffer, pH 6.4. ISA reader (SpectraMax platereader, Molecula Measurements were taken using a 3D MRI device. Activity calculation: 2-o-Nitrophenol (Sigma-Aldrich) in MES buffer at pH 6.4 The molar extinction coefficient of 33444-25G was determined to be 0.5998 x 10 - 6 M -1 xcm -1 One lactase unit (LAU) is the enzyme activity of 1 n of ONPG per minute. The total volume of the microtubules is 200 μl. Using the iterator plate, lactase activity per ml of enzyme sample was calculated using the following formula: Calculated: TIFF0007612445000004.tif26170

[0242] Specific activity calculations for BIF917 shown in SEQ ID NO:1 Determination of BIF917 concentration: Quantification of the target enzyme (BIF917) and cleavage products was performed using Criterion's unstained SDS-PAGE. The stain-free precast of any kD was used. Togel 4-20% Tris-HCl, 18 wells (Comb #345-0418) Serva Tris-Glycine / SDS buffer (BioRad cat. #4 2529). The gel was run at the following parameters: 200V, 120mA. , 25W, 50 minutes. BSA (1.43mg / ml) (Sigma-Aldrich, c At. #500-0007) was used as a protein standard. on Stain Free Imager (BioRad) on Image Lab s The results were used with the BioRad software to determine the bands correlated with tryptophan content. The intensity of the light was used for quantification. The specific LAU activity of BIF917 was determined in two independent fermentations (as described in Method 1). was determined from the crude fermentate (ultrafiltration concentrate) at five different dilutions (see Table 1). . The specific activity of BIF917 is 21.3 LAU / mg or 0.0213 LAU / ppm. I found out that... Table 1: Determination of BIF917 specific activity TIFF0007612445000005.tif121169

[0243] Working Example Example 1 Determination of β-galactosidase activity of BIF truncation mutants 8 different variants: BIF_917, BIF_995, BIF_1068, BIF_ 1172, BIF_1241, BIF1326, BIF_1400 and BIF_147 8 was constructed as described in Method 1 and purified as described in Method 2 (See Figure 2). As described in Method 3 above, all truncation mutants were synthesized in the presence and absence of cellobiose. The β-galactosidase activity was determined. result Ratio of transgalactosylation activity (Abs420 +セロビオース / Abs420 -セ ロビオース )*100) was calculated from the β-galactosidase activity measured for each mutant. The results are shown in Figure 3. Mutants with lengths of 1241 residues or less showed more than 100% transgalactosidase activity. This indicates that these mutants are mainly transgalactosylated. Mutants longer than 1241 residues show less than 100% transgalactosylation. These mutants showed hydrolytic activity, indicating that they were primarily hydrolyzed. BIF_917 and BIF_995 showed the highest transgalactosylation rate of approximately 250%. It has metabolic activity.

[0244] Example 2 GOS produced in yogurt matrix The evaluation of BIF enzymes in GOS production was tested in a pseudo-yogurt product. Batch tests were performed with 98.60% (w / v) fresh pasteurized low-fat milk (Mi ni-malk, Arla Foods, Denmark) and 1.4% (w / v ) Yogurt blend consisting of Nutrilac YQ-5075 whey ingredient (Arla) The assay was carried out in 96-well microtiter plates using the following materials: Nutrilac The mixture was stirred for 20 h to completely hydrate YQ-5075, and then diluted with 20 mM phosphorus. The pH was adjusted to 6.5 by adding sodium phosphate pH 6.5. This milk was plain and The lactose concentration was determined to be 5.5% (w / v), which was 5.3% (w / v) in solution. The following factors are relevant in this example: 1% (w / v) lactose = 0.9587% (w / w) lactose. 90 μl of milk base (substrate) was mixed with 10 μl The mixture was mixed with purified enzyme, sealed with tape and incubated at 43°C for 3 hours. The reaction was stopped with 100 μl of 10% Na2CO3. Samples were stored at -20°C. HPLC method Galactooligosaccharides (GOS), lactose, glucose, and galactose The samples were analyzed using a Dionex ICS 3000. The IC parameters were as follows: mobile phase: 150 mM NaOH, flow rate: : Isochratic, 0.25ml / min, Column: Carbopac PA1, Column temperature: room temperature, injection volume: 10 μl, detector: PAD, integration: manual Sample preparation: 100-fold dilution with Milli-Q water (0.1 ml sample + 9.9 ml 1) water) and filtered through a 45 μm syringe filter. Quantitative analysis: Peak area of ​​standard substance Percentage of peak area of ​​sample containing GOS syrup (Vivanal GOS , Friesland Campina) was used as a standard for quantification of GOS. In this embodiment, the term "GOS" refers to galactooligosaccharides with a degree of polymerization of 3 or more. It is defined as being. result In milk-based substrates in BIF_917, BIF_995, and BIF_1326 The quantification of GOS produced in the short mutant BIF_91 is shown in Figure 4. 7 and BIF_995 produced 0.1% (w / v) compared to 0.1% (w / v) produced by BIF1326. The results showed that the GOS production was significantly higher than that of the control group (approximately 1.2% (w / v) at 95% confidence interval). (determined by mean T test).

[0245] Example 3 Degradation patterns of truncated mutants A library covering the region between BIF1230 and BIF1325 was The order was placed with neART (Regensburg, Germany) (see Table 2). Obtained). Truncation mutants were generated as described in Method 1. The resulting peptides were analyzed by DSD-PAGE. Simply Blue Safestain (Invitrogen, at# LC6060) (Figure 5). result Surprisingly, the target bands appeared in various amounts at the end of the fermentation, and the final bands In Ross, most of the mutants were proteolytically modified.

[0246] This mutant produced three distinct bands of varying intensity. The mutation was assessed using chromosome cloning. This mutation corresponds to the end of SEQ ID NO:1. BIF917, which corresponds to the 1′ end of SEQ ID NO:2; and BIF995, which corresponds to the 1′ end of SEQ ID NO:2. There is a C-terminal truncation in BIF1068 that corresponds to the end of EQ ID NO:3. Protein bands excised from the gel (indicated by arrows in Figure 5) were analyzed by mass spectrometry. As a preparation for analysis, it is digested with three different enzymes. In particular, peptides are arginine (R) except when proline (P) is on the carboxyl side. It specifically binds to the carboxyl side of lysine (K) residues. The peptides are tyrosine (Y), phenylalanine (P), and phenylalanine (Y) except when proline (P) is on the carboxyl side. Carboxyls of nylalanine (F), tryptophan (W), and leucine (L) residues Glu-C preferentially binds to the ammonium dicarboxylate buffer at pH 8. In the case of phosphate buffer, the carboxyl side of glutamyl (E) is cleaved, but the hydrolysis is When performed with H8 it cleaves on the carboxyl side of an aspartic acid (D).

[0247] To detect the C-terminus, 40% 18 Except for 100 ml water as the digestion buffer, Select the desired protein for analysis using the Basic Method for Protein Characterization (A2963). The resulting peptides are prepared by proteolytic cleavage. 8O Water and 16 O The principle is that both water and protein are incorporated and appear as a pair. is not cleaved, but is simply the "last peptide" left in the protein, , 16 The C-terminus appears as a single peptide with O-water. are mapped using analytics. Table 2 Mutants TIFF0007612445000006.tif166150

[0248] TIFF0007612445000007.tif249168

[0249] TIFF0007612445000008.tif173151

[0250] Example 4 In situ enzymatically produced GOS in milk bases and yogurt In this embodiment, the term "GOS" refers to galactooligosaccharides having a degree of polymerization (DP) of 3 or more. It is defined as a ride.

[0251] The evaluation of GOS generated by BIF917 and BIF995 was performed using different set styles. The peptides were then incubated with β-galactosidase in yogurt. The yogurt fermentation was carried out by adding a specific yogurt medium to the yogurt base. Along with the process, a transgalactosylation reaction also takes place.

[0252] Initial yogurt (set style) batch experiment with 100ml milk base (yogurt The milk base was 98.60% (w / v) fresh pasteurized Natural (non-organic) low-fat milk (Mini-malk 0.5% fat, Arla Food s Amba, Denmark) and 1.4% (w / v) Nutrilac YQ -5075 Whey Ingredients (Arla Foods Ingredients, Denm ark) and had a lactose concentration of 5.5% (w / v), which corresponds to 5.3% (w / w). (1% (w / v) lactose = 0.9587% (w / w) lactose in solution) The mixture was incubated at 4°C for 2 h to fully hydrate the Nutrilac YQ-5075. The mixture was left stirring gently for 10 hours. In the initial experiment, Lactobacillus delbrueckii subtilis bulgaricus (Lactobacillus delbrueckii subsp bulgaricus and Streptococcus thermophilus coccus thermophilus) (DuPont Nutrition B iosciences, Denmark) MIX 485LYO was used. The initial dilution of the medium was 10 g of YO-MIX 485L. YO with 400ml of UHT conventional (non-organic) milk (Let-malk 1.5% fat, A The experiment was carried out by adding 1.43 ml of 100 ml of 100% ethanol to 100% ethanol (Rla Foods Amba, Denmark). The diluted medium was added to 1 liter of milk base. 100 ml of milk base was added to 25 The enzyme was added at various concentrations (10, 20, and 40 ppm) in 0.0 ml blue cap bottles. m, which corresponds to 0.213, 0.426, and 0.853 LAU in method 4) Yogurt fermentation was carried out at 43°C, and after 10 hours, it was rapidly cooled on ice. The fermentation was always carried out twice and the yogurt sugar / oligosaccharide composition was added the next day after the fermentation. The fermented yogurt samples were always analyzed by HPLC on the same day (see HPLC method below). The yogurt was stored at 4°C. The results of the initial yogurt experiments are shown in Table 3. Increasing the concentration of either 995 from 10 ppm to 40 ppm reduced the amount of 995 in the final yogurt. The concentration of GOS increased and the amount of DP2 (containing lactose) decreased. The differences in performance were within the variance of the HPLC measurements, which is consistent with the results of the studies they investigated. It can be concluded that they function similarly within the dosage range. Table 3 In fermented yogurt treated with increasing doses of BIF917 and BID995 The content of DP2 saccharides (mainly lactose) and GOS (DP3+) in all the samples. Results were calculated as the mean of three independent experiments. TIFF0007612445000009.tif67143

[0253] To evaluate the effect on the GOS concentration achieved in the final yogurt, set-stir The yogurt had a higher lactose concentration of 7.5% w / v (1% w / v La (In the solution, the amount of lactose is 0.9423% w / w, which corresponds to 7.1% w / w.) The order used was: 1. Mix all powder ingredients (listed in Table 4) and stir the dry blend thoroughly at 4-5°C. It was added to the milk / water and left to hydrate at 4°C for 20 hours. 2. The milk base was preheated to 65°C (P1) 3. The milk base was homogenized at 65°C / 200bar. 4. The milk base was pasteurized at 95°C for 6 minutes. (P3) 5. The milk base was cooled to 5°C. (K2) Table 4. SET yogurt composition in % (w / w) TIFF0007612445000010.tif67142

[0254] The milk base is then heated to 43°C (K1). bulgaricus (Lactobacillus delbrueckii subsp bulgaricus and Streptococcus thermophilus coccus thermophilus)(DuPont Nutrition Bi The dilution of YO-MIX 495 medium (Osciences, Denmark) was Fermentation temperature: 250ml milk base in 500ml blue cap bottles The enzyme was added to a final yogurt concentration of 1% (v / v). Mix 495 at 20 DCU per 100 liters (Danisco Culture 1 DCU is measured as 1000 colony forming units. The total number of cells was 100 million. Three samples were prepared for each test. Fermentation was carried out at pH 4. The reaction was carried out at 43°C for 6 h and then stopped by rapid cooling to 5°C. The yeast composition was analyzed by HPLC the day after fermentation (see HPLC method below). Gurt samples were always stored at 4°C.

[0255] The results of this yogurt experiment are shown in Table 5. As shown in Table 3, the initial 5.5% (w / v) Compared to the GOC achieved with lactose, a higher initial lactose (7.5% w / v ) produced higher total GOS in yogurt. A final GOS concentration of 2.945% was achieved using 25 ppm BIF The GOS produced using 917 was 2.662%. Kluyveromyces lactis (GODO-YNL2 The conventional hydrolytic β-galactosidase from Godo Shusei Co., Ltd. (Japan) is 25 ppm. The lactose concentration of 2.127% was reduced by 0.335% GOS at the dose of 1.0. A small amount was observed in blank yogurt to which the same amount of water was added instead of the enzyme. Table 5. Effect of increasing doses of BIF917 and Kluveromyces lactis (K In fermented yogurt treated with β-gal of Luyveromyces lactis, Content of DP2 saccharides (mainly lactose) and GOS (DP3+) in TIFF0007612445000011.tif67155

[0256] To test the effect of acidity on yogurt fermentation in BIF917, Lactobacillus delbrueckii subsp. bulgaricus eckii subsp bulgaricus) and Streptococcus sermov Streptococcus thermophilus (DuPont N Three different The fermentation time was relatively slow in YO-MIX 495; YO-MIX 495 has a relatively fast fermentation time; a relatively long induction period and strong acidity Fermented YO-MIX 601. All fermentations contained the same amount of BIF917 (25 ppm). In addition to examining the effect of temperature on BIF917, YO-M Fermentation of IX 495 with 25 ppm BIF917 at 43°C, 45°C, and 47°C Set-style yogurt with an initial lactose concentration of 7.5% (W / v) was produced. The following procedure was used to prepare the lactose solution: (1% w / v lactose = 0.9423% w / v (Since it is in solution, this corresponds to 7.1% w / w.) 1. Mix all powder ingredients (listed in Table 6) and stir the dry blend thoroughly at 4-5°C. It was added to the milk / water and left to hydrate at 4°C for 20 hours. 2. The milk base was preheated to 65°C (P1) 3. The milk base was homogenized at 65°C / 200bar. 4. The milk base was sterilized at 95°C for 6 minutes. (P3) 5. The milk base was cooled to 5°C. (K2) 6. Heat the milk base to 43°C (K1). and 601601 (DuPont Nutrition Biosciences, De nmark) is diluted at fermentation temperature. 7. Add 100ml of milk base into a 250ml blue cap bottle and add the enzymes. The final yogurt mix was added to the 1% (v / v) variable concentration component. 8. Add 100 ml of YO-MIX 495, 485, or 601 to the starting medium. The cells were added at a concentration of 20 DCU (Danisco Culture Units) per 100 ml. For each trial, three samples were prepared. 9. Fermentation was carried out at pH 4.6 and 43°C (temperature tests were carried out at 43°C, 45°C, and The reaction was then stopped by rapid cooling to 5°C. 10. Yogurt sugar / oligosaccharide composition was analyzed by HPLC the day after fermentation. (See HPLC method below.) Fermented yogurt samples were always stored at 4°C. Table 6. SET yogurt composition in % (w / w) TIFF0007612445000012.tif67142

[0257] The results of the yogurt experiment are shown in Table 7. Different YO-MI with different acidity profiles It can be seen that the X medium does not significantly affect the final GOS acidity. The GOS produced by YO-mix485 was the most abundant GOS in the yogurt produced by YO-mix485. The high GOS concentration (3.300%) is within the quantitative variation range. Exposure to 5°C and 47°C had no significant effect on the amount of GOS produced in either yogurt. No significant changes were observed (Student's T-test with 95% confidence level): 3 at 43°C. 258% w / v, 3.375% w / v at 45°C and 3.236% at 47°C. and temperature for in situ generation of GOS in yogurt under the conditions evaluated. It can be concluded that the effect of BIF917 is potent. Table 7 TIFF0007612445000013.tif240170 HPLC method All reagents used were of analytical grade. D-(+)-lactose (min 9 9%, no.17814), D-(+)-glucose (min 99.5%, no G 8270-100G, batch# 036K0137), and D-(+)-galactosidase Toss(min 99%, no G0750-25G, batch# 031M00 43V) was obtained from Sigma (St. Louis, Mo, USA). inal GOS Syrup (Prod. No. 502675 Batch#6495 66) is the Friesland Campina Domo (Amersfoort, The samples were obtained from the company AB (The Netherlands) and contained 57% gallon water on a dry weight basis (DM). Lacto-oligosaccharide, 21% anhydrous lactose, 20% anhydrous glucose, and 0 Contains .8% anhydrous galactose.

[0258] Sample preparation All standards: Lactose, glucose, galactose and GOS in double distilled water (ddH20) and filtered through a 0.45 μm syringe filter. A set of standards was prepared ranging in concentration from 10 to 200,000 ppm.

[0259] For the quantitative evaluation of a set of sugars in a yogurt / milk matrix, the above-mentioned Daled was added to milk and yogurt samples as an internal control. Whole milk and yogurt samples containing galactose were sanitized by heating at 95°C for 10 min. All milk samples were plated in 96-well MTP plates (Corning , NY, USA), diluted a minimum of 20-fold, and diluted to 0.20 μM prior to analysis. The cells were filtered through a 96-well filter (Corning filter plate , PVDF hydrophile membrane, NY, USA). 500 Samples containing more than 0.00 ppm (5% w / v) lactose were heated at 30°C. All yogurt samples were weighed and tested using Ultra Turbo. rax TP18 / 10(Janke & Kunkel Ika-labortech nik, Bie & Berntsen, Denmark) for a few minutes. The cells were diluted 10-fold in ddH2O before homogenization. After heat inactivation, samples were further diluted in 96-well MTP plates prior to analysis. The mixture was filtered through a 0.20 μm 96-well plate (Corning filter plate, PVDF hydrophile membrane, NY, US A) All samples were analyzed in 96-well MTP plates sealed with tape.

[0260] Equipment Galactooligosaccharides (GOS), lactose, glucose, and galactose The quantitative analysis of the samples was performed using HPLC. Ent analysis bump, WPS-3000TSL Thermostatted autosampler, TC C-3000SD thermostatted column oven and RI-101 reflectance index detector Dionex Ultimate with a 300-μm sieve (Shodex, JM Science) 3000 HPLC system (Thermo Fisher Scienti fic). Chromeleon Data System Software (Version 6.80, DU 10A Build 2826, 171948) was used to obtain and analyze the data. I did.

[0261] Chromatographic conditions The sample was then eluted at 70°C on an analytical guard column (Carbo-Ag + neutral, A J0-4491, Phenomenex, The Netherlands) RSO oligosaccharide column, Ag + 4% cross-linked (Phenomenex, The analysis was performed by HPLC using a column (The Netherlands). Elute with double distilled water at a flow rate of 3 ml / min (0.45 μM regenerated cellulose membrane). The flow was then filtered and purged with helium gas. A uniform flow rate of 0.3 ml / min was maintained throughout the analysis. The total run time was 37 min and the injection volume was 10 μL. The autosampler compartment with the thermostat was maintained at 30°C to ensure dissolution of the lactose. Elution is monitored with a refractive index detector and quantification is performed relative to a given standard. According to the manufacturer's declaration for this product, Vivinal G OS Syrup (Friesland Food Domo, The Netherlands ands) are used as standards for quantifying GOS. .

[0262] Array List >SEQ ID NO:1(BIF_917) vedatrsdsttqmsstpevvyssavdskqnrtsdfdanwk fmlsdsvqaqdpafddsawqqvdlphdysitqkysqsnea esaylpggtgwyrksftidrdlagkriainfdgvymnatv wfngvklgthpygyspfsfdltgnakfggentivvkvenr lpssrwysgsgiyrdvtltvtdgvhvgnngvaiktpslat qnggdvtmnlttkvandteaaanitlkqtvfpkggktdaa igtvttasksiaagasadvtstitaaspklwsiknpnlyt vrtevlnggkvldtydteygfrwtgfdatsgfslngekvk lkgvsmhhdqgslgavanrraierqveilqkmgvnsirtt hnpaakalidvcnekgvlvveevfdmwnrskngntedygk wfgqaiagdnavlggdkdetwakfdltstinrdrnapsvi mwslgnemmegisgsvsgfpatsaklvawtkaadstrpmt ygdnkikanwnesntmgdnltanggvvgtnysdganydki rtthpswaiygsetasainsrgiynrttggaqssdkqlts ydnsavgwgavassawydvvqrdfvagtyvwtgfdylgep tpwngtgsgavgswpspknsyfgivdtagfpkdtyyfyqs qwnddvhtlhilpawnenvvakgsgnnvpvvvytdaakvk lyftpkgstekrligeksftkkttaagytyqvyegsdkds tahknmyltwnvpwaegtisaeaydennrlipegstegna svtttgkaaklkadadrktitadgkdlsyievdvtdangh ivpdaanrvtfdvkgagklvgvdngsspdhdsyqadnrka fsgkvlaivqstkeageitvtakadglqsstvkiattavp fried >SEQ ID NO:2(BIF_995) vedatrsdsttqmsstpevvyssavdskqnrtsdfdanwk fmlsdsvqaqdpafddsawqqvdlphdysitqkysqsnea esaylpggtgwyrksftidrdlagkriainfdgvymnatv wfngvklgthpygyspfsfdltgnakfggentivvkvenr lpssrwysgsgiyrdvtltvtdgvhvgnngvaiktpslat qnggdvtmnlttkvandteaaanitlkqtvfpkggktdaa igtvttasksiaagasadvtstitaaspklwsiknpnlyt vrtevlnggkvldtydteygfrwtgfdatsgfslngekvk lkgvsmhhdqgslgavanrraierqveilqkmgvnsirtt hnpaakalidvcnekgvlvveevfdmwnrskngntedygk wfgqaiagdnavlggdkdetwakfdltstinrdrnapsvi mwslgnemmegisgsvsgfpatsaklvawtkaadstrpmt ygdnkikanwnesntmgdnltanggvvgtnysdganydki rtthpswaiygsetasainsrgiynrttggaqssdkqlts ydnsavgwgavassawydvvqrdfvagtyvwtgfdylgep tpwngtgsgavgswpspknsyfgivdtagfpkdtyyfyqs qwnddvhtlhilpawnenvvakgsgnnvpvvvytdaakvk lyftpkgstekrligeksftkkttaagytyqvyegsdkds tahknmyltwnvpwaegtisaeaydennrlipegstegna svtttgkaaklkadadrktitadgkdlsyievdvtdangh ivpdaanrvtfdvkgagklvgvdngsspdhdsyqadnrka fsgkvlaivqstkeageitvtakadglqsstvkiattavp gtstektvrsfyysrnyyvktgnkpilpsdvevrysdgts drqnvtwdavsddqiakagsfsvagtvaggqkisvrvtmid crazy >SEQ ID NO:3(BIF_1068) Vedatrsdsttqmsstpevvyssavdskqnrtsdfdanwk fmlsdsvqaqdpafddsawqqvdlphdysitqkysqsnea esaylpggtgwyrksftidrdlagkriainfdgvymnatv wfngvklgthpygyspfsfdltgnakfggentivvkvenr lpssrwysgsgiyrdvtltvtdgvhvgnngvaiktpslat qnggdvtmnlttkvandteaaanitlkqtvfpkggktdaa igtvttasksiaagasadvtstitaaspklwsiknpnlyt vrtevlnggkvldtydteygfrwtgfdatsgfslngekvk lkgvsmhhdqgslgavanrraierqveilqkmgvnsirtt hnpaakalidvcnekgvlvveevfdmwnrskngntedygk wfgqaiagdnavlggdkdetwakfdltstinrdrnapsvi mwslgnemmegisgsvsgfpatsaklvawtkaadstrpmt ygdnkikanwnesntmgdnltanggvvgtnysdganydki rtthpswaiygsetasainsrgiynrttggaqssdkqlts ydnsavgwgavassawydvvqrdfvagtyvwtgfdylgep tpwngtgsgavgswpspknsyfgivdtagfpkdtyyfyqs qwnddvhtlhilpawnenvvakgsgnnvpvvvytdaakvk lyftpkgstekrligeksftkkttaagytyqvyegsdkds tahknmyltwnvpwaegtisaeaydennrlipegstegna svtttgkaaklkadadrktitadgkdlsyievdvtdangh ivpdaanrvtfdvkgagklvgvdngsspdhdsyqadnrka fsgkvlaivqstkeageitvtakadglqsstvkiattavp gtstektvrsfyysrnyyvktgnkpilpsdvevrysdgts drqnvtwdavsddqiakagsfsvagtvaggqkisvrvtmid eigallnysastpvgtpavlpgsrpavlpdgtvtsanfav hwtkpadtvyntagtvkvpgtatvfgkefkvtatirvq >SEQ ID NO:4(BIF_1172) vedatrsdsttqmsstpevvyssavdskqnrtsdfdanwk fmlsdsvqaqdpafddsawqqvdlphdysitqkysqsnea esaylpggtgwyrksftidrdlagkriainfdgvymnatv wfngvklgthpygyspfsfdltgnakfggentivvkvenr lpssrwysgsgiyrdvtltvtdgvhvgnngvaiktpslat qnggdvtmnlttkvandteaaanitlkqtvfpkggktdaa igtvttasksiaagasadvtstitaaspklwsiknpnlyt vrtevlnggkvldtydteygfrwtgfdatsgfslngekvk lkgvsmhhdqgslgavanrraierqveilqkmgvnsirtt hnpaakalidvcnekgvlvveevfdmwnrskngntedygk wfgqaiagdnavlggdkdetwakfdltstinrdrnapsvi mwslgnemmegisgsvsgfpatsaklvawtkaadstrpmt ygdnkikanwnesntmgdnltanggvvgtnysdganydki rtthpswaiygsetasainsrgiynrttggaqssdkqlts ydnsavgwgavassawydvvqrdfvagtyvwtgfdylgep tpwngtgsgavgswpspknsyfgivdtagfpkdtyyfyqs qwnddvhtlhilpawnenvvakgsgnnvpvvvytdaakvk lyftpkgstekrligeksftkkttaagytyqvyegsdkds tahknmyltwnvpwaegtisaeaydennrlipegstegna svtttgkaaklkadadrktitadgkdlsyievdvtdangh ivpdaanrvtfdvkgagklvgvdngsspdhdsyqadnrka fsgkvlaivqstkeageitvtakadglqsstvkiattavp gtstektvrsfyysrnyyvktgnkpilpsdvevrysdgts drqnvtwdavsddqiakagsfsvagtvaggqkisvrvtmid eigallnysastpvgtpavlpgsrpavlpdgtvtsanfav hwtkpadtvyntagtvkvpgtatvfgkefkvtatirvqrs qvtigssvsgnalrltqnipadkqsdtldaikdgsttvda ntggganpsawtnwayskaghntaeitfeyateqqlgqiv myffrdsnavrfpdagktkiqi >SEQ ID NO:5(BIF_1241) vedatrsdsttqmsstpevvyssavdskqnrtsdfdanwk fmlsdsvqaqdpafddsawqqvdlphdysitqkysqsnea esaylpggtgwyrksftidrdlagkriainfdgvymnatv wfngvklgthpygyspfsfdltgnakfggentivvkvenr lpssrwysgsgiyrdvtltvtdgvhvgnngvaiktpslat qnggdvtmnlttkvandteaaanitlkqtvfpkggktdaa igtvttasksiaagasadvtstitaaspklwsiknpnlyt vrtevlnggkvldtydteygfrwtgfdatsgfslngekvk lkgvsmhhdqgslgavanrraierqveilqkmgvnsirtt hnpaakalidvcnekgvlvveevfdmwnrskngntedygk wfgqaiagdnavlggdkdetwakfdltstinrdrnapsvi mwslgnemmegisgsvsgfpatsaklvawtkaadstrpmt ygdnkikanwnesntmgdnltanggvvgtnysdganydki rtthpswaiygsetasainsrgiynrttggaqssdkqlts ydnsavgwgavassawydvvqrdfvagtyvwtgfdylgep tpwngtgsgavgswpspknsyfgivdtagfpkdtyyfyqs qwnddvhtlhilpawnenvvakgsgnnvpvvvytdaakvk lyftpkgstekrligeksftkkttaagytyqvyegsdkds tahknmyltwnvpwaegtisaeaydennrlipegstegna svtttgkaaklkadadrktitadgkdlsyievdvtdangh ivpdaanrvtfdvkgagklvgvdngsspdhdsyqadnrka fsgkvlaivqstkeageitvtakadglqsstvkiattavp gtstektvrsfyysrnyyvktgnkpilpsdvevrysdgts drqnvtwdavsddqiakagsfsvagtvaggqkisvrvtmid eigallnysastpvgtpavlpgsrpavlpdgtvtsanfav hwtkpadtvyntagtvkvpgtatvfgkefkvtatirvqrs qvtigssvsgnalrltqnipadkqsdtldaikdgsttvda ntggganpsawtnwayskaghntaeitfeyateqqlgqiv myffrdsnavrfpdagktkiqisadgknwtdlaatetiaa qessdrvkpytydfapvgatfvkvtvtnadtttpsgvvca glteielctat >SEQ ID NO:6(BIF_1326) vedatrsdsttqmsstpevvyssavdskqnrtsdfdanwk fmlsdsvqaqdpafddsawqqvdlphdysitqkysqsnea esaylpggtgwyrksftidrdlagkriainfdgvymnatv wfngvklgthpygyspfsfdltgnakfggentivvkvenr lpssrwysgsgiyrdvtltvtdgvhvgnngvaiktpslat qnggdvtmnlttkvandteaaanitlkqtvfpkggktdaa igtvttasksiaagasadvtstitaaspklwsiknpnlyt vrtevlnggkvldtydteygfrwtgfdatsgfslngekvk lkgvsmhhdqgslgavanrraierqveilqkmgvnsirtt hnpaakalidvcnekgvlvveevfdmwnrskngntedygk wfgqaiagdnavlggdkdetwakfdltstinrdrnapsvi mwslgnemmegisgsvsgfpatsaklvawtkaadstrpmt ygdnkikanwnesntmgdnltanggvvgtnysdganydki rtthpswaiygsetasainsrgiynrttggaqssdkqlts ydnsavgwgavassawydvvqrdfvagtyvwtgfdylgep tpwngtgsgavgswpspknsyfgivdtagfpkdtyyfyqs qwnddvhtlhilpawnenvvakgsgnnvpvvvytdaakvk lyftpkgstekrligeksftkkttaagytyqvyegsdkds tahknmyltwnvpwaegtisaeaydennrlipegstegna svtttgkaaklkadadrktitadgkdlsyievdvtdangh ivpdaanrvtfdvkgagklvgvdngsspdhdsyqadnrka fsgkvlaivqstkeageitvtakadglqsstvkiattavp gtstektvrsfyysrnyyvktgnkpilpsdvevrysdgts drqnvtwdavsddqiakagsfsvagtvagqkisvrvtmid eigallnysastpvgtpavlpgsrpavlpdgtvtsanfav hwtkpadtwyntagtvkvpgtatvfgkefkvtatirvqrs qvtigssvsgnalrltqnipadkqsdtldaikdgsttvda ntggganpsawtnwayskaghntaeitfeyateqqlgqiv myffrdsnavrfpdagktkiqisadgknwtdlaatetiaa qessdrvkpytydfapvgatfvkvtvtnadttpsgvvca glteielktatskfvtntsaalssltvngtkvsdsvlaag syntpaiiadvkaegegnasvtvlpahdnvirvitesedh vtrktftinlgteqef >SEQ ID NO:7ビフィドバクテリーム·ビフィダム(Bifidobacte rium bifidum) グリコシドヒドロラエシュクレーア qnrtsdfdanwkfmlsdsvqaqdpafddsawqqvdlphdy sitqkysqsneaesaylpggtgwyrksftidrdlagkria infdgwymnatvwfngvklgthpygyspfsfdltgnakfg gentivvkvenrlpssrwysgsgiyrdvtltvtdgvhvgn ngvaiktpslatqnggdvtmnlttkvandteaaanitlkq tvfpkggktdaaigtvttasksiaagasadvsttitaasp klwsiknpnlytvrtevlnggkvldtydteygfrwtgfda tsgfslngekvklkgvsmhhdqgslgavanrraierqvei lqkmgvnsirtthnpaakalidvcnekgvlvveevfdmwn rskngntedygkwfgqaiagdnavlggdkdetwakfdlts tinrdrnapsvimwslgnemmegisgsvsgfpatsaklva wtkaadstrpmty >SEQ ID NO:8 full length coding nucleotide sequence gcagttgaagatgcaacaagaagcgatagcacaacacaaa tgtcatcaacaccggaagttgtttattcatcagcggtcga tagcaaacaaaatcgcacaagcgattttgatgcgaactgg aaatttatgctgtcagatagcgttcaagcacaagatccgg catttgatgattcagcatggcaacaagttgatctgccgca tgattatagcatcacacagaaatatagccaaagcaatgaa gcagaatcagcatatcttccgggaggcacaggctggtata gaaaaagctttacaattgatagagatctggcaggcaaacg cattgcgattaattttgatggcgtctatatgaatgcaaca gtctggtttaatggcgttaaactgggcacacatccgtatg gctattcaccgttttcatttgatctgacaggcaatgcaaa atttggcggagaaaacacaattgtcgtcaaagttgaaaat agactgccgtcatcaagatggtattcaggcagcggcattt atagagatgttacactgacagttacagatggcgttcatgt tggcaataatggcgtcgcaattaaaacccgtcactggca acacaaaatggcggagatgtcaatgaacctgacaacaa aagtcgcgaatgatacagaagcagcgaacattacact gaaacagacagtttttccgaaggcggaaaaacggatgca gcaattggcacagttacaacagcatcaaaatcaattgcag caggcgcatcagcagatgttacaagcacaattacagc aagcccgaaactgtggtcaattaaaaacccgaacctgtat acagttagaacagaagttctgaacggaggcaaagttctgg atacattgatacagaatgctttcgctgacaggctt tgatgcaacatcaggcttttcactgaatggcgaaaaagtc aaactgaaaggcgttagcatgcatcatgatcaaggctcac ttggcgcagttgcaatagacgcgcaattgaaagacaagt cgaaatcctgcaaaaaatgggcgtcaatagcattcgcaca acacataatccggcagcaaaagcactgattgatgtctgca atgaaaaaggcgttctggttgtcgaagaagtctttgatat gtggaaccgcagcaaaaatggcaacggaagatatggc aaatggtttggccaagcaattgcaggcgataatgcagttc tgggaggcgataaagatgaaacatgggcgaaatttgatct tacatcaacaattaaccgcgatagaaatgcaccgtcagtt attatgtggtcactgggcaatgaaatgatggaaggcattt caggctcagtttcaggctttccggcaacatcagcaaaact ggttgcatggacaaaagcagcagattcaacaagaccgatg acatatggcgataacaaaattaaagcgaactggaacgaat caaatacaatgggcgataatctgacagcaaatggcggagt tgttggcacaaattattcagatggcgcaaactatgataaa attcgtacaacacatccgtcatgggcaatttatggctcag aaacagcatcagcgattaatagccgtggcatttataatag aacaacaggcggagcacaatcatcagataaacagctgaca agctatgataattcagcagttggctggggagcagttgcat catcagcatggtatgatgttgttcagagagattttgtcgc aggcacatatgtttggacaggatttgattatctgggcgaa ccgacaccgtggaatggcacaggctcaggcgcagttggct catggccgtcaccgaaaaaatagctattttggcatcgttga tacagcaggctttccgaaagatacatattatttttatcag agccagtggaatgatgatgttcatacactgcatattcttc cggcatggaatgaaaatgttgttgcaaaaggctcaggcaa taatgttccggttgtcgtttatacagatgcagcgaaagtg aaactgtttttacaccgaaaggctcaacaagaaaaaagac tgatcggcgaaaaatcatttacaaaaaaaaacaacagcggc aggctatacatatcaagtctatgaaggcagcgataaagat tcaacagcgcataaaaacatgtatctgacatggaatgttc cgtgggcagaaggcacaatttcagcggaagcgtatgatga aataatcgcctgattccggaaggcagcacagagaggcaac gcatcagttacaacaacaggcaaagcagcaaactgaaag cagatgcggatcgcaaaacaattacagcggatggcaaaga tctgtcatatattgaagtcgatgtcacagatgcaaatggc catattgttccggatgcagcaaatagagtcacatttgatg ttaaaggcgcaggcaaactggttggcgttgataatggctc atcaccggatcatgattcatatcaagcggataaaccgcaaa gcattttcaggcaaagtcctggcaattgttcagtcaacaa aagaagcaggcgaaattacagttacagcaaaagcagatgg cctgcaatcaagcacagtaaaaattgcaacaacagcagtt ccgggaacaagcacagaaaaacagtccgcagcttttatt acagccgcaactattatgtcaaaacaggcaacaaaccgat tctgccgtcagatgttgaagttcgctattcagatggaaca agcgatagacaaaacgttacatgggatgcagtttcagatg atcaaattgcaaaagcaggctcattttcagttgcaggcac agttgcaggccaaaaaattagcgttcgcgtcacaatgatt gatgaaattggcgcactgctgaattattcagcaagcacac cggttggcacaccggcagttcttccgggatcaagaccggc agtcctgccggatggcacagtcacatcagcaaattttgca gtccattggacaaaaccggcagatacagtctataatacag caggcacagtcaaagtaccgggaacagcaacagtttttgg caaagaatttaaagtcacagcgacaattagagttcaaaga agccaagttacaattggctcatcagtttcaggaaatgcac tgagactgacacaaaatattccggcagataaacaatcaga tacactggatgcgattaaagatggctcaacaacagttgat gcaaatacaggcggaggcgcaaatccgtcagcatggacaa attgggcatattcaaaagcaggccataacacagcggaaat tacatttgaatatgcgacagaacaacaactgggccagatc gtcatgtatttttttcgcgatagcaatgcagttagatttc cggatgctggcaaaacaaaaattcagatcagcgcagatgg caaaaattggacagatctggcagcaacagaaacaattgca gcgcaagaatcaagcgatagagtcaaaccgtatacatatg attttgcaccggttggcgcaacatttgttaaagtgacagt cacaaacgcagatacaacaacaccgtcaggcgttgtttgc gcaggcctgacagaaattgaactgaaaacagcgacaagca aatttgtcacaaatacatcagcagcactgtcatcacttac agtcaatggcacaaaagtttcagattcagttctggcagca ggctcatataacacaccggcaattatcgcagatgttaaag cggaaggcgaaggcaatgcaagcgttacagtccttccggc acatgataatgttattcgcgtcattacagaaagcgaagat catgtcacacgcaaaacatttacaatcaacctgggcacag aacaagaatttccggctgattcagatgaaagagattatcc ggcagcagatatgacagtcacagttggctcagaacaaaca tcaggcacagcaacagaaggaccgaaaaaatttgcagtcg atggcaacacatcaacatattggcatagcaattggacacc gacaacagttaatgatctgtggatcgcgtttgaactgcaa aaaccgacaaaactggatgcactgagatatcttccgcgtc cggcaggctcaaaaaatggcagcgtcacagaatataaagt tcaggtgtcagatgatggaacaaactggacagatgcaggc tcaggcacatggacaacggattatggctggaaactggcgg aatttaatcaaccggtcacaacaaaacatgttagactgaa agcggttcatacatatgcagatagcggcaacgataaattt atgagcgcaagcgaaattagactgagaaaagcggtcgata caacggatatttcaggcgcaacagttacagttccggcaaa actgacagttgatagagttgatgcagatcatccggcaaca tttgcaacaaaagatgtcacagttacactgggagatgcaa cactgagatatggcgttgattatctgctggattatgcagg caatacagcagttggcaaagcaacagtgacagttagaggc attgataaatattcaggcacagtcgcgaaaacatttacaa ttgaactgaaaaatgcaccggcaccggaaccgacactgac atcagttagcgtcaaaacaaaaccgagcaaactgacatat gttgtcggagatgcatttgatccggcaggcctggttctgc aacatgatagacaagcagatagacctccgcaaccgctggt tggcgaacaagcggatgaacgcggactgacatgcggcaca agatgcgatagagttgaacaactgcgcaaacatgaaaata gagaagcgcatagaacaggcctggatcatctggaatttgt tggcgcagcagatggcgcagttggagaacaagcaacattt aaagtccatgtccatgcagatcagggagatggcagacatg atgatgcagatgaacgcgatattgatccgcatgttccggt cgatcatgcagttggcgaactggcaagagcagcatgccat catgttattggcctgagagtcgatacacatagacttaaag caagcggctttcaaattccggctgatgatatggcagaaat c gatcgcattacaggctttcatcgttttgaacgccatgtc >SEQ ID NO:9 Nucleotide sequence encoding BIF_917 gttgaagatgcaacaagaagcgatagcacaacacaaatgt catcaacaccggaagttgtttattcatcagcggtcgatag caaacaaaatcgcacaagcgattttgatgcgaactggaaa tttatgctgtcagatagcgttcaagcacaagatccggcat ttgatgattcagcatggcaacaagttgatctgccgcatga ttatagcatcacacagaaatatagccaaagcaatgaagca gaatcagcatatcttccgggaggcacaggctggtatagaa aaagctttacaattgatagagatctggcaggcaaacgcat tgcgattaattttgatggcgtctatatgaatgcaacagtc tggtttaatggcgttaaactgggcacacatccgtatggct attcaccgttttcatttgatctgacaggcaatgcaaaatt tggcggagaaaacacaattgtcgtcaaagttgaaaataga ctgccgtcatcaagatggtattcaggcagcggcatttata gagatgttacactgacagttacagatggcgttcatgttgg caataatggcgtcgcaattaaaacaccgtcactggcaaca caaaatggcggagatgtcacaatgaacctgacaacaaaag tcgcgaatgatacagaagcagcagcgaacattacactgaa acagacagtttttccgaaaggcggaaaaacggatgcagca attggcacagttacaacagcatcaaaatcaattgcagcag gcgcatcagcagatgttacaagcacaattacagcagcaag cccgaaactgtggtcaattaaaaacccgaacctgtataca gttagaacagaagttctgaacggaggcaaagttctggata catatgatacagaatatggctttcgctggacaggctttga tgcaacatcaggcttttcactgaatggcgaaaaagtcaaa ctgaaaggcgttagcatgcatcatgatcaaggctcacttg gcgcagttgcaaatagacgcgcaattgaaagacaagtcga aatcctgcaaaaaatgggcgtcaatagcattcgcacaaca cataatccggcagcaaaagcactgattgatgtctgcaatg aaaaaggcgttctggttgtcgaagaagtctttgatatgtg gaaccgcagcaaaaatggcaacacggaagattatggcaaa tggtttggccaagcaattgcaggcgataatgcagttctgg gaggcgataaagatgaaacatgggcgaaatttgatcttac atcaacaattaaccgcgatagaaatgcaccgtcagttatt atgtggtcactgggcaatgaaatgatggaaggcatttcag gctcagtttcaggctttccggcaacatcagcaaaactggt tgcatggacaaaagcagcagattcaacaagaccgatgaca tatggcgataacaaaattaaagcgaactggaacgaatcaa atacaatgggcgataatctgacagcaaatggcggagttgt tggcacaaattattcagatggcgcaaactatgataaaatt cgtacaacacatccgtcatgggcaatttatggctcagaaa cagcatcagcgattaatagccgtggcatttataatagaac aacaggcggagcacaatcatcagataaacagctgacaagc tatgataattcagcagttggctggggagcagttgcatcat cagcatggtatgatgttgttcagagagattttgtcgcagg cacatatgtttggacaggatttgattatctgggcgaaccg acaccgtggaatggcacaggctcaggcgcagttggctcat ggccgtcaccgaaaaatagctattttggcatcgttgatac agcaggctttccgaaagatacatattatttttatcagagc cagtggaatgatgatgttcatacactgcatattcttccgg catggaatgaaaatgttgttgcaaaaggctcaggcaataa tgttccggttgtcgtttatacagatgcagcgaaagtgaaa ctgtattttacaccgaaaggctcaacagaaaaaagactga tcggcgaaaaatcatttacaaaaaaaacaacagcggcagg ctatacatatcaagtctatgaaggcagcgataaagattca acagcgcataaaaacatgtatctgacatggaatgttccgt gggcagaaggcacaatttcagcggaagcgtatgatgaaaa taatcgcctgattccggaaggcagcacagaaggcaacgca tcagttacaacaacaggcaaagcagcaaaactgaaagcag atgcggatcgcaaaacaattacagcggatggcaaagatct gtcatatattgaagtcgatgtcacagatgcaaatggccat attgttccggatgcagcaaatagagtcacatttgatgtta aaggcgcaggcaaactggttggcgttgataatggctcatc accggatcatgattcatatcaagcggataaccgcaaagca ttttcaggcaaagtcctggcaattgttcagtcaacaaaag aagcaggcgaaattacagttacagcaaaagcagatggcct gcaatcaagcacagttaaaattgcaacaacagcagttccg ggaacaagcacagaaaaaaca >Nucleotide sequence encoding SEQ ID NO:10 BIF_995 gttgaagatgcaacaagaagcgatagcacaacacaaatgt catcaacaccggaagttgtttattcatcagcggtcgatag caaacaaaatcgcacaagcgattttgatgcgaactggaaa tttatgctgtcagatagcgttcaagcacaagatccggcat ttgatgattcagcatggcaacaagttgatctgccgcatga ttatagcatcacacagaaatatagccaaagcaatgaagca gaatcagcatatcttccgggaggcacaggctggtatagaa aaagctttacaattgatagagatctggcaggcaaacgcat tgcgattaattttgatggcgtctatatgaatgcaacagtc tggtttaatggcgttaaactgggcacacatccgtatggct attcaccgttttcatttgatctgacaggcaatgcaaaatt tggcggagaaaacacaattgtcgtcaaagttgaaaataga ctgccgtcatcaagatggtattcaggcagcggcatttata gagatgttacactgacagttacagatggcgttcatgttgg caataatggcgtcgcaattaaaacaccgtcactggcaaca caaaatggcggagatgtcacaatgaacctgacaacaaaag tcgcgaatgatacagaagcagcagcgaacattacactgaa acagacagtttttccgaaaggcggaaaaacggatgcagca attggcacagttacaacagcatcaaaatcaattgcagcag gcgcatcagcagatgttacaagcacaattacagcagcaag cccgaaactgtggtcaattaaaaacccgaacctgtataca gttagaacagaagttctgaacggaggcaaagttctggata catatgatacagaatatggctttcgctggacaggctttga tgcaacatcaggcttttcactgaatggcgaaaaagtcaaa ctgaaaggcgttagcatgcatcatgatcaaggctcacttg gcgcagttgcaaatagacgcgcaattgaaagacaagtcga aatcctgcaaaaaatgggcgtcaatagcattcgcacaaca cataatccggcagcaaaagcactgattgatgtctgcaatg aaaaaggcgttctggttgtcgaagaagtctttgatatgtg gaaccgcagcaaaaatggcaacacggaagattatggcaaa tggtttggccaagcaattgcaggcgataatgcagttctgg gaggcgataaagatgaaacatgggcgaaatttgatcttac atcaacaattaaccgcgatagaaatgcaccgtcagttatt atgtggtcactgggcaatgaaatgatggaaggcatttcag gctcagtttcaggctttccggcaacatcagcaaaactggt tgcatggacaaaagcagcagattcaacaagaccgatgaca tatggcgataacaaaattaaagcgaactggaacgaatcaa atacaatgggcgataatctgacagcaaatggcggagttgt tggcacaaattattcagatggcgcaaactatgataaaatt cgtacaacacatccgtcatgggcaatttatggctcagaaa cagcatcagcgattaatagccgtggcatttataatagaac aacaggcggagcacaatcatcagataaacagctgacaagc tatgataattcagcagttggctggggagcagttgcatcat cagcatggtatgatgttgttcagagagattttgtcgcagg cacatatgtttggacaggatttgattatctgggcgaaccg acaccgtggaatggcacaggctcaggcgcagttggctcat ggccgtcaccgaaaaatagctattttggcatcgttgatac agcaggctttccgaaagatacatattatttttatcagagc cagtggaatgatgatgttcatacactgcatattcttccgg catggaatgaaaatgttgttgcaaaaggctcaggcaataa tgttccggttgtcgtttatacagatgcagcgaaagtgaaa ctgtattttacaccgaaaggctcaacagaaaaaagactga tcggcgaaaaatcatttacaaaaaaaacaacagcggcagg ctatacatatcaagtctatgaaggcagcgataaagattca acagcgcataaaaacatgtatctgacatggaatgttccgt gggcagaaggcacaatttcagcggaagcgtatgatgaaaa taatcgcctgattccggaaggcagcacagaaggcaacgca tcagttacaacaacaggcaaagcagcaaaactgaaagcag atgcggatcgcaaaacaattacagcggatggcaaagatct gtcatatattgaagtcgatgtcacagatgcaaatggccat attgttccggatgcagcaaatagagtcacatttgatgtta aaggcgcaggcaaactggttggcgttgataatggctcatc accggatcatgattcatatcaagcggataaccgcaaagca ttttcaggcaaagtcctggcaattgttcagtcaacaaaag aagcaggcgaaattacagttacagcaaaagcagatggcct gcaatcaagcacagttaaaattgcaacaacagcagttccg ggaacaagcacagaaaaaacagtccgcagcttttattaca gccgcaactattatgtcaaaacaggcaacaaaccgattct gccgtcagatgttgaagttcgctattcagatggaacaagc gatagacaaaacgttacatgggatgcagtttcagatgatc aaattgcaaaagcaggctcattttcagttgcaggcacagt tgcaggccaaaaaattagcgttcgcgtcacaatgattgat gaaattggcgcactg >SEQ ID NO:11 Nucleotide sequence encoding BIF_1068 gttgaagatgcaacaagaagcgatagcacaacacaaatgt catcaacaccggaagttgtttattcatcagcggtcgatag caaacaaaatcgcacaagcgattttgatgcgaactggaaa tttatgctgtcagatagcgttcaagcacaagatccggcat ttgatgattcagcatggcaacaagttgatctgccgcatga ttatagcatcacacagaaatatagccaaagcaatgaagca gaatcagcatatcttccgggaggcacaggctggtatagaa aaagctttacaattgatagagatctggcaggcaaacgcat tgcgattaattttgatggcgtctatatgaatgcaacagtc tggtttaatggcgttaaactgggcacacatccgtatggct attcaccgttttcatttgatctgacaggcaatgcaaaatt tggcggagaaaacacaattgtcgtcaaagttgaaaataga ctgccgtcatcaagatggtattcaggcagcggcatttata gagatgttacactgacagttacagatggcgttcatgttgg caataatggcgtcgcaattaaaacaccgtcactggcaaca caaaatggcggagatgtcacaatgaacctgacaacaaaag tcgcgaatgatacagaagcagcagcgaacattacactgaa acagacagtttttccgaaaggcggaaaaacggatgcagca attggcacagttacaacagcatcaaaatcaattgcagcag gcgcatcagcagatgttacaagcacaattacagcagcaag cccgaaactgtggtcaattaaaaacccgaacctgtataca gttagaacagaagttctgaacggaggcaaagttctggata catatgatacagaatatggctttcgctggacaggctttga tgcaacatcaggcttttcactgaatggcgaaaaagtcaaa ctgaaaggcgttagcatgcatcatgatcaaggctcacttg gcgcagttgcaaatagacgcgcaattgaaagacaagtcga aatcctgcaaaaaatgggcgtcaatagcattcgcacaaca cataatccggcagcaaaagcactgattgatgtctgcaatg aaaaaggcgttctggttgtcgaagaagtctttgatatgtg gaaccgcagcaaaaatggcaacacggaagattatggcaaa tggtttggccaagcaattgcaggcgataatgcagttctgg gaggcgataaagatgaaacatgggcgaaatttgatcttac atcaacaattaaccgcgatagaaatgcaccgtcagttatt atgtggtcactgggcaatgaaatgatggaaggcatttcag gctcagtttcaggctttccggcaacatcagcaaaactggt tgcatggacaaaagcagcagattcaacaagaccgatgaca tatggcgataacaaaattaaagcgaactggaacgaatcaa atacaatgggcgataatctgacagcaaatggcggagttgt tggcacaaattattcagatggcgcaaactatgataaaatt cgtacaacacatccgtcatgggcaatttatggctcagaaa cagcatcagcgattaatagccgtggcatttataatagaac aacaggcggagcacaatcatcagataaacagctgacaagc tatgataattcagcagttggctggggagcagttgcatcat cagcatggtatgatgttgttcagagagattttgtcgcagg cacatatgtttggacaggatttgattatctgggcgaaccg acaccgtggaatggcacaggctcaggcgcagttggctcat ggccgtcaccgaaaaatagctattttggcatcgttgatac agcaggctttccgaaagatacatattatttttatcagagc cagtggaatgatgatgttcatacactgcatattcttccgg catggaatgaaaatgttgttgcaaaaggctcaggcaataa tgttccggttgtcgtttatacagatgcagcgaaagtgaaa ctgtattttacaccgaaaggctcaacagaaaaaagactga tcggcgaaaaatcatttacaaaaaaaacaacagcggcagg ctatacatatcaagtctatgaaggcagcgataaagattca acagcgcataaaaacatgtatctgacatggaatgttccgt gggcagaaggcacaatttcagcggaagcgtatgatgaaaa taatcgcctgattccggaaggcagcacagaaggcaacgca tcagttacaacaacaggcaaagcagcaaaactgaaagcag atgcggatcgcaaaacaattacagcggatggcaaagatct gtcatatattgaagtcgatgtcacagatgcaaatggccat attgttccggatgcagcaaatagagtcacatttgatgtta aaggcgcaggcaaactggttggcgttgataatggctcatc accggatcatgattcatatcaagcggataaccgcaaagca ttttcaggcaaagtcctggcaattgttcagtcaacaaaag aagcaggcgaaattacagttacagcaaaagcagatggcct gcaatcaagcacagttaaaattgcaacaacagcagttccg ggaacaagcacagaaaaaacagtccgcagcttttattaca gccgcaactattatgtcaaaacaggcaacaaaccgattct gccgtcagatgttgaagttcgctattcagatggaacaagc gatagacaaaacgttacatgggatgcagtttcagatgatc aaattgcaaaagcaggctcattttcagttgcaggcacagt tgcaggccaaaaaattagcgttcgcgtcacaatgattgat gaaattggcgcactgctgaattattcagcaagcacaccgg ttggcacaccggcagttcttccgggatcaagaccggcagt cctgccggatggcacagtcacatcagcaaattttgcagtc cattggacaaaaccggcagatacagtctataatacagcag gcacagtcaaagtaccgggaacagcaacagtttttggcaa agaatttaaagtcacagcgacaattagagttcaa >SEQ ID NO:12 Nucleotide sequence encoding BIF_1172 gttgaagatgcaacaagaagcgatagcacaacacaaatgt catcaacaccggaagttgtttattcatcagcggtcgatag caaacaaaatcgcacaagcgattttgatgcgaactggaaa tttatgctgtcagatagcgttcaagcacaagatccggcat ttgatgattcagcatggcaacaagttgatctgccgcatga ttatagcatcacacagaaatatagccaaagcaatgaagca gaatcagcatatcttccgggaggcacaggctggtatagaa aaagctttacaattgatagagatctggcaggcaaacgcat tgcgattaattttgatggcgtctatatgaatgcaacagtc tggtttaatggcgttaaactgggcacacatccgtatggct attcaccgttttcatttgatctgacaggcaatgcaaaatt tggcggagaaaacacaattgtcgtcaaagttgaaaataga ctgccgtcatcaagatggtattcaggcagcggcatttata gagatgttacactgacagttacagatggcgttcatgttgg caataatggcgtcgcaattaaaacaccgtcactggcaaca caaaatggcggagatgtcacaatgaacctgacaacaaaag tcgcgaatgatacagaagcagcagcgaacattacactgaa acagacagtttttccgaaaggcggaaaaacggatgcagca attggcacagttacaacagcatcaaaatcaattgcagcag gcgcatcagcagatgttacaagcacaattacagcagcaag cccgaaactgtggtcaattaaaaacccgaacctgtataca gttagaacagaagttctgaacggaggcaaagttctggata catatgatacagaatatggctttcgctggacaggctttga tgcaacatcaggcttttcactgaatggcgaaaaagtcaaa ctgaaaggcgttagcatgcatcatgatcaaggctcacttg gcgcagttgcaaatagacgcgcaattgaaagacaagtcga aatcctgcaaaaaatgggcgtcaatagcattcgcacaaca cataatccggcagcaaaagcactgattgatgtctgcaatg aaaaaggcgttctggttgtcgaagaagtctttgatatgtg gaaccgcagcaaaaatggcaacacggaagattatggcaaa tggtttggccaagcaattgcaggcgataatgcagttctgg gaggcgataaagatgaaacatgggcgaaatttgatcttac atcaacaattaaccgcgatagaaatgcaccgtcagttatt atgtggtcactgggcaatgaaatgatggaaggcatttcag gctcagtttcaggctttccggcaacatcagcaaaactggt tgcatggacaaaagcagcagattcaacaagaccgatgaca tatggcgataacaaaattaaagcgaactggaacgaatcaa atacaatgggcgataatctgacagcaaatggcggagttgt tggcacaaattattcagatggcgcaaactatgataaaatt cgtacaacacatccgtcatgggcaatttatggctcagaaa cagcatcagcgattaatagccgtggcatttataatagaac aacaggcggagcacaatcatcagataaacagctgacaagc tatgataattcagcagttggctggggagcagttgcatcat cagcatggtatgatgttgttcagagagattttgtcgcagg cacatatgtttggacaggatttgattatctgggcgaaccg acaccgtggaatggcacaggctcaggcgcagttggctcat ggccgtcaccgaaaaatagctattttggcatcgttgatac agcaggctttccgaaagatacatattatttttatcagagc cagtggaatgatgatgttcatacactgcatattcttccgg catggaatgaaaatgttgttgcaaaaggctcaggcaataa tgttccggttgtcgtttatacagatgcagcgaaagtgaaa ctgtattttacaccgaaaggctcaacagaaaaaagactga tcggcgaaaaatcatttacaaaaaaaacaacagcggcagg ctatacatatcaagtctatgaaggcagcgataaagattca acagcgcataaaaacatgtatctgacatggaatgttccgt gggcagaaggcacaatttcagcggaagcgtatgatgaaaa taatcgcctgattccggaaggcagcacagaaggcaacgca tcagttacaacaacaggcaaagcagcaaaactgaaagcag atgcggatcgcaaaacaattacagcggatggcaaagatct gtcatatattgaagtcgatgtcacagatgcaaatggccat attgttccggatgcagcaaatagagtcacatttgatgtta aaggcgcaggcaaactggttggcgttgataatggctcatc accggatcatgattcatatcaagcggataaccgcaaagca ttttcaggcaaagtcctggcaattgttcagtcaacaaaag aagcaggcgaaattacagttacagcaaaagcagatggcct gcaatcaagcacagttaaaattgcaacaacagcagttccg ggaacaagcacagaaaaaacagtccgcagcttttattaca gccgcaactattatgtcaaaacaggcaacaaaccgattct gccgtcagatgttgaagttcgctattcagatggaacaagc gatagacaaaacgttacatgggatgcagtttcagatgatc aaattgcaaaagcaggctcattttcagttgcaggcacagt tgcaggccaaaaaattagcgttcgcgtcacaatgattgat gaaattggcgcactgctgaattattcagcaagcacaccgg ttggcacaccggcagttcttccgggatcaagaccggcagt cctgccggatggcacagtcacatcagcaaattttgcagtc cattggacaaaaccggcagatacagtctataatacagcag gcacagtcaaagtaccgggaacagcaacagtttttggcaa agaatttaaagtcacagcgacaattagagttcaaagaagc caagttacaattggctcatcagtttcaggaaatgcactga gactgacacaaaatattccggcagataaacaatcagatac actggatgcgattaaagatggctcaacaacagttgatgca aatacaggcggaggcgcaaatccgtcagcatggacaaatt gggcatattcaaaagcaggccataacacagcggaaattac atttgaatatgcgacagaacaacaactgggccagatcgtc atgtatttttttcgcgatagcaatgcagttagatttccgg atgctggcaaaacaaaaattcagatc >SEQ ID NO:13 Nucleotide sequence encoding BIF_1241 gttgaagatgcaacaagaagcgatagcacaacacaaatgt catcaacaccggaagttgtttattcatcagcggtcgatag caaacaaaatcgcacaagcgattttgatgcgaactggaaa tttatgctgtcagatagcgttcaagcacaagatccggcat ttgatgattcagcatggcaacaagttgatctgccgcatga ttatagcatcacacagaaatatagccaaagcaatgaagca gaatcagcatatcttccgggaggcacaggctggtatagaa aaagctttacaattgatagagatctggcaggcaaacgcat tgcgattaattttgatggcgtctatatgaatgcaacagtc tggtttaatggcgttaaactgggcacacatccgtatggct attcaccgttttcatttgatctgacaggcaatgcaaaatt tggcggagaaaacacaattgtcgtcaaagttgaaaataga ctgccgtcatcaagatggtattcaggcagcggcatttata gagatgttacactgacagttacagatggcgttcatgttgg caataatggcgtcgcaattaaaacaccgtcactggcaaca caaaatggcggagatgtcacaatgaacctgacaacaaaag tcgcgaatgatacagaagcagcagcgaacattacactgaa acagacagtttttccgaaaggcggaaaaacggatgcagca attggcacagttacaacagcatcaaaatcaattgcagcag gcgcatcagcagatgttacaagcacaattacagcagcaag cccgaaactgtggtcaattaaaaacccgaacctgtataca gttagaacagaagttctgaacggaggcaaagttctggata catatgatacagaatatggctttcgctggacaggctttga tgcaacatcaggcttttcactgaatggcgaaaaagtcaaa ctgaaaggcgttagcatgcatcatgatcaaggctcacttg gcgcagttgcaaatagacgcgcaattgaaagacaagtcga aatcctgcaaaaaatgggcgtcaatagcattcgcacaaca cataatccggcagcaaaagcactgattgatgtctgcaatg aaaaaggcgttctggttgtcgaagaagtctttgatatgtg gaaccgcagcaaaaatggcaacacggaagattatggcaaa tggtttggccaagcaattgcaggcgataatgcagttctgg gaggcgataaagatgaaacatgggcgaaatttgatcttac atcaacaattaaccgcgatagaaatgcaccgtcagttatt atgtggtcactgggcaatgaaatgatggaaggcatttcag gctcagtttcaggctttccggcaacatcagcaaaactggt tgcatggacaaaagcagcagattcaacaagaccgatgaca tatggcgataacaaaattaaagcgaactggaacgaatcaa atacaatgggcgataatctgacagcaaatggcggagttgt tggcacaaattattcagatggcgcaaactatgataaaatt cgtacaacacatccgtcatgggcaatttatggctcagaaa cagcatcagcgattaatagccgtggcatttataatagaac aacaggcggagcacaatcatcagataaacagctgacaagc tatgataattcagcagttggctggggagcagttgcatcat cagcatggtatgatgttgttcagagagattttgtcgcagg cacatatgtttggacaggatttgattatctgggcgaaccg acaccgtggaatggcacaggctcaggcgcagttggctcat ggccgtcaccgaaaaatagctattttggcatcgttgatac agcaggctttccgaaagatacatattatttttatcagagc cagtggaatgatgatgttcatacactgcatattcttccgg catggaatgaaaatgttgttgcaaaaggctcaggcaataa tgttccggttgtcgtttatacagatgcagcgaaagtgaaa ctgtattttacaccgaaaggctcaacagaaaaaagactga tcggcgaaaaatcatttacaaaaaaaacaacagcggcagg ctatacatatcaagtctatgaaggcagcgataaagattca acagcgcataaaaacatgtatctgacatggaatgttccgt gggcagaaggcacaatttcagcggaagcgtatgatgaaaa taatcgcctgattccggaaggcagcacagaaggcaacgca tcagttacaacaacaggcaaagcagcaaaactgaaagcag atgcggatcgcaaaacaattacagcggatggcaaagatct gtcatatattgaagtcgatgtcacagatgcaaatggccat attgttccggatgcagcaaatagagtcacatttgatgtta aaggcgcaggcaaactggttggcgttgataatggctcatc accggatcatgattcatatcaagcggataaccgcaaagca ttttcaggcaaagtcctggcaattgttcagtcaacaaaag aagcaggcgaaattacagttacagcaaaagcagatggcct gcaatcaagcacagttaaaattgcaacaacagcagttccg ggaacaagcacagaaaaaacagtccgcagcttttattaca gccgcaactattatgtcaaaacaggcaacaaaccgattct gccgtcagatgttgaagttcgctattcagatggaacaagc gatagacaaaacgttacatgggatgcagtttcagatgatc aaattgcaaaagcaggctcattttcagttgcaggcacagt tgcaggccaaaaaattagcgttcgcgtcacaatgattgat gaaattggcgcactgctgaattattcagcaagcacaccgg ttggcacaccggcagttcttccgggatcaagaccggcagt cctgccggatggcacagtcacatcagcaaattttgcagtc cattggacaaaaccggcagatacagtctataatacagcag gcacagtcaaagtaccgggaacagcaacagtttttggcaa agaatttaaagtcacagcgacaattagagttcaaagaagc caagttacaattggctcatcagtttcaggaaatgcactga gactgacacaaaatattccggcagataaacaatcagatac actggatgcgattaaagatggctcaacaacagttgatgca aatacaggcggaggcgcaaatccgtcagcatggacaaatt gggcatattcaaaagcaggccataacacagcggaaattac atttgaatatgcgacagaacaacaactgggccagatcgtc atgtatttttttcgcgatagcaatgcagttagatttccgg atgctggcaaaacaaaaattcagatcagcgcagatggcaa aaattggacagatctggcagcaacagaaacaattgcagcg caagaatcaagcgatagagtcaaaccgtatacatatgatt ttgcaccggttggcgcaacatttgttaaagtgacagtcac aaacgcagatacaacaacaccgtcaggcgttgtttgcgca ggcctgacagaaattgaactgaaaacagcgaca >SEQ ID NO:14 Nucleotide sequence encoding BIF_1326 gttgaagatgcaacaagaagcgatagcacaacacaaatgt catcaacaccggaagttgtttattcatcagcggtcgatag caaacaaaatcgcacaagcgattttgatgcgaactggaaa tttatgctgtcagatagcgttcaagcacaagatccggcat ttgatgattcagcatggcaacaagttgatctgccgcatga ttatagcatcacacagaaatatagccaaagcaatgaagca gaatcagcatatcttccgggaggcacaggctggtatagaa aaagctttacaattgatagagatctggcaggcaaacgcat tgcgattaattttgatggcgtctatatgaatgcaacagtc tggtttaatggcgttaaactgggcacacatccgtatggct attcaccgttttcatttgatctgacaggcaatgcaaaatt tggcggagaaaacacaattgtcgtcaaagttgaaaataga ctgccgtcatcaagatggtattcaggcagcggcatttata gagatgttacactgacagttacagatggcgttcatgttgg caataatggcgtcgcaattaaaacaccgtcactggcaaca caaaatggcggagatgtcacaatgaacctgacaacaaaag tcgcgaatgatacagaagcagcagcgaacattacactgaa acagacagtttttccgaaaggcggaaaaacggatgcagca attggcacagttacaacagcatcaaaatcaattgcagcag gcgcatcagcagatgttacaagcacaattacagcagcaag cccgaaactgtggtcaattaaaaacccgaacctgtataca gttagaacagaagttctgaacggaggcaaagttctggata catatgatacagaatatggctttcgctggacaggctttga tgcaacatcaggcttttcactgaatggcgaaaaagtcaaa ctgaaaggcgttagcatgcatcatgatcaaggctcacttg gcgcagttgcaaatagacgcgcaattgaaagacaagtcga aatcctgcaaaaaatgggcgtcaatagcattcgcacaaca cataatccggcagcaaaagcactgattgatgtctgcaatg aaaaaggcgttctggttgtcgaagaagtctttgatatgtg gaaccgcagcaaaaatggcaacacggaagattatggcaaa tggtttggccaagcaattgcaggcgataatgcagttctgg gaggcgataaagatgaaacatgggcgaaatttgatcttac atcaacaattaaccgcgatagaaatgcaccgtcagttatt atgtggtcactgggcaatgaaatgatggaaggcatttcag gctcagtttcaggctttccggcaacatcagcaaaactggt tgcatggacaaaagcagcagattcaacaagaccgatgaca tatggcgataacaaaattaaagcgaactggaacgaatcaa atacaatgggcgataatctgacagcaaatggcggagttgt tggcacaaattattcagatggcgcaaactatgataaaatt cgtacaacacatccgtcatgggcaatttatggctcagaaa cagcatcagcgattaatagccgtggcatttataatagaac aacaggcggagcacaatcatcagataaacagctgacaagc tatgataattcagcagttggctggggagcagttgcatcat cagcatggtatgatgttgttcagagagattttgtcgcagg cacatatgtttggacaggatttgattatctgggcgaaccg acaccgtggaatggcacaggctcaggcgcagttggctcat ggccgtcaccgaaaaatagctattttggcatcgttgatac agcaggctttccgaaagatacatattatttttatcagagc cagtggaatgatgatgttcatacactgcatattcttccgg catggaatgaaaatgttgttgcaaaaggctcaggcaataa tgttccggttgtcgtttatacagatgcagcgaaagtgaaa ctgtattttacaccgaaaggctcaacagaaaaaagactga tcggcgaaaaatcatttacaaaaaaaacaacagcggcagg ctatacatatcaagtctatgaaggcagcgataaagattca acagcgcataaaaacatgtatctgacatggaatgttccgt gggcagaaggcacaatttcagcggaagcgtatgatgaaaa taatcgcctgattccggaaggcagcacagaaggcaacgca tcagttacaacaacaggcaaagcagcaaaactgaaagcag atgcggatcgcaaaacaattacagcggatggcaaagatct gtcatatattgaagtcgatgtcacagatgcaaatggccat attgttccggatgcagcaaatagagtcacatttgatgtta aaggcgcaggcaaactggttggcgttgataatggctcatc accggatcatgattcatatcaagcggataaccgcaaagca ttttcaggcaaagtcctggcaattgttcagtcaacaaaag aagcaggcgaaattacagttacagcaaaagcagatggcct gcaatcaagcacagttaaaattgcaacaacagcagttccg ggaacaagcacagaaaaaacagtccgcagcttttattaca gccgcaactattatgtcaaaacaggcaacaaaccgattct gccgtcagatgttgaagttcgctattcagatggaacaagc gatagacaaaacgttacatgggatgcagtttcagatgatc aaattgcaaaagcaggctcattttcagttgcaggcacagt tgcaggccaaaaaattagcgttcgcgtcacaatgattgat gaaattggcgcactgctgaattattcagcaagcacaccgg ttggcacaccggcagttcttccgggatcaagaccggcagt cctgccggatggcacagtcacatcagcaaattttgcagtc cattggacaaaaccggcagatacagtctataatacagcag gcacagtcaaagtaccgggaacagcaacagtttttggcaa agaatttaaagtcacagcgacaattagagttcaaagaagc caagttacaattggctcatcagtttcaggaaatgcactga gactgacacaaaatattccggcagataaacaatcagatac actggatgcgattaaagatggctcaacaacagttgatgca aatacaggcggaggcgcaaatccgtcagcatggacaaatt gggcatattcaaaagcaggccataacacagcggaaattac atttgaatatgcgacagaacaacaactgggccagatcgtc atgtatttttttcgcgatagcaatgcagttagatttccgg atgctggcaaaacaaaaattcagatcagcgcagatggcaa aaattggacagatctggcagcaacagaaacaattgcagcg caagaatcaagcgatagagtcaaaccgtatacatatgatt ttgcaccggttggcgcaacatttgttaaagtgacagtcac aaacgcagatacaacaacaccgtcaggcgttgtttgcgca ggcctgacagaaattgaactgaaaacagcgacaagcaaat ttgtcacaaatacatcagcagcactgtcatcacttacagt caatggcacaaaagtttcagattcagttctggcagcaggc tcatataacacaccggcaattatcgcagatgttaaagcgg aaggcgaaggcaatgcaagcgttacagtccttccggcaca tgataatgttattcgcgtcattacagaaagcgaagatcat gtcacacgcaaaacatttacaatcaacctgggcacagaac aagaattt >SEQ ID NO:15 Forward primer for generation of BIF mutants GGGTA ACTAGT GGAAGATGCAACAAGAAG >SEQ ID NO:16 BIF917 reverse primer GCGCTTAATTAATTATGTTTTTTCTGTGCTTGTTC >SEQ ID NO:17 BIF995 reverse primer GCGCTTAATTAATTACAGTGCGCCAATTTCATCAATCA >SEQ ID NO:18 BIF1068 reverse primer GCGCTTAATTAATTATTGAACTCTAATTGTCGCTG >SEQ ID NO:19 BIF1241 reverse primer GCGCTTAATTAATTATGTCGCTGTTTTCAGTTCAAT >SEQ ID NO:20 Reverse primer of BIF1326 GCGCTTAATTAATTAAAATTCTTGTTCTGTGCCCA >SEQ ID NO:21 Reverse primer of BIF1478 GCGCTTAATTAATTATTCTCAGTCTAATTTCGCTTGCGC >SEQ ID NO:22 Bifidobacterium bifidum terium bifidum) BIF1750 vedatrsdsttqmsstpevvyssavdskqnrtsdfdanwk fmlsdsvqaqdpafddsawqqvdlphdysitqkysqsnea esaylpggtgwyrksftidrdlagkriainfdgvymnatv wfngvklgthpygyspfsfdltgnakfggentivvkvenr lpssrwysgsgiyrdvtltvtdgvhvgnngvaiktpslat qnggdvtmnlttkvandteaaanitlkqtvfpkggktdaa igtvttasksiaagasadvtstitaaspklwsiknpnlyt vrtevlnggkvldtydteygfrwtgfdatsgfslngekvk lkgvsmhhdqgslgavanrraierqveilqkmgvnsirtt hnpaakalidvcnekgvlvveevfdmwnrskngntedygk wfgqaiagdnavlggdkdetwakfdltstinrdrnapsvi mwslgnemmegisgsvsgfpatsaklvawtkaadstrpmt ygdnkikanwnesntmgdnltanggvvgtnysdganydki rtthpswaiygsetasainsrgiynrttggaqssdkqlts ydnsavgwgavassawydvvqrdfvagtyvwtgfdylgep tpwngtgsgavgswpspknsyfgivdtagfpkdtyyfyqs qwnddvhtlhilpawnenvvakgsgnnvpvvvytdaakvk lyftpkgstekrligeksftkkttaagytyqvyegsdkds tahknmyltwnvpwaegtisaeaydennrlipegstegna svtttgkaaklkadadrktitadgkdlsyievdvtdangh ivpdaanrvtfdvkgagklvgvdngsspdhdsyqadnrka fsgkvlaivqstkeageitvtakadglqsstvkiattavp gtstektvrsfyysrnyyvktgnkpilpsdvevrysdgts drqnvtwdavsddqiakagsfsvagtvaggqkisvrvtmid eigallnysastpvgtpavlpgsrpavlpdgtvtsanfav hwtkpadtvyntagtvkvpgtatvfgkefkvtatirvqrs qvtigssvsgnalrltqnipadkqsdtldaikdgsttvda ntggganpsawtnwayskaghntaeitfeyateqqlgqiv myffrdsnavrfpdagktkiqisadgknwtdlaatetiaa qessdrvkpytydfapvgatfvkvtvtnadtttpsgvvca glteielktatskfvtntsaalssltvngtkvsdsvlaag syntpaiiadvkaegegnasvtvlpahdnvirvitesedh vtrktftinlgteqefpadsderdypaadmtvtvgseqts gtategpkkfavdgntstywhsnwtpttvndlwiafelqk ptkldalrylprpagskngsvteykvqvsddgtnwtdags gtwttdygwklaefnqpvttkhvrlkavhtyadsgndkfm saseirlrkavdttdisgatvtvpakltvdrvdadhpatf atkdvtvtlgdatlrygvdylldyagntavgkatvtvrgi dkysgtvaktftielknapapeptltsvsvktkpskltyv vgdafdpaglvlqhdrqadrppqplvgeqadergltcgtr cdrveqlrkhenreahrtgldhlefvgaadgavgeqatfk vhvhadqgdgrhddaderdidphvpvdhavgelaraachh viglrvdthrlkasgfqipaddmaeidritgfhrferhvg >SEQ ID NO:23 Bifidobacterium bifidum Signal sequence of extracellular lactase from Tertium bifidum DSM 20215. Vrskklwisllfalaliftmafgstssaqa

Claims

1. A transgalactosylation active enzyme, A galactosylation activity ratio of at least 100% is calculated by dividing the Abs420 (absorbance at 420 nm) in the presence of the receptor by the Abs420 in the absence of the receptor and multiplying by 100. ＋セロビオース / Abs420 －セロビオース ) *100), a transgalactosylating active enzyme consisting of a polypeptide.

2. 2. The transgalactosylating active enzyme of claim 1, which comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

2.

3. 2. The active transgalactosylating enzyme of claim 1, which comprises an amino acid sequence having at least 96.5% sequence identity to the amino acid sequence of SEQ ID NO:

2.

4. 2. The active transgalactosylating enzyme of claim 1, which comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO:

2.

5. 2. The transgalactosylating active enzyme of claim 1, which consists of the amino acid sequence of SEQ ID NO:

2.

6. 6. The transgalactosylation active enzyme according to any one of claims 1 to 5, wherein the polypeptide has a transgalactosylation activity ratio of more than 150%.

7. A nucleic acid capable of encoding a transgalactosylation active enzyme according to any one of claims 1 to 6.

8. 8. An expression vector comprising a nucleic acid according to claim 7 or capable of expressing an active transgalactosylation enzyme according to any one of claims 1 to 6.

9. A cell capable of expressing an active transgalactosylation enzyme according to any one of claims 1 to 6.

10. 10. A method for expressing a transgalactosylation active enzyme according to any one of claims 1 to 6, said method comprising the steps of obtaining a cell according to claim 9 and expressing said polypeptide from said cell.

11. The method of claim 10, further comprising the step of purifying the transgalactosylation active enzyme.

12. A composition comprising a transgalactosylation active enzyme according to any one of claims 1 to 6 and lactose.

13. The composition of claim 12, wherein the composition is a food composition.

14. The composition of claim 13, wherein the food composition is a dairy product.

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