WO2009119538A1 - Novel polypeptide having epimerase activity, method for production of the same, and use of the same - Google Patents

Abstract

The object aims to obtain a novel polypeptide capable of catalyzing a reaction for altering the conformation of a hydroxy group in an oligosaccharide and provide an enzymatic synthesis method for an oligosaccharide in which the conformation of a hydroxy group is altered. Disclosed is any one polypeptide selected from the following polypeptides (1) and (2): (1) a polypeptide comprising an amino acid sequence depicted in any one of SEQ ID NOs:1 to 4; and (2) a polypeptide which comprises an amino acid sequence having the deletion, substitution or addition of one or several amino acid residues in an amino acid sequence depicted in any one of SEQ ID NOs:1 to 4 and can catalyze a reaction for altering the conformation of a position-2 hydroxy group in a reducing terminal sugar residue in an oligosaccharide in which the reducing terminal sugar residue is bound via a ß-1,4 bond.

Description

Novel polypeptide having epimerase activity, method for producing the same and method for using the same

The present invention relates to a novel polypeptide and use thereof, and more specifically, the 2-position hydroxyl group of the reducing terminal sugar residue of an oligosaccharide in which the reducing terminal sugar residue is linked by a β-1,4 bond. Polypeptide that catalyzes a reaction that changes the conformation of the polypeptide, a method for producing the same, a polynucleotide that encodes the polypeptide and a method for using the same, and the conformation of the hydroxyl group at the 2-position of the reducing end sugar residue using the polypeptide The present invention relates to a method for producing oligosaccharides and prebiotics in which the reducing terminal sugar residue having a changed locus is bound by a β-1,4 bond.

Carbohydrate is a main energy source of living organisms and is a biosynthetic precursor that is converted into biological components (fatty acids, triglycerides, amino acids, nucleic acids). Carbohydrates also play an important role as structural components of connective tissue, neural tissue, bacterial cell walls, and nucleic acids. Furthermore, it has recently been clarified that sugar chains possessed by glycoproteins play an important role in various information transmissions in vivo, and are attracting attention in genetic engineering, protein engineering, sugar chain engineering, and the like.

Various natural oligosaccharides or artificial oligosaccharides have been reported as functional saccharides that are different from carbohydrates in glycoproteins involved in in vivo signal transmission as carbohydrates as energy sources, and pharmaceuticals using these And / or health foods have been actively developed (see, for example, Patent Documents 1 and 2).

In recent years, oligosaccharides having various physiological functions have attracted particular attention as prebiotics. As a result of many researches and developments aimed at synthesizing such functional oligosaccharides, various oligosaccharides have been discovered, synthesized, used as research materials for application development, and some are produced industrially. There are some (see, for example, Patent Document 3). Currently, more than half of foods for specified health use in Japan contain oligosaccharides as functional ingredients.

An example of a functional oligosaccharide is trehalose (α-D-glucopyranosyl-α-D-glucopyranoside) widely used in foods and cosmetics. Trehalose is produced by using amylose from starch using isoamylase, and then starting with amylosyl trehalose as an intermediate by the action of malto-oligosyl trehalose synthase, and this intermediate is further converted into maltooligosyl trehalose trehalohydrolase. It is produced by hydrolysis using In this production method, it is necessary to collect amylose as a reaction residue and repeat the same reaction as described above (see Non-Patent Document 1).

Trehalose uses maltose phosphorylase produced by a bacterium belonging to the genus Paenibacillus in the deep sea to produce β-D-glucose-1-phosphate in the presence of maltose and phosphate, and glucose is added to the reaction solution to add trehalose. It is also produced by the reverse reaction of phosphorylase (see Patent Document 4). This production method is also complicated because it requires an enzyme reaction of 2 to 4 stages, as in the above method using starch as a raw material.

As another example of functional oligosaccharides, there can be mentioned isomaltoligosaccharides used as prebiotics and low-calorie products. Isomaltooligosaccharides are produced from starch by a three-stage enzymatic reaction using Bacillus α-amylase, soybean β-amylase and Aspergillus niger α-glucosidase. The oligosaccharide produced by this enzyme reaction is generally a mixture of (α-1,2, α-1,3, α-1,6) glycosidic linked saccharides with a degree of polymerization of 2-6 and contained in the mixture. It is difficult to control the composition of oligosaccharides (see Non-Patent Document 2).

Another example of functional oligosaccharide is epilactose. Epilactose is produced using a reverse reaction of β-1,4-galactosidase derived from Bacillus circulans ATCC 31382 strain (see Non-Patent Document 3). However, in this production method, galactosyl mannose having β-1,3 bond, β-1,4 bond and β-1,6 bond is produced at about 1: 3: 1, so that pure epilactose is obtained. It is necessary to remove these by-products together with the remaining substrate by a complicated purification operation.

As another method for producing epilactose found by the present inventors, there is a method using cellobiose 2-epimerase (hereinafter referred to as CE) (see Non-Patent Document 4). CE (EC 5.1.3.11) is an enzyme having an activity of catalyzing a reaction that changes the conformation of the hydroxyl group at the 2-position of the reducing terminal sugar residue of cellobiose, and is an obligate anaerobic rumen bacterium Luminocox albus. (Ruminococcus albus) (hereinafter, R. albus) 7 strains (stored as ATCC 27210) have been suggested to exist (see Non-Patent Document 5). We have described R.I. The gene coding for CE from albus NE1 was successfully cloned and its nucleotide sequence was determined. The enzyme was an intracellular enzyme. Cellobiose reported that O-β-D-glucopyranosyl- (1 → 4) -D-mannose In addition to producing (Glc-Man) (FIG. 1), Glc-Glc-Man (O-β-D-glucopyranosyl- (1 → 4)-) which is a rare cellooligosaccharide from cellotriose (Glc-Glc-Glc) O-β-D-glucopyranosyl- (1 → 4) -D-mannose) (FIG. 2) is converted from cellotetraose (Glc-Glc-Glc-Glc) to Glc-Glc-Glc-Man (O-β-D -Glucopyranosyl- (1 → 4) -OD-glucopyranosyl- (1 → 4) -O-β-D-glucopyranosyl- (1 → 4) -D-mannose) It has been clarified that this is achieved (see Non-Patent Document 4).

The present inventors are R.I. Proving that CE from albus NE1 strain produces epilactose (O-β-D-galactopyranosyl- (1 → 4) -D-mannose) (Fig. 3) from lactose (Gal-Glc) A patent application has been filed separately (PCT / JP2007 / 001253).

Up to 30 types of epimerase, which is an isomerase, have been reported so far. Most epimerases use saccharides modified with nucleotides, phosphates, acyl groups, etc. as substrates, while those using unmodified sugars as substrates are aldose 1-epimerase (EC 5.1.3.3). , Maltose 1-epimerase (EC 5.1.3.21) and the aforementioned CE (EC 5.1.3.11). Among these three types of epimerases, the CE is the only epimerase that acts on the hydroxyl group at the 2-position of the reducing end sugar residue.

CE can be expected to be highly useful in the production of oligosaccharides because it does not produce by-products because it catalyzes a reaction that changes the conformation within the substrate molecule. Thus, epimerase, which is an isomerase, can be expected to have many advantages and usefulness for the synthesis of rare oligosaccharides due to its unique enzyme activity. The epimerase acting on the hydroxyl group at the 2-position of the group is only one type of CE (see Non-Patent Document 4).

JP-A-8-256730 (published on October 8, 1996) JP 2003-47402 A (published February 18, 2003) JP 2-16992 A (published January 19, 1990) WO2005 / 003343 (released on January 13, 2005) Toshiyuki Sugimoto et al., Journal of Japanese Society for Agricultural Chemistry, 1995, Vol. 72, 915-922 Tomoei Kanno, Monthly Food Chemical, 1998, Vol. 10, pp. 61-66 Miyasato, M .; and Ajisaka, K .; Biosci. Biotechnol. Biochem. 2004, 68, 2086-2090 S. Ito et al. Biochem. Biophys. Res. Commun. , 2007, 360, 640-645 T.A. R. Tyler and J. M.M. Leaferwood, Arch. Biochem. Acta, 1967, 119, 363-367.

An object of the present invention is to obtain a novel polypeptide that catalyzes a reaction that changes the conformation of a hydroxyl group of an oligosaccharide, and to provide an enzymatic synthesis method of the oligosaccharide in which the conformation of the hydroxyl group is changed.

The present inventors newly acquired a gene encoding epimerase that catalyzes a reaction that changes the hydroxyl conformation of the oligosaccharide, and further changes the hydroxyl conformation of the oligosaccharide encoded by the gene. Polypeptides that catalyze the reaction to be obtained were obtained, and the enzyme chemical properties were elucidated, and the following inventions were completed.

(I) (1) a polypeptide comprising the amino acid sequence represented by any of SEQ ID NOs: 1 to 4, or (2) one or more amino acids in the amino acid sequence represented by any of SEQ ID NOs: 1 to 4 Is a conformation of the hydroxyl group at the 2-position of the reducing end sugar residue of an oligosaccharide consisting of an amino acid sequence deleted, substituted or added, and the reducing end sugar residue linked by a β-1,4 bond A polypeptide that catalyzes a reaction that changes.

(Ii) a polypeptide having the physicochemical properties shown in (A) to (G) below;
(A) The apparent molecular weight by SDS-PAGE is 42 to 44 kDa,
(B) having the amino acid sequence shown in SEQ ID NO: 9 at the N-terminus,
(C) specifically catalyzing a reaction that changes the conformation of the hydroxyl group at the 2-position of the reducing end sugar residue of the oligosaccharide to which the reducing end sugar residue is bonded by a β-1,4 bond;
(D) When the enzyme activity at pH 7.5 is 100%, the enzyme activity is 80% or more at pH 7.0 to 7.5,
(E) After holding at 4 ° C. for 20 hours at pH 5.0 to 8.0, the enzyme activity remains 80% or more,
(F) Optimum temperature: Shows an enzyme activity of 80% or more at 15-30 ° C., assuming that the enzyme activity at 25 ° C. is 100%, and (G) Temperature stability: after holding at 50 ° C. for 1 hour, The enzyme activity at 30 ° C. remains at 80% or more.

(Iii) a polypeptide having the physicochemical properties shown in (A) to (G) below;
(A) The apparent molecular weight by SDS-PAGE is 43 to 45 kDa,
(B) having the amino acid sequence shown in SEQ ID NO: 10 at the N-terminus,
(C) specifically catalyzing a reaction that changes the conformation of the hydroxyl group at the 2-position of the reducing end sugar residue of the oligosaccharide to which the reducing end sugar residue is bonded by a β-1,4 bond;
(D) When the enzyme activity at pH 7.5 is 100%, the enzyme activity is 80% or more at pH 7.0 to 8.0,
(E) After holding at 4 ° C. for 20 hours at pH 4.0 to 8.0, the enzyme activity remains at 80% or more,
(F) When the enzyme activity at 25 ° C. is 100%, the enzyme activity is 80% or more at 10 to 45 ° C., and (G) the enzyme activity at 30 ° C. is 80 after holding at 40 ° C. for 1 hour. % Or more remain.

(Iv) a polypeptide having the physicochemical properties shown in (A) to (G) below;
(A) The apparent molecular weight by SDS-PAGE is 46 to 48 kDa,
(B) having the amino acid sequence shown in SEQ ID NO: 11 at the N-terminus,
(C) specifically catalyzing a reaction that changes the conformation of the hydroxyl group at the 2-position of the reducing end sugar residue of the oligosaccharide to which the reducing end sugar residue is bonded by a β-1,4 bond;
(D) When the enzyme activity at pH 7.5 is 100%, the enzyme activity is 80% or more at pH 7.0 to 9.0,
(E) After holding at 4 ° C. for 20 hours at pH 4.0 to 8.0, the enzyme activity remains at 80% or more,
(F) When the enzyme activity at 25 ° C. is 100%, the enzyme activity is 80% or more at 15 to 35 ° C., and (G) the enzyme activity at 30 ° C. is 80 after holding at 40 ° C. for 1 hour. % Or more remain.

(V) a polypeptide having the physicochemical properties shown in (A) to (G) below;
(A) The apparent molecular weight by SDS-PAGE is 44 to 46 kDa,
(B) having the amino acid sequence shown in SEQ ID NO: 12 at the N-terminus,
(C) specifically catalyzing a reaction that changes the conformation of the hydroxyl group at the 2-position of the reducing end sugar residue of the oligosaccharide to which the reducing end sugar residue is bonded by a β-1,4 bond;
(D) When the enzyme activity at pH 7.5 is 100%, the enzyme activity is 80% or more at pH 7.0 to 7.5,
(E) After holding at 4 ° C. for 20 hours at pH 5.0 to 8.0, the enzyme activity remains 80% or more,
(F) When the enzyme activity at 25 ° C. is 100%, the enzyme activity is 80% or more at 20 to 45 ° C., and (G) the enzyme activity at 30 ° C. is 80 after holding at 50 ° C. for 1 hour. % Or more remain.

(Vi) (1) a polynucleotide encoding a polypeptide comprising the amino acid sequence represented by any of SEQ ID NOs: 1 to 4, or (2) one of the amino acid sequences represented by any of SEQ ID NOs: 1 to 4 Alternatively, the oligosaccharide having the amino acid sequence in which a plurality of amino acids are deleted, substituted, or added, and the reducing terminal sugar residue of the oligosaccharide having the reducing terminal sugar residue linked by a β-1,4 bond is positioned at position 2 A polynucleotide encoding a polypeptide that catalyzes a reaction that changes the conformation of a hydroxyl group.

(Vii) (1) a polynucleotide comprising the nucleotide sequence represented by any of SEQ ID NOs: 5 to 8, or (2) a complementary sequence of the nucleotide sequence represented by any of SEQ ID NOs: 5 to 8 and stringent conditions Catalyzing a reaction that changes the conformation of the hydroxyl group at the 2-position of the reducing end sugar residue of an oligosaccharide consisting of a base sequence that hybridizes with and having a reducing end sugar residue bonded by a β-1,4 bond The polynucleotide according to (vi), which is any one of the polynucleotides encoding the polypeptide.

(Viii) A recombinant vector comprising the polynucleotide according to (vi) or (vii).

(Ix) A transformant into which the recombinant vector according to (viii) has been introduced.

(X) A method for producing a polypeptide that catalyzes a reaction that changes the conformation of the hydroxyl group at the 2-position of the reducing end sugar residue of an oligosaccharide in which the reducing end sugar residue is linked by a β-1,4 bond. A step of culturing the transformant according to (ix), a step of causing the transformant to produce the polypeptide, and recovering the polypeptide from the transformant or a culture supernatant of the transformant Comprising the step of:

(Xi) A method for producing an oligosaccharide in which the reducing end sugar residue in which the conformation of the hydroxyl group at the 2-position of the reducing end sugar residue is changed is bound by a β-1,4 bond, A step of incubating the polypeptide according to any one of (v) and an oligosaccharide in which a reducing terminal sugar residue is bonded by a β-1,4 bond; and a reducing terminal sugar residue of the oligosaccharide on the polypeptide. A method comprising producing the oligosaccharide by catalyzing a reaction that changes the conformation of the hydroxyl group at the 2-position of the group.

(Xii) a method for producing a prebiotic comprising an oligosaccharide in which the conformation of the hydroxyl group at the 2-position of the reducing end sugar residue is changed by a β-1,4 bond to the reducing end sugar residue. A step of incubating the polypeptide according to any one of claims 1 to 5 with a material containing an oligosaccharide in which a reducing terminal sugar residue is bound by a β-1,4 bond; and A method comprising producing a prebiotic by catalyzing a reaction that changes a conformation of a hydroxyl group at the 2-position of a reducing terminal sugar residue of a sugar.

The polypeptide provided by the present invention (1) catalyzes a reaction that changes the internal conformation of a substrate molecule and does not require any reactant other than oligosaccharide. (2) Since there is no reaction by-product, purification of the target product is easy, and it is suitable for mass production of functional oligosaccharides. (3) Substrate specificity for non-reducing end sugars Since it is not strict, it has advantages such as being able to synthesize oligosaccharides that cannot be synthesized by conventional methods.

Therefore, if the polypeptide provided by the present invention is used, not only the cellooligosaccharide or epilactose in which the conformation of the 2-position hydroxyl group of the reducing end sugar residue is changed, but also the stereo of the 2-position hydroxyl group of the desired reducing end sugar residue. Oligosaccharides can be synthesized easily and in large quantities in which the reducing end sugar residue having a changed conformation is bound by a β-1,4 bond. Thereby, the oligosaccharide which has a physiological effect, a prebiotic, a synbiotic, and the functional food containing these can be provided at low cost.

FIG. 5 is a scheme showing a reaction catalyzed by the polypeptide of the present invention to isomerize the hydroxyl group at the 2-position of glucose at the reducing end of cellobiose and convert it to Glc-Man. 1 is a scheme showing a reaction catalyzed by the polypeptide of the present invention to isomerize the hydroxyl group at the 2-position of the reducing end glucose of cellotriose and convert it to Glc-Glc-Man. It is a scheme showing a reaction catalyzed by the polypeptide of the present invention to isomerize the hydroxyl group at the 2-position of glucose at the reducing end of lactose and convert it to epilactose. CE-NE1 (registration number BAF81108), C.I. phytofermentans-derived AGE-like protein (Cp AGE-like, registration number ABX42625), C.I. saccharolyticus-derived AGE-like protein (Cs AGE-like, registration number ABP65941); It is a figure which shows the result of carrying out multiple alignment of the amino acid sequence of fragilis origin AGE-like protein (Bf AGE-like, accession number BAD47600) using the Clustal W program. Bold amino acids in the sequence indicate two conserved regions, * below the sequence indicates amino acids that are conserved in all sequences, and ● indicates a putative catalytic residue involved in the AGE reaction of Anabaena. It is a figure which shows the photograph of the gel which carried out CBB dyeing | staining of SDS-PAGE after CE-NE1 and the purified recombinant enzyme (mD1, mD2, mD3, mR6). Lane 1 is CE-NE1 (control), lane 2 is mD1, lane 3 is mD2, lane 4 is mD3, and lane 5 is mR6. FIG. 3 is a diagram showing the result of multiple alignment of amino acid sequences of mD1, mD2, mD3, and mR6 using the Clustal W program for comparison with the amino acid sequence of CE-NE1. Bold amino acids in the sequence indicate amino acids conserved in CE-NE1. It is a figure which shows the result of having analyzed the reaction product by TLC, after performing enzyme reaction which used cellobiose as a substrate about CE-NE1 and the purified recombinant enzyme (mD1, mD2, mD3, mR6). Lane S is a reaction product using no enzyme, Lane 1 is CE-NE1 (control), Lane 2 is mD1, Lane 3 is mD2, Lane 4 is mD3, and Lane 5 is mR6. It is a figure which shows the result of having analyzed the reaction product by TLC after performing enzyme reaction which used cellotriose as a substrate about CE-NE1 and the refined recombinant enzyme (mD1, mD2, mD3, mR6). Lane S is a reaction product using no enzyme, Lane 1 is CE-NE1 (control), Lane 2 is mD1, Lane 3 is mD2, Lane 4 is mD3, and Lane 5 is mR6. FIG. 3 is a diagram showing the results of analyzing the reaction product by TLC after performing an enzyme reaction using CE-NE1 and purified recombinant enzymes (mD1, mD2, mD3, mR6) using cellotetraose as a substrate. Lane S is a reaction product using no enzyme, Lane 1 is CE-NE1 (control), Lane 2 is mD1, Lane 3 is mD2, Lane 4 is mD3, and Lane 5 is mR6. It is a figure which shows the result of having analyzed the reaction product by TLC after performing enzyme reaction which used lactose as a substrate about CE-NE1 and the purified recombinant enzyme (mD1, mD2, mD3, mR6). Lane S is a reaction product using no enzyme, Lane 1 is CE-NE1 (control), Lane 2 is mD1, Lane 3 is mD2, Lane 4 is mD3, and Lane 5 is mR6. FIG. 4 is a diagram showing the results of TLC analysis of a reaction product after CE-NE1 and purified recombinant enzymes (mD1, mD2, mD3, mR6) were subjected to an enzyme reaction using 4β-mannobiose as a substrate. Lane S is a reaction product using no enzyme, Lane 1 is CE-NE1 (control), Lane 2 is mD1, Lane 3 is mD2, Lane 4 is mD3, and Lane 5 is mR6. FIG. 3 is a diagram showing the results of analyzing the reaction product by TLC after performing an enzyme reaction using CE-NE1 and purified recombinant enzymes (mD1, mD2, mD3, mR6) using globotriose as a substrate. Lane S is a reaction product using no enzyme, Lane 1 is CE-NE1 (control), Lane 2 is mD1, Lane 3 is mD2, Lane 4 is mD3, and Lane 5 is mR6. It is a figure which shows the result of having analyzed the reaction product by TLC, after performing the enzyme reaction which used cellobiose and glucosyl mannose as a substrate about purified recombinant enzyme mR6. Lane 1 is a product of cellobiose only, lane 2 is a product of cellobiose + mR6, lane 3 is a product of glucosylmannose only, and lane 4 is a product of glucosylmannose + mR6. It is a figure which shows the result of having analyzed the reaction product by TLC after performing the enzyme reaction which used the monosaccharide for the refine | purified recombinant enzyme (mD1, mD2, mD3, mR6). Lane 1 is glucose, lane 2 is mannose, lane 3 is galactose, lane 4 is fructose, lane 5 is xylose, lane 6 is arabinose as a substrate, the left side of each lane has no enzyme added, and the right side has an enzyme added reaction product. Analysis was conducted. It is a figure which shows the result of having analyzed the reaction product by TLC after performing the enzyme reaction which used monosaccharide as alpha-2 saccharide | sugar about the refined recombinant enzyme (mD1, mD2, mD3, mR6). Lane 1 was cordierbiose, lane 2 was nigerose, lane 3 was maltose, lane 4 was isomaltose as a substrate, and the left side of each lane was analyzed without any enzyme, and the right side was analyzed for the reaction product with the enzyme added. It is a figure which shows the result of having analyzed the reaction product by TLC after performing the enzyme reaction which used the monosaccharide as (beta) -2 saccharide | sugar about the purified recombinant enzyme (mD1, mD2, mD3, mR6). Lane 1 was sophorose, lane 2 was laminaribiose, lane 3 was cellobiose, lane 4 was gentiobiose as a substrate, and the left side of each lane was analyzed without any enzyme and the right side was analyzed for the reaction product. CE-NE1 and purified recombinant enzymes (mD1, mD2, mD3, mR6) were subjected to an enzymatic reaction using (A) cellobiose and (B) lactose as substrates, and then the reaction product was subjected to acid hydrolysis, and then TLC It is a figure which shows the result analyzed by. Lane Std is a standard monosaccharide ((A) glucose and mannose, (B) galactose and mannose), lane 1 is CE-NE1 (control), lane 2 is mD1, lane 3 is mD2, lane 4 is mD3, lane 5 Is a reaction product using mR6. It is a figure which shows optimal pH about CE-NE1 and the purified recombinant enzyme (mD1, mD2, mD3, mR6). The vertical axis of the graph shows the relative value when the enzyme activity at pH 7.5 is 100%, and the horizontal axis of the graph shows the pH. It is a figure which shows pH stability about CE-NE1 and the purified recombinant enzyme (mD1, mD2, mD3, mR6). The vertical axis of the graph shows the relative activity when the enzyme activity without the retention treatment is 100%, and the horizontal axis of the graph shows the pH. It is a figure which shows optimal temperature about CE-NE1 and the purified recombinant enzyme (mD1, mD2, mD3, mR6). The vertical axis of the graph shows the relative activity when the enzyme activity at 25 ° C. is 100%, and the horizontal axis of the graph shows the reaction temperature. It is a figure which shows temperature stability about CE-NE1 and the purified recombinant enzyme (mD1, mD2, mD3, mR6). The vertical axis of the graph shows the relative activity when the enzyme activity without the retention treatment is 100%, and the horizontal axis of the graph shows the reaction temperature. It is a figure which shows the result of having made the refined recombinant enzyme (mR6) react with commercially available milk, and analyzing the reaction product by TLC. Lane S is standard lactose, Lane 1 is 1 hour after reaction at 30 ° C, Lane 2 is 2 hours after reaction at 30 ° C, Lane 3 is 3 hours after reaction at 30 ° C, and Lane 4 is 4 hours after reaction at 30 ° C. Lane 5 is the reaction product after 24 hours of reaction at room temperature.

(I) Polypeptide The present invention relates to (1) a polypeptide comprising the amino acid sequence represented by any one of SEQ ID NOs: 1 to 4, or (2) 1 in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 4. 2 of the reducing terminal sugar residue of an oligosaccharide having an amino acid sequence in which one or a plurality of amino acids are deleted, substituted, or added, and the reducing terminal sugar residue is linked by a β-1,4 bond. Provided is a polypeptide that catalyzes a reaction that changes the conformation of a hydroxyl group.

The present invention also provides polypeptides having the physicochemical properties shown in (A) to (G) below:
(A) The apparent molecular weight by SDS-PAGE is 42-44 kDa,
(B) having the amino acid sequence shown in SEQ ID NO: 9 at the N-terminus,
(C) specifically catalyzing a reaction that changes the conformation of the hydroxyl group at the 2-position of the reducing end sugar residue of the oligosaccharide to which the reducing end sugar residue is bonded by a β-1,4 bond;
(D) When the enzyme activity at pH 7.5 is 100%, the enzyme activity is 80% or more at pH 7.0 to 7.5,
(E) After holding at 4 ° C. for 20 hours at pH 5.0 to 8.0, the enzyme activity remains 80% or more,
(F) When the enzyme activity at 25 ° C. is defined as 100%, the enzyme activity is 80% or more at 15 to 30 ° C., and (G) the enzyme activity at 30 ° C. is 80% after holding at 50 ° C. for 1 hour. % Or more remain.

The apparent molecular weight by SDS-PAGE in (A) above varies slightly depending on the degree of post-translational modification in the polypeptide used and the concentration of the gel used in SDS-PAGE. One skilled in the art will readily appreciate that it can be about 38 kDa to 48 kDa. In addition, the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 provided by the present invention is an example of a polypeptide having the physicochemical properties represented by the above (A) to (G), and based on the sequence information. The calculated molecular weight is 45425.5. Further, if the position of a single band shown in lane 2 of FIG. 5 is accurately represented, the apparent molecular weight by SDS-PAGE is 43.2 kDa.

Furthermore, the present invention also provides polypeptides having the physicochemical properties shown in (A) to (G) below:
(A) The apparent molecular weight by SDS-PAGE is 43 to 45 kDa,
(B) having the amino acid sequence shown in SEQ ID NO: 10 at the N-terminus,
(C) specifically catalyzing a reaction that changes the conformation of the hydroxyl group at the 2-position of the reducing end sugar residue of the oligosaccharide to which the reducing end sugar residue is bonded by a β-1,4 bond;
(D) When the enzyme activity at pH 7.5 is 100%, the enzyme activity is 80% or more at pH 7.0 to 8.0,
(E) After holding at 4 ° C. for 20 hours at pH 4.0 to 8.0, the enzyme activity remains at 80% or more,
(F) Optimum temperature: Shows an enzyme activity of 80% or more at 10 to 45 ° C. when the enzyme activity at 25 ° C. is 100%, and (G) the enzyme at 30 ° C. after holding at 40 ° C. for 1 hour. The activity remains 80% or more.

The apparent molecular weight by SDS-PAGE in (A) above varies slightly depending on the degree of post-translational modification in the polypeptide used and the concentration of the gel used in SDS-PAGE, so the actual molecular weight of the polypeptide is about One skilled in the art will readily understand that it can be between 39 kDa and 49 kDa. The polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 provided by the present invention is an example of a polypeptide having the physicochemical properties represented by the above (A) to (G), and based on the sequence information. The calculated molecular weight is 48346.9. Further, when the position of a single band shown in lane 3 of FIG. 5 is accurately represented, the apparent molecular weight by SDS-PAGE is 44.3 kDa.

The present invention further provides polypeptides having physicochemical properties shown in A) to (G) below:
(A) The apparent molecular weight by SDS-PAGE is 46 to 48 kDa,
(B) having the amino acid sequence shown in SEQ ID NO: 11 at the N-terminus,
(C) specifically catalyzing a reaction that changes the conformation of the hydroxyl group at the 2-position of the reducing end sugar residue of the oligosaccharide to which the reducing end sugar residue is bonded by a β-1,4 bond;
(D) When the enzyme activity at pH 7.5 is 100%, the enzyme activity is 80% or more at pH 7.0 to 9.0,
(E) After holding at 4 ° C. for 20 hours at pH 4.0 to 8.0, the enzyme activity remains at 80% or more,

(F) Optimum temperature: Shows an enzyme activity of 80% or more at 15 to 35 ° C. when the enzyme activity at 25 ° C. is 100%, and (G) the enzyme at 30 ° C. after holding at 40 ° C. for 1 hour. The activity remains 80% or more.

The apparent molecular weight by SDS-PAGE varies slightly depending on the degree of post-translational modification in the polypeptide used and the concentration of the gel used for SDS-PAGE. Therefore, the actual molecular weight of the polypeptide is about 42 kDa to 52 kDa. Those skilled in the art will readily understand that this is possible. Further, the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 3 provided by the present invention is an example of a polypeptide having physicochemical properties represented by the above (A) to (G), and based on the sequence information. The calculated molecular weight is 46964.0. Further, if the position of a single band shown in lane 4 of FIG. 5 is accurately represented, the apparent molecular weight by SDS-PAGE is 46.7 kDa.

The present invention also provides polypeptides having the physicochemical properties shown in A) to (G) below:
(A) The apparent molecular weight by SDS-PAGE is 44 to 46 kDa,
(B) having the amino acid sequence shown in SEQ ID NO: 12 at the N-terminus,
(C) specifically catalyzing a reaction that changes the conformation of the hydroxyl group at the 2-position of the reducing end sugar residue of the oligosaccharide to which the reducing end sugar residue is bonded by a β-1,4 bond;
(D) When the enzyme activity at pH 7.5 is 100%, the enzyme activity is 80% or more at pH 7.0 to 7.5,
(E) After holding at 4 ° C. for 20 hours at pH 5.0 to 8.0, the enzyme activity remains 80% or more,
(F) When the enzyme activity at 25 ° C. is 100%, the enzyme activity is 80% or more at 20 to 45 ° C., and (G) the enzyme activity at 30 ° C. is 80 after holding at 50 ° C. for 1 hour. % Or more remain.

The apparent molecular weight by SDS-PAGE varies slightly depending on the degree of post-translational modification in the polypeptide used and the concentration of the gel used for SDS-PAGE. Therefore, the actual molecular weight of the polypeptide is about 40 kDa to 50 kDa. Those skilled in the art will readily understand that this is possible. Further, the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4 provided by the present invention is an example of a polypeptide having the physicochemical properties represented by the above (A) to (G), and based on the sequence information. The calculated molecular weight is 49177.6. Further, when the position of a single band shown in lane 5 of FIG. 5 is accurately represented, the apparent molecular weight by SDS-PAGE is 45.4 kDa.

Any of the above polypeptides is a polypeptide that catalyzes a reaction that changes the conformation of the hydroxyl group at the 2-position of the reducing end sugar residue of an oligosaccharide in which the reducing end sugar residue is bonded by a β-1,4 bond. It is. Hereinafter, as used herein, “2-epimerase activity” means an activity that catalyzes a reaction that changes the conformation of the 2-position hydroxyl group of a reducing terminal sugar residue of an oligosaccharide. “Epimerization” is intended to change the conformation of the hydroxyl group at the 2-position of the reducing terminal sugar residue of the oligosaccharide. Therefore, the polypeptide of the present invention exhibits 2-epimerase activity with respect to an oligosaccharide in which the reducing terminal sugar residue is bound by a β-1,4 bond, and the oligosaccharide can be 2-epimerized. It is a polypeptide.

The polypeptide of the present invention acts on an oligosaccharide in which a reducing terminal sugar residue is bonded by a β-1,4 bond, and the reducing terminal sugar residue is a β-1,2 bond, a β-1,3 bond, Substrate specificity that does not act on oligosaccharides linked by any of β-1,6 bond, α-1,2 bond, α-1,3 bond, α-1,4 bond, α-1,6 bond Have sex.

An oligosaccharide having 2 to 15 sugar residues is intended as an oligosaccharide in which a reducing terminal sugar residue is bound by a β-1,4 bond as a substrate for the polypeptide of the present invention. The number of sugar residues is preferably 2 to 5, specifically cellobiose, cellotriose, cellotetraose, cellopentaose, lactose, glucosyl mannose, 4β-mannobiose (O-β-D-mannopyranosyl- (1 → 4) -OD-mannose) or globotriose (O-α-D-galactopyranosyl- (1 → 4) -O-β-D-galactopyranosyl- (1 → 4) -D- Glucose) and the like.

The 2-epimerase activity possessed by the polypeptide of the present invention is a 2-epimerized oligosaccharide produced by an enzymatic reaction using an oligosaccharide having a reducing terminal sugar residue bound by a β-1,4 bond as a substrate. Can be expressed qualitatively or quantitatively. For example, the oligosaccharide in the reaction solution is analyzed by chromatography such as HPLC or TLC, the reaction product peak is identified by comparing the retention time or mobility with the standard product, and the peak area ratio or spot intensity with the standard product The activity can be measured by calculating the reaction product concentration from In the case of an unknown reaction product that does not have a standard product, for example, the reaction product is decomposed into constituent sugars by acid decomposition, then separated by chromatography, and then analyzed by analyzing the concentration of reducing end sugar residues. Can be measured.

As described above, the present invention provides a polypeptide having 2-epimerase activity with respect to an oligosaccharide in which a reducing terminal sugar residue is bound by a β-1,4 bond. The polypeptide of the present invention has 2-epimerase activity against cellobiose, but also has 2-epimerase activity against cellooligosaccharides other than lactose or cellobiose. The polypeptide of the present invention having such properties is very useful for the synthesis of novel oligosaccharides or the mass production of rare oligosaccharides.

(D) in the physicochemical properties possessed by the polypeptide of the present invention is a property relating to so-called “optimal pH”. When the enzyme activity at 30 ° C. and pH 7.5 is 100%, the enzyme activity is 80% or more. Means a pH indicating Further, (E) in the physicochemical properties of the polypeptide of the present invention is a property relating to so-called “pH stability”. After holding the enzyme at 4 ° C. for 20 hours at various pHs, at 30 ° C. and pH 7.5. The pH range where the enzyme activity of 80% or more remains. Furthermore, (F) in the physicochemical properties possessed by the polypeptide of the present invention is a property relating to the so-called “optimum temperature”, which is 80% or more when the enzyme activity at 25 ° C. and pH 7.5 is 100%. The reaction temperature showing enzyme activity is meant. (G) in the physicochemical properties of the polypeptide of the present invention is a property related to so-called “temperature stability”, and the enzyme activity at 30 ° C. and pH 7.5 after holding the enzyme at various temperatures for 1 hour. Means a temperature range in which 80% or more remains. The buffer solution required for the reaction solution is preferably a sodium phosphate buffer solution or a glycylglycine-NaOH buffer solution, and the buffer solution concentration is preferably 20 to 100 mM.

As used herein, “polypeptide” is used interchangeably with “peptide” or “protein”. A polypeptide “fragment” is intended to be a polypeptide comprising a partial amino acid sequence of the polypeptide.

Further, the polypeptide of the present invention may be a polypeptide containing an amino acid sequence useful for purification of the polypeptide. Examples of amino acid sequences useful for polypeptide purification include amino acid sequences that encode epitope-tagged polypeptides such as His, Myc, and Flag.

In addition, the polypeptide of the present invention may be any polypeptide in which amino acids are peptide-bonded, but is not limited thereto, and is a polypeptide having various modifying groups such as phosphate groups and sugar chains. There may be.

Furthermore, the present invention also provides a variant of a polypeptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 1 to 4.

As used herein with respect to a polypeptide, a “variant” consists of an amino acid sequence in which one or more amino acids are substituted, deleted, or added in the amino acid sequences of SEQ ID NOs: 1-4. Also contemplated are polypeptides that retain 2-epimerase activity for oligosaccharides in which the reducing terminal sugar residue is linked by a β-1,4 linkage. The amino acid mutation site and number in the present invention are not particularly limited as long as a polypeptide having 2-epimerase activity against an oligosaccharide in which the reducing terminal sugar residue is bound by a β-1,4 bond is provided. In terms of the number of amino acids, it is 1 to 80, preferably 1 to 40, more preferably 1 to 20, and still more preferably 1 to 10. If the allowable range of modification is expressed by the degree of identity of the amino acid sequence, the amino acid sequence of the protein of the present invention is 80% or more, preferably 90% or more, more preferably 95% with respect to the amino acid sequence shown in SEQ ID NO: 1. % Of identity is sufficient.

It is well known in the art that some amino acids in the amino acid sequence of a polypeptide can be easily modified without significantly affecting the structure or function of the polypeptide.

Preferred variants have conservative or non-conservative amino acid substitutions, deletions or additions. Silent substitution, deletion and addition are preferred, and conservative substitution is particularly preferred. These do not change the 2-epimerase activity for oligosaccharides in which the reducing terminal sugar residue of the polypeptide of the present invention is bound by a β-1,4 bond. Typical conservative substitutions include mutual substitution between the hydrophobic amino acids Ala, Val, Leu, and Ile, mutual substitution of the hydroxyl amino acids Ser and Thr, mutual substitution of the acidic residues Asp and Glu, amides Type amino acids Asn and Gln, mutual substitution of basic amino acids Lys and Arg, mutual substitution of aromatic amino acids Phe and Tyr, and the like.

Those skilled in the art can easily mutate one or several amino acids in the amino acid sequence of a polypeptide using known techniques. For example, a mutant in which one or several amino acids are substituted, deleted, or added can be prepared by a known point mutation introduction method or a PCR method using DNA. Furthermore, the object can be achieved by random mutation. Furthermore, if the method for measuring 2-epimerase activity described above is used, does the produced mutant have 2-epimerase activity for an oligosaccharide in which the desired reducing terminal sugar residue is bound by a β-1,4 bond? It can easily be determined.

(II) Polynucleotide The present invention encodes the polypeptide of the present invention, that is, the polypeptide having 2-epimerase activity for the oligosaccharide in which the reducing terminal sugar residue described above is linked by β-1,4 bond. A polynucleotide is provided.

Based on the description in the present specification, a person skilled in the art can obtain the entire amino acid sequence of the polypeptide of the present invention and the entire base sequence (or ORF or a part thereof) of the polynucleotide encoding the polypeptide. . For example, the polynucleotide of the present invention can be obtained as follows.

First, DNA or RNA that serves as a template for cloning the polynucleotide of the present invention is prepared. The template DNA or RNA is not particularly limited, and may be derived from, for example, a single organism or may be derived from a plurality of organisms. Therefore, for example, metagenomic DNA or RNA prepared from a natural sample such as lumen or soil by a known method can be used.

“Metagenome” refers to a genome directly collected from the natural world without separating and culturing (micro) organisms. In recent years, in order to investigate the ecology of environmental microorganisms and to acquire genes with useful functions of uncultured microorganisms, research on microbial ecology by metagenomic techniques has attracted attention. The “metagenomic technique” is a technique for expressing a target trait (for example, enzyme activity) by directly recombining DNA or RNA without separating (cultivating) (micro) organisms from nature and performing large-scale sequencing.

The metagenomic method is most effective when the sequences of the similar gene and the target gene are known to some extent, and the full-length target functional gene can be isolated and expressed by PCR. Eschenfeldt et al. Isolated two 2,5-diketo-D-gluconate reductases from difficult-to-cultivate microbial DNA in soil (WH Eschenfeldt et al., Appl. Environ. Microbiol., 2001). 67, 4206-4214), Bell et al. Also isolated a novel lipase from environmental biomass (PJL Bell et al., Microbiology, 2002, 148, 2283-2291). ). Similarly, Uchiyama and Watanabe perform metagenomic walking by PCR to isolate chitinase (T. Uchiyama and T. Watanabe, Biotechniques, 2006, Vol. 41, 183-188).

The primer for cloning the polynucleotide of the present invention preferably comprises a base sequence capable of specifically amplifying the polynucleotide of the present invention. As an example, a part or all of a base sequence characteristic of each base sequence is selected from base sequences encoding the polypeptide of the present invention, for example, the base sequences shown in any of SEQ ID NOs: 5 to 8. Can be used as a primer.

The polynucleotide of the present invention can be obtained from recombinant DNA obtained by a known method such as PCR using the above template DNA or RNA and the above primer. Nucleotide sequence information from several recombinant DNA isolates can be combined to provide the coding sequence for all amino acids of the polypeptides of the invention, as well as upstream and downstream nucleotide sequences.

The polynucleotide of the present invention can also be produced by a chemical synthesis method such as a phosphoramidide method, or using a commercially available DNA synthesizer.

A preferred example of the polynucleotide of the present invention is a polynucleotide comprising the base sequence shown in any of SEQ ID NOs: 5 to 8. The present invention also provides a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a complementary sequence of the base sequence represented by any of SEQ ID NOs: 5 to 8. “Stringent hybridization conditions” include, for example, a hybridization solution (50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhart. Solution, 10% dextran sulfate, and 20 μg / ml denatured sheared salmon sperm DNA), followed by overnight incubation at 42 ° C. followed by washing the filter in 0.1 × SSC at about 65 ° C. Intended. Further, in terms of the identity (%) of the base sequence, it is 70% or more, preferably 80% or more, more preferably 90% or more, and still more preferably 95% or more with respect to the base sequences of SEQ ID NOs: 5 to 8, respectively. Any nucleic acid having a base sequence having identity may be used.

The polynucleotide of the present invention may exist in the form of DNA (for example, cDNA or genomic DNA) or RNA (for example, mRNA). DNA can be double-stranded or single-stranded. Single-stranded DNA or RNA can be the coding strand (also known as the sense strand) or it can be the non-coding strand (also known as the antisense strand). The nucleic acid may be labeled with an enzyme such as horseradish peroxidase (HRPO), a radioisotope, a fluorescent substance, a chemiluminescent substance, or the like.

The polynucleotide of the present invention is obtained by adding a base sequence encoding a marker protein such as His tag, FLAG tag, or GFP added to the polypeptide of the present invention to the 5 ′ side or the 3 ′ side. Also good. In addition, the polynucleotide of the present invention may have other base sequences if necessary. Other base sequences include enhancer sequences, promoter sequences, ribosome binding sequences, base sequences used for the purpose of copy number amplification, base sequences encoding signal peptides, base sequences encoding other polypeptides, poly A Additional sequences, splicing sequences, replication origins, base sequences of genes that serve as selection markers, untranslated region sequences, and the like are included.

(III) Vector The present invention provides a vector comprising the polynucleotide of the present invention.

The vector of the present invention may be in any form such as circular or linear as long as it contains the above-described polynucleotide of the present invention, and even a vector used for in vitro translation can be used for recombinant expression. The vector to be used may be used. Examples thereof include a recombinant expression vector into which a cDNA of a polynucleotide encoding the polypeptide of the present invention has been inserted.

The vector of the present invention may be used by selecting an appropriate vector according to the host to be used, and various viruses such as bacteriophage, baculovirus, retrovirus, vaccinia virus can be used in addition to the plasmid. is there. In particular, the vector of the present invention is preferably capable of autonomous replication in a host, and is preferably in the form of plasmid DNA or phage DNA. Examples of vectors for introducing nucleic acid into E. coli include plasmid DNA such as pBR322, pUC18, pBluescript II, commercially available expression vectors pET-23d (manufactured by Novagen), pET-28a (manufactured by Novagen), etc. , M13, λgtII, and other phage DNAs. Examples of vectors for introduction into yeast include YEp13 and YCp50.

In addition to the polynucleotide of the present invention, the recombinant vector may have other nucleotide sequences if necessary. Other base sequences include an enhancer sequence, a promoter sequence, a ribosome binding sequence, a base sequence used for the purpose of amplification of copy number, a base sequence encoding a signal peptide, a base sequence encoding another polypeptide, poly A Additional sequences, splicing sequences, replication origins, base sequences of genes that serve as selection markers, and the like.

The expression vector preferably contains at least one selectable marker gene. Examples of selectable marker genes include drug resistance genes such as ampicillin resistance gene, tetracycline resistance gene, neomycin resistance gene, kana machine resistance gene, chloramphenicol resistance gene, and intracellular biosynthesis of nutrients such as amino acids and nucleic acids. Examples include genes involved, fluorescent protein genes such as GFP, and luciferase genes.

A method for producing a recombinant expression vector includes, but is not limited to, a method using a plasmid, phage, cosmid or the like. The specific type of vector is not particularly limited, and a vector that can be expressed in a host cell can be appropriately selected. That is, according to the type of the host cell, a promoter sequence is appropriately selected in order to reliably express the polynucleotide of the present invention, and a vector in which this and the polynucleotide of the present invention are incorporated into various plasmids or the like is used as an expression vector. Good.

When constructing a vector using the polypeptide of the present invention, an arbitrary base sequence and a selection marker, a translation start codon and a translation stop codon are added using an appropriate synthetic DNA adapter, or an appropriate base sequence is used. It is also possible to newly generate or eliminate restriction enzyme cleavage sequences. These are within the scope of work normally performed by those skilled in the art. Experimental manuals describing various gene recombination operations in detail, including Sambrook et al. (See Molecular Cloning, a Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory, New York, 1989). Can be done based on.

When the vector of the present invention is used, the polynucleotide can be introduced into an organism or cell, and the polypeptide of the present invention can be expressed in the organism or cell. Furthermore, if the vector of the present invention is used in a cell-free protein synthesis system, the polypeptide of the present invention can be synthesized. Thus, it can be said that the vector of the present invention should contain at least a polynucleotide encoding the polypeptide of the present invention. That is, it should be noted that vectors other than expression vectors are also included in the technical scope of the present invention.

(IV) Transformant The present invention provides a transformant into which the polynucleotide of the present invention has been introduced. That is, the transformant of the present invention is a transformant capable of expressing and producing the polypeptide of the present invention. In the transformant of the present invention, the polypeptide of the present invention is preferably stably expressed, but may be transiently expressed.

As used herein, “transformants” are intended to include not only cells, tissues or organs but also individual organisms, but cells (especially prokaryotic cells, fungi (eg, The transformant in the present invention can be prepared by introducing the polynucleotide or vector of the present invention into an appropriate host cell. Suitable microorganisms include Escherichia bacteria (more preferably Escherichia coli), Bacillus bacteria (more preferably Bacillus subtilis), and Corynebacterium genus. Bacteria, bacteria belonging to the genus Brevibacterium, Serratia ia) genus bacteria, Pseudomonas genus bacteria, Arthrobacter genus bacteria, Erwinia genus bacteria, Methylobacterium genus bacteria, Rhodobacter genus bacteria, Streptomyces (c) Streptomyces (c) Examples include microorganisms such as microorganisms of the genus Zymomonas, yeasts of the genus Saccharomyces, etc. Particularly preferred host cells are Escherichia coli and Bacillus subtilis.

Introduction of the polynucleotide or vector of the present invention into a host cell can be carried out by methods known to those skilled in the art. Examples of methods for introducing a vector or the like into a host cell include the calcium phosphate method, electroporation method, spheroplast method, liposome method, DEAE dextran method, lithium acetate method, junction transfer method, methods using calcium ions, and the like. It is done.

(V) Method for Producing Polypeptide The present invention provides a method for producing the polypeptide of the present invention.

For example, after culturing a host transformed with the expression vector, a fraction containing the polypeptide (transformed cells, culture supernatant, or a mixture thereof) is collected, and a conventional method (for example, Salting-out method, ultrafiltration method, isoelectric point precipitation method, gel filtration method, electrophoresis method, ion exchange chromatography, various affinity chromatography such as hydrophobic chromatography and antibody chromatography, chromatofocusing method, adsorption chromatography And the desired polypeptide can be recovered and purified in accordance with reverse phase chromatography and the like. When the fraction contains transformed cells, it is possible to recover and purify the target polypeptide by combining cell disruption with the above-mentioned conventional technique.

The fraction isolated in the above purification process is analyzed using, for example, a bioassay specific to the polypeptide of the present invention (for example, enzymatic reaction using lactose or cellooligosaccharide as a substrate and analysis of the product). By doing so, the presence of the polypeptide of the present invention can be confirmed.

Also, for example, the polypeptide of the present invention can be purified by affinity chromatography in combination with the conventional purification method described above. For example, among the polypeptides of the present invention, those having a His tag can be efficiently obtained by affinity chromatography using a nickel column, those having a Myc tag using an anti-Myc antibody, and those having a Flag tag using an anti-Flag antibody. Purification is realized. When the polypeptide of the present invention is expressed in a form to which a tag or other functional polypeptide is attached, the tag or functional polypeptide added using an appropriate protease (thrombin, trypsin, etc.) is cleaved, The protein of the invention can be recovered.

The polypeptide of the present invention can be prepared in a single form or in a form to which a tag or functional polypeptide is added, but is not limited thereto, and the protein used in the present invention is further limited. It is also possible to convert into various forms. For example, various chemical modifications to proteins, binding to polymers such as polyethylene glycol, binding to insoluble carriers, encapsulation in liposomes, and the like can be considered by various techniques known to those skilled in the art.

The polypeptide of the present invention may be obtained by an organic chemical synthesis method such as Fmoc method (fluorenylmethyloxycarbonyl method) or tBoc method (t-butyloxycarbonyl method), or a suitable commercially available peptide synthesizer. However, by means of gene recombination technology, the nucleic acid, particularly DNA incorporated into an expression vector, can be converted into a suitable expression system using a suitable host cell selected from prokaryotes or eukaryotes. It is preferable to manufacture by introducing.

(VI) Method for Producing Oligosaccharide The present invention provides a method for producing a 2-epimerized oligosaccharide in which a reducing terminal sugar residue is bound by a β-1,4 bond.

The method for producing an oligosaccharide of the present invention uses the polypeptide of the present invention. In one embodiment, the method of the invention comprises incubating a polypeptide of the invention with an oligosaccharide having a reducing terminal sugar residue attached by a β-1,4 linkage. The oligosaccharide is not particularly limited, but is preferably cellooligosaccharide (cellobiose, cellotriose, cellotetraose, etc.), lactose, glucosyl mannose, 4β-mannobiose or globotriose.

The conditions for the catalytic reaction in the method of the present invention are not particularly limited, but can be set in consideration of the optimum conditions described above according to the polypeptide of the present invention to be used. In addition, the polypeptide of the present invention can be used for a catalytic reaction in a purified state, but can also be used in a state of being immobilized on a column or in the state of the above transformant.

The oligosaccharide produced in the method of the present invention is purified at a mass production level by a known method such as HPLC, silica gel, activated carbon column chromatography or the like. Purification can be confirmed by measuring 2-epimerase activity by the method described above.

(VII) Method for Producing Prebiotics The present invention provides a method for producing prebiotics comprising 2-epimerized oligosaccharides in which the reducing terminal sugar residues are linked by β-1,4 bonds.

“Prebiotics” means “indigestible substances that have a positive impact on the health of the host by improving the balance of the intestinal flora (the group of microorganisms that live in the digestive tract (mainly anaerobic flora))” (GR Gibson and MB RobertFroid, J. Nutr., 1995, Vol. 125, pages 1401-1141, or MB Robertfroid, J. Nutr., 2007, No. 133830S-137737S. ) And as used herein, it is useful to propagate useful microorganisms (eg, probiotics such as lactic acid bacteria and bifidobacteria) in the intestinal environment of animals (including humans) Substances that promote the improvement of the internal environment are intended. Typical “prebiotics” include indigestible substances such as oligosaccharides, whey fermented products by propionic acid bacteria, and dietary fiber. Oligosaccharides serve as food for probiotics, Stores intestinal bacteria and aids their growth.

The effects of “prebiotics” include absorption of minerals, suppression of blood cholesterol and neutral fat levels, prevention of arteriosclerosis, suppression of blood glucose levels, improvement of diabetes, improvement of obesity, activity of intestinal motility , Improvement of constipation, activation of immunity, prevention of infection, prevention of cancer, suppression of blood ammonia level, improvement of hepatic encephalopathy due to decreased liver function, promotion of vitamin synthesis by enteric bacteria, various minerals Include, but are not limited to, promoting the absorption of ulcer and improving the symptoms of ulcerative colitis. In addition, recent studies have shown that the intake of “prebiotics” improves the absorption of minerals (eg, Ca ²⁺ , Mg ²⁺ , Zn ²⁺ , Fe ^3+, etc.), especially Ca ²⁺ The absorption promoting effect is attracting attention. An oligosaccharide may have a mineral absorption promoting effect. The mechanism of action is that oligosaccharides promote the absorption of various minerals by widening the intercellular tissue gap (so-called tight junction (TJ)) between cells inside the small or large intestine. (T. Suzuki and H. Hara, Life Sci. 2006, 79401-79410).

The present inventors have shown that epilactose, which is a stereoisomer (epimer) of the hydroxyl group at the reducing terminal sugar residue of lactose, has an intestinal environment improving action, lipid metabolism improving action, mineral absorption promoting action as prebiotics. Have been found to have a function such as low calorie properties and have filed a patent application separately (International Patent Application No. PCT / JP2007 / 001253). The polypeptide of the present invention is capable of producing epilactose from lactose, and therefore the polypeptide of the present invention comprises a prebiotic comprising a 2-epimerized oligosaccharide in which a reducing terminal sugar residue is linked by a β-1,4 bond. Can be used to make ticks.

The method for producing prebiotics of the present invention uses the above polypeptide. In one embodiment, the method of the invention comprises incubating the polypeptide with a material comprising an oligosaccharide in which the reducing terminal sugar residue is linked by a β-1,4 bond. The material containing the oligosaccharide is not particularly limited. For example, milk that is a natural raw material containing lactose, particularly livestock milk such as cow, goat or sheep, can be used as it is or after being defatted. In addition, whey (milk) fraction recovered from livestock milk, processed milk and milk beverages such as low fat milk, low protein milk, skim / deproteinized milk or low lactose milk are also compositions containing the above oligosaccharides. In this case, prebiotics containing epilactose can be produced inexpensively and easily.

Hereinafter, the present invention will be described in more detail by way of examples. However, the present invention is not limited to these examples, and various modifications are possible within the scope shown in the claims and the above embodiments. Embodiments obtained by appropriately combining technical means disclosed in different embodiments are also included in the technical scope of the present invention.

Hereinafter, analysis of the base sequence is pDRAW32 AcaClone Software (http://www.acaclone.com/), homology search is BLAST (SF Altschul et al., J. Mol. Biol., 1990, No. 1). 215, 403-410), and CLUSTAL W (ver. 1.83) (JD Thompson et al., Nucl. Acids. Res., 1994) for multiple alignment of base sequences and amino acid sequences. Volume 22, pages 4673-4680) were used.

<Example 1> Cloning and sequencing of gene from metagenomic DNA and RNA Cloning of CE gene from metagenomic DNA and RNA and determination of nucleotide sequence were performed according to the following method. In addition, a submarine type electrophoresis layer Mupid-ex (ADVANCE, Tokyo, Japan) is used for the agarose gel electrophoresis apparatus, and a Seake GTG Agarose (Lonza, Rockland, ME) is used for the agarose for electrophoresis. did. Base sequence analysis was performed by the die terminator method, and was performed by ABI PRISM 310 (Applied Biosystems, Foster City, CA) or CEQ 8000 (Beckman Coulter, Fullerton, CA). The thermal cycler used was T-gradient (Biometra, Gottingen, Germany) or TaKaRa Thermalcycler Dice (Takara Bio, Kyoto, Japan).

(1) Extraction of DNA from rumen contents The rumen contents were collected from a sheep using a cannula. About 6 g of fresh lumen content is diluted into 45 mL of 0.1% (w / v) Tween 80 [3 g / L Na ₂ CO ₃ , 0.34 g / L KH ₂ PO ₄ , 0.34 g / L NaCl, 0.34 g / L (NH ₄ ) ₂ SO ₄ , 7.5 mg / L MnSO ₄ .H ₂ O, 24.8 mg / L CaCl ₂ .2H ₂ O, 38.4 mg / L MgSO ₄ .7H ₂ O, 7.5 mg / L FeSO ₄ · 7H ₂ O, 7.5 mg / L ZnSO ₄ · 7H ₂ O, 0.75 mg / L CoCl ₂ · 7H ₂ O, 0.5 g / L L-cysteine · HCl · H ₂ O, 1 mg / L resazurin (pH 6.8)], and rumen bacteria were detached from the plant surface at 4 ° C. The suspension was filtered through a 40 μm nylon mesh and the filtrate was centrifuged at 500 × g. The collected supernatant was centrifuged (13,400 × g, 1 minute, 4 ° C.) to recover the rumen bacterial precipitate. DNA was extracted from this precipitate using Isoplant II (Nippongene).

(2) Extraction of RNA from rumen contents and synthesis of single-stranded cDNA About 0.2 g of sheep rumen contents collected in the same manner as in (1) above were frozen in liquid nitrogen, and a multi-bead shocker (Yasui Kikai) ), And crushing at 2000 rpm for 10 seconds was repeated four times. Thereafter, 1 mL of Trizol reagent (Invitrogen) was added, and crushing at 2000 rpm for 10 seconds at room temperature was repeated twice. 200 μL of chloroform was added to the cell lysate, stirred well, allowed to stand at room temperature for 2 to 3 minutes, and then centrifuged (20000 × g, 10 minutes, room temperature). 500 μL of isopropanol was added to the supernatant, stirred well, allowed to stand at room temperature for 10 minutes, and then centrifuged (20000 × g, 10 minutes, 4 ° C.). The obtained RNA precipitate was dissolved in 100 μL of RNase-free water and purified using RNeasy (Qiagen). The purified mRNA was concentrated using MICROB Express (Ambion). Single-stranded cDNA was synthesized from the concentrated mRNA using SuperScript III 1st Strand cDNA Synthesis kit (Invitrogen).

(3) Amplification of Epimerase Gene Partial Fragment N-acetyl-D-glucosamine 2-epimerase (N-acetyl-D-glucosamine 2-epimerase, hereinafter referred to as AGE) is obtained by N-acetyl-D-glucosamine being 2-epimerized to form N -An enzyme that produces acetyl-D-mannosamine, and the three-dimensional structure is already known in pigs (registration number PDB 1F3P) and cyanobacteria Anabaena (registration number PDB 2GZ6). Therefore, for the purpose of designing a common primer suitable for amplification of the epimerase gene, R.I. CE-NE1 (registration number BAF81108) derived from albus, AGE-like protein (Cp AGE-like, registration number ABX42625) derived from Clostridium phytofermentans ICDg strain, Caldicelluloslpital saccharoltics (Caldric DS03) Using the Clustal W program, the amino acid sequences of the strain-derived AGE-like protein (Cs AGE-like, registration number EAP43970) and Bacteroides fragilis YCH 46-derived AGE-like protein (Bf AGE-like, registration number BAD47600) are used. Multiple aligned.

As a result of multiple alignment (FIG. 4), two storage areas (represented by bold face bodies) were observed. A black circle (●) shown above the sequence indicates a putative catalytic residue involved in the AGE reaction of Anabaena (YC Lee et al., J. Mol. Biol., 2007, Vol. 367). 895-908). Using the nucleotide sequence of this conserved region, univ-f (SEQ ID NO: 13) and univ-r (SEQ ID NO: 14) were designed as common primers.

When the extracted DNA of (1) was used as a sample, 100 ng DNA, 1 U TaKaRa Ex Taq polymerase (Takara Bio), 1 × PCR buffer (2 mM MgCl ₂ , 0.25 mM dNTP) and 0.25 μM each PCR was performed with 25 μL of the PCR reaction solution prepared with the primers univ-f and univ-r. In addition, when the single-stranded DNA of (2) is used as a sample, 100 ng of DNA, 12.5 μL of Ampdirect Plus (Shimadzu), 1 U of TaKaRa Ex Taq polymerase (Takara Bio) or Nova Taq Hot Start DNA polymerase (Merck) and PCR with 25 μL of the PCR reaction solution prepared with 6.25 pmol of each of the primers univ-f and univ-r. The DNA thermal cycler was set to be cooled at 4 ° C. after 30 cycles of 94 ° C. for 30 seconds, 48 ° C. for 30 seconds, 72 ° C. for 30 seconds, followed by extension reaction at 72 ° C. for 4.5 minutes. Four types of fragments (md1, md2, md3, md6) amplified by PCR reaction were separated by electrophoresis (1.5% agarose gel), purified using QIAquick PCR purification kit (Qiagen), and the bases The sequence was determined.

(4) Acquisition of full-length gene from epimerase gene partial fragment The following primers for inverse PCR and nested PCR based on the nucleotide sequences of md1 to 3 and md6 amplified in (3) Was designed and synthesized.

The extracted DNA of (1) was digested with EcoRI, HindIII, and PstI for 2 hours, respectively, and self-circulation (self-ligation) reaction was performed overnight using DNA Ligation Convenience kit (Nippongene). Prepared with 100 ng of self-ligated DNA, 1 U of TaKaRa Ex Taq polymerase (Takara Bio) and 1 × PCR buffer (2 mM MgCl ₂ , each containing 0.2 mM dNTP, 20 pmol of each primer set for inverse PCR) Inverse PCR was performed using the PCR reaction solution. Next, 1.0 μL of inverse PCR reaction product, 1 U TaKaRa Ex Taq polymerase (Takara Bio) and 1 × PCR buffer (2 mM MgCl ₂ , 0.2 mM dNTP each, 20 pmol of each primer set for nested PCR) ) Nested PCR was performed with a PCR reaction solution prepared by 25 μL. In both PCR reactions, the DNA thermal cycler is set to be set at 40 ° C for 30 cycles at 94 ° C, 30 seconds at 60 ° C, 2 minutes at 72 ° C, followed by extension reaction at 72 ° C for 5 minutes and then cooled at 4 ° C. did. The amplified fragments by nested PCR were separated by electrophoresis (1.0% agarose gel) and recovered using Recochip (Takara Bio). Each recovered amplified fragment was subcloned into a pGEM-T vector (Promega), and the nucleotide sequence was determined.

Based on the determined base sequence, the following primers were further designed.
md1; md1F-S1 (SEQ ID NO: 31) and md1F-A1 (SEQ ID NO: 32)
md2; md2F-S1 (SEQ ID NO: 33) and md2F-A1 (SEQ ID NO: 34)
md3; md3F-S1 (SEQ ID NO: 35) and md3F-A1 (SEQ ID NO: 36)
mr6; mr6F-S1 (SEQ ID NO: 37) and mr6F-A1 (SEQ ID NO: 38)

A PCR reaction solution prepared by 100 μg of the extracted DNA (1), 1 U TaKaRa Ex Taq polymerase, and 1 × PCR buffer solution (containing ₂ mM MgCl ₂ , 0.2 mM dNTP, 20 pmol of each of the above primer sets), and further PCR Went. The DNA thermal cycler was set to be cooled at 4 ° C. after 35 cycles of 94 ° C. for 30 seconds, 56 ° C. for 30 seconds, and 72 ° C. for 2 minutes, followed by 72 ° C. for 5 minutes. The amplified DNA fragments were subcloned into the pGEM-T vector to obtain recombinant plasmids pGEM-md1, pGEM-md2, pGEM-md3 and pGEM-mr6 containing full length md1, md2, md3, mr6, respectively. The base sequence was determined.

The base sequence of md1 and the deduced amino acid sequence of the protein encoded by it (denoted as mD1) are shown in SEQ ID NO: 5 and SEQ ID NO: 1, and the base sequence of the ORF contained in md2 and the protein encoded by it (denoted as mD2) The amino acid sequences are SEQ ID NO: 6 and SEQ ID NO: 2, the nucleotide sequence of the ORF contained in md3 and the deduced amino acid sequence of the protein encoded by it (denoted as mD3) are shown in SEQ ID NO: 7 and SEQ ID NO: 3, and the ORF contained in mr6 SEQ ID NO: 8 and SEQ ID NO: 4 show the deduced amino acid sequences of the nucleotide sequences of and the protein encoded thereby (denoted as mR6), respectively.

<Example 2> Construction and purification of protein mD1, mD2, mD3, mR6 expression system (1) Construction of expression plasmid Construction of an expression vector for expressing proteins mD1, mD2, mD3, mR6 using E. coli as a host Therefore, the following primers were designed.

mD1; mD1-F (SEQ ID NO: 39) and mD1-R (SEQ ID NO: 40)
mD2; mD2-F (SEQ ID NO: 41) and mD2-R (SEQ ID NO: 42)
mD3; mD3-F (SEQ ID NO: 43) and mD3-R (SEQ ID NO: 44)
mR6; mR6-F (SEQ ID NO: 45) and mR6-R (SEQ ID NO: 46)

mD1-F and mD1-R and pGEM-md1, mD2-F and mD2-R and pGEM-md2, mD3-F and mD3-R and pGEM-md3, mR6-F and mR6-R PCR was performed in combination with pGEM-mr6, and restriction enzyme recognition sites were introduced at both ends of each ORF (md1: NdeI-XhoI, md2: NdeI-EcoRI, md3: NdeI-XhoI, md6: NdeI-XhoI). PCR was performed by 25 cycles of reaction at 98 ° C. for 10 seconds, 55 ° C. for 2 seconds, and 72 ° C. for 1.5 minutes. Each amplified fragment was recovered from the reaction solution and subcloned into the EcoRV site of pBluescript II SK (+) (Stratagene). After confirming that no mutation was introduced into the inserted fragment, it was inserted into the E. coli expression vector pET-23a (+) (Novagen) using the introduced restriction enzyme recognition site, and the expression vectors pET-md1, pET -Md2, pET-md3 and pET-mr6 were obtained.

(2) Induction of protein expression pET-md1, pET-md2, pET-md3 and pET-mr6 were transformed into E. coli. E. coli BL21 (DE3) was introduced, and a transformant was selected on an LB agar medium containing ampicillin having a final concentration of 100 μg / mL, and pre-cultured overnight at 37 ° C. in the same liquid medium 10 mL. 200 mL of the same liquid medium placed in a 500 mL Erlenmeyer flask was inoculated with 0.5 mL of the preculture solution (total 800 mL), and cultured with shaking at 37 ° C. and 160 rpm. When the absorbance at 600 nm reaches 0.6, isopropyl-β-D-thiogalactoside (isopropyl-β-D-thiogalactoside) is added to a final concentration of 0.1 mM to induce expression of the target protein. Further, shaking culture was performed at 20 ° C. for 20 hours.

(3) Purification of recombinant enzyme All the following operations were performed at 4 ° C.
The cells were collected from 800 mL of the culture solution of (2) by centrifugation at 4000 × g for 10 minutes. The cells were suspended in 25 mL of buffer A (20 mM MES-NaOH (pH 6.0), 1 mM EDTA, 1 mM dithiothreitol) containing 1 mM phenylmethylsulfonyl fluoride, and then Ultrasonic UD. -201 (TOMY) was used for crushing. After disrupting the cells, centrifugation was performed at 20000 × g for 20 minutes, and the resulting supernatant was used as a crude enzyme solution.

The crude enzyme solution was added to a coupled column of TOYOPEARL CM-650M (Tosoh Bioscience) and DEAE Sepharose Fast Flow (GE Healthcare Bio-Sciences) equilibrated with buffer A in advance. After washing with buffer A, the TOYOPEARL CM-650M column was separated, and the protein adsorbed on the DEAE-Sepharose column was eluted with a 0 to 500 mM NaCl linear concentration gradient and fractionated to 50 by 5 mL. Each fraction was subjected to SDS-PAGE, and the fraction in which the recombinant protein was most eluted was collected as a purified fraction, and then dialyzed in buffer A.

The enzyme solution after dialysis was concentrated using VIVASPIN 20 (VIVASCIENCE). An equal amount of glycerol was added to the concentrate and stirred well, and then stored as a purified recombinant enzyme at −20 ° C. until use.

<Example 3> Properties of recombinant enzyme (1) SDS-polyacrylamide gel electrophoresis of recombinant enzyme (SDS-PAGE)
SDS-PAGE was performed according to the method of Laemmli (Nature, 1970, Vol. 227, pages 680-685). MiniProtean III (Bio-Rad) was used for the electrophoresis apparatus. The separation gel had an acrylamide concentration of 10% (gel thickness of 0.75 mm), and electrophoresis was performed at a constant current of 15 mA. The gel after electrophoresis was stained using Bio-Safe CBB G-250 Stain (Bio-Rad). SDS-PAGE Standard, Low range (Bio-Rad) was used as the standard protein. Control CE-NE1 was prepared according to the method described in Non-Patent Document 1.

All of the obtained purified recombinant enzymes, mD1, mD2, mD3 and mR6 gave a single band by SDS-PAGE. The electrophoresis pattern of each recombinant enzyme is shown in FIG. Lane 1 is CE-NE1 (control), lane 2 is mD1, lane 3 is mD2, lane 4 is mD3, and lane 5 is mR6.

(2) N-terminal amino acid sequence of the recombinant enzyme After the purified protein was subjected to SDS-PAGE, it was transferred to a PVDF membrane (Immobilon-P, Millipore) using a Mini Transfer Blot Elephrophoretic Transfer Cell (Bio-Rad). . Transcription was performed using a blotting buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, 10% methanol) at a constant voltage of 100 V for 1 hour. After transcription, the protein was stained with CBB, the band was cut out and washed with water and methanol three times. The N-terminal amino acid sequence of the obtained band was analyzed using a protein sequencer. As a result, the N-terminal amino acid sequences of mD1, mD2, mD3, and mR6 were identified as MFVEEIKKDLVED (SEQ ID NO: 9), MDLKTMSEQMKEH (SEQ ID NO: 10), MKNEVVYKQL (SEQ ID NO: 11), and MVQTMIKEMQ (SEQ ID NO: 12), respectively. These amino acid sequences completely matched the N-terminal sequence of each full-length deduced amino acid sequence (SEQ ID NO: 1, 2, 3, 4).

(3) Molecular weight After the purified recombinant enzymes mD1, mD2, mD3, and mR6 were stained by SDS-PAGE, the gel was photographed with a commercially available digital camera and migrated using ImageJ 1.36b (National Institutes of Health). The molecular weight was calculated from the degree of migration of SDS-PAGE Standard, Low range (Bio-Rad) on the same gel. As a result, the molecular weights of mD1, mD2, mD3, and mR6 were estimated to be about 43.2, 44.3, 46.7, and 45.4 kDa, respectively. The estimated molecular weight was calculated using Compute pI / Mw (http://kr.expasy.org/tools/pi_tool.html) based on the deduced amino acid sequences of mD1, mD2, mD3, and mR6. As a result, the molecular weights of mD1, mD2, mD3, and mR6 are 45425.5 (SEQ ID NO: 1; number of amino acids, 389), 48346.9 (SEQ ID NO: 2; number of amino acids, 412), 46964.0 (SEQ ID NO: 3; amino acids) 405), 49177.6 (SEQ ID NO: 4; number of amino acids, 423), which was close to the results of SDS-PAGE. According to Non-Patent Document 1, R.I. The molecular weight of CE-NE1 of albus NE1 is around 43.1 kDa (calculated molecular weight, 45217.4; number of amino acids, 389).

(4) Isoelectric point The isoelectric point (pI) based on the deduced amino acid sequence was calculated using Compute pI / Mw. As a result, the isoelectric points of mD1, mD2, mD3, and mR6 were calculated as pH 4.99, 4.83, 4.82, and 4.89, respectively. According to Non-Patent Document 1, R.I. The calculated isoelectric point of CE-NE1 of albus NE1 is pH 4.69.

(5) Amino acid sequence homology A homology (identity) search was performed (blastp, http: //www.ncbi.nlm.) With known ones having high homology with the deduced amino acid sequences of mD1, mD2, mD3, and mR6. nih.gov/blast/Blast.cgi).

mD1 is derived from Coprococcus eutactus ATCC 27759-derived hypothetical protein (AGE-like) (registration number EDP27130) and 65%, Fecalibacterium prasunithizine AG (Faecalactiplatinium ApitrophizinMig. No. EDP22108) and 60%, and C.I. Phytofermentans ISDg-derived AGE (registration number ABX42625) showed 59% identity. R. Albus NE1 strain and ATCC 27210 strain CE (registration numbers BAF81108 and BAF81109) showed 51% identity.

MD2 is C.I. eutactus ATCC 27759-derived hypothetical protein (like AGE) (registration number EDP27130) and 55%, C.I. Phytofermentans ISDg-derived AGE-like protein (registration number ABX42625) and 55%, F.I. Prausnitzii M21 / 2-derived hypothetical protein (AGE-like) (registration number ABP6594) and 50%, C.I. Saccharolyticus DSM8903-derived AGE-like protein (registration number EDP27130) showed 45% identity. R. Albus NE1 and ATCC 27210 CE (BAF81108 and BAF81109) showed 44% identity.

MD3 is C.I. eutactus ATCC 27759-derived hypothetical protein (AGE) (registration number EDP22108), 54%, F.E. Prausnitzii M21 / 2-derived hypothetical protein (AGE-like) (registration number ABP6594) and 51%, C.I. Phytofermentans ISDg-derived AGE-like protein (registration number ABX42625) showed 50% identity. R. Albus NE1 and ATCC27210 CE (registration numbers BAF81108 and BAF81109) showed 46% identity.

MR6 is a Bacteroides uniformis ATCC 8492 derived hypothetical protein (AGE) (registration number EDO52282) and 57%. fragilis NCTC 9343-derived hypothetical protein (AGE-like) (registration number CAH06520) and 56%. fragilis YCH46-derived AGE-like protein (registration number BAD47600) was 56%, and Parabacteroides disstasonis ATCC8503-derived AGE-like protein (registration number ABR41852) was 54% identical. R. Albus NE1 and ATCC 27210 CE (registration numbers BAF81108 and BAF81109) showed 38% identity.

FIG. 6 shows multiple alignment of the deduced amino acid sequence of CE-NE1 (registration number BAF81108) and the deduced amino acid sequences of mD1, mD2, mD3, and mR6. The deduced amino acid sequences of mD1, mD2, mD3, and mR6 showed 50.9, 45.5, 46.6, and 40.9% identity, respectively, with that of CE-NE1.

(6) Quantitative measurement of 2-epimerase activity After mixing 40 μL of 250 mM sodium phosphate buffer (pH 7.5), 10 μL of 0.2 M D-lactose (or a suitable substrate) and 40 μL of water, The reaction was started by adding 10 μL of the diluted enzyme solution. The reaction temperature was 30 ° C. After 10 minutes, 100 μL of 0.1N HCl was added and mixed, and then immediately boiled for 2 minutes to stop the reaction. After the enzyme reaction under the above conditions, 300 μL of water was added and centrifuged at 13,000 × g for 5 minutes. 90 μL of the supernatant was mixed with 10 μL of 2 mg / mL maltitol (Hayashibara) as an internal standard and subjected to HPLC with the following settings.

HPLC system: LC-2000 Plus (JASCO)
Guard column: SUGAR SP-G (6.0 mm × 50 mm, Shodex)
Separation column: SUGAR SP0810 (8.0 mm × 300 mm, Shodex)
Eluent: Water Flow rate: 0.8 mL / min Column temperature: 80 ° C.
Detection: vaporized light scattering detector (ELD2000ES; Alltech Associates, Deerfield, IL), temperature 115 ° C., gas flow rate 3.2 l / min

A calibration curve was prepared using epilactose (Sigma). It calculated relatively from the area of the peak derived from epilactose (elution time) and the peak derived from maltitol (elution time). Protein was quantified according to the Bradford method (MM Bradford, Anal. Biochem., 1976, 72, 248-254). A calibration curve was prepared using bovine serum albumin.

(7) Substrate specificity The substrate specificity of mD1, mD2, mD3, and mR6 was examined by thin layer chromatography (TLC). 15 μL of 20 mM glycylglycine-NaOH (pH 7.5), 20 μL of each substrate solution (0.1 M) and 5 μL of purified enzyme solution (about 0.5 μg) were mixed and reacted at 30 ° C. overnight. 1 μL of the reaction solution was spotted on a TLC aluminum sheet (Silica gel 60, Merck) and developed using a solvent system of 2-propanol / 1-butanol / water = 7: 6: 2. After drying, the presence or absence of the product was verified by the anisaldehyde method.

FIG. 7 shows the results when cellobiose was used as a substrate, FIG. 8 shows the results of cellotriose, FIG. 9 shows the results of cellotetraose, FIG. 10 shows the results of lactose, and FIG. 11 shows the results of 4β-mannobiose. The results are shown in FIG. mD1, mD2, mD3 produced products from cellobiose, cellotriose, cellotetraose, lactose, 4β-mannobiose and globotriose. In the case of mR6, the product was detected from cellobiose, cellotriose, lactose, 4β-mannobiose and globotriose, and no product was detected from cellotetraose. The product mobility was identical to CE-NE1. Therefore, Glc-Man (O-β-D-glucopyranosyl- (1 → 4) -D-mannose) is obtained from cellobiose, and Glc-Glc-Man (O-β-D-glucopyranosyl- (1 → 4) is obtained from cellotriose. -O-β-D-glucopyranosyl- (1 → 4) -D-mannose) is obtained from cellotetraose by Glc-Glc-Glc-Man (O-β-D-glucopyranosyl- (1 → 4) -O— β-D-glucopyranosyl-O-β-D-glucopyranosyl- (1 → 4) -D-mannose) is derived from lactose by epilactose (O-β-D-galactopyranosyl- (1 → 4) -D -Mannose) is Man-Glc (O-β-D-mannopyranosyl- (1 → 4) -D-glucose) from 4β-mannobiose and O-α- from globotriose. - galactopyranosyl - (1 → 4) -O-β-D- galactopyranosyl - (1 → 4) -D- mannose, was inferred to have respectively generated.

Under the same experimental conditions, a test was performed using cellobiose and glucosyl mannose, which is an epimer of the hydroxyl group at the 2-position, as a substrate. The results using mR6 are shown in FIG. When cellobiose was used as a substrate, glucosyl mannose was produced, and when glucosyl mannose was used as a substrate, cellobiose was produced. Therefore, it has been clarified that the polypeptide of the present invention exhibits epimerase activity with respect to a sugar having a hydroxyl group at the 2-position at either the axial position or the equivalent position.

Under the same experimental conditions, glucose, mannose, galactose, fructose, xylose, arabinose, cordobiose (α-1,2), nigerose (α-1,3), maltose (α-1,4), isomaltose (α-1 6), sophorose (β-1,2), laminaribiose (β-1,3), and gentiobiose (β-1,6) as substrates, all of mD1, mD2, mD3 and mD6 are generated No object was given (FIGS. 14 to 16).

(8) Identification of reaction product a. Purification of reaction products Among the substrates for which reaction products were detected in (7) above, products were identified for cellobiose, cellotriose and lactose. 50 mg of cellobiose, cellotriose or lactose was dissolved in 1 mL of 5 mM glycyglycine-NaOH (pH 7.5), 5 μL (about 2 μg) of enzyme solution was added and reacted at 30 ° C. overnight. The plate was developed twice by TLC plate (Silica gel 60 F ₂₅₄ , Merck) (2-propanol / 1-butanol / water = 12: 3: 4). The silica gel in the region containing the reaction product on the TLC plate was scraped and extracted with 30 mL of water. This was centrifuged at 8000 × g for 10 minutes, the supernatant was filtered with a 0.22 μm syringe filter (ADVANTEC), and the filtrate was concentrated with a freeze dryer FD-1000 (EYELA).

b. Identification by acid hydrolysis Dissolve the reaction product from purified cellobiose and lactose (about 1 mg) in 100 μL of water, add an equal volume of 8M trifluoroacetic acid (TFA), treat at 100 ° C. for 3 hours, Concentrated to dryness. The operation of adding a small amount of 2-propanol → re-drying was repeated three times to completely remove TFA. Then, it melt | dissolved in 100 microliters water, and analyzed 1 microliter by TLC on the same conditions as said a. As a result, glucose and mannose were detected in the product from cellobiose, galactose and mannose were detected in the product from lactose, and the composition ratio was estimated to be 1: 1 from the color intensity (FIG. 17). From these results, the oligosaccharide produced from cellobiose is Glc-Man, the oligosaccharide produced from lactose is epilactose, and mD1, mD2, mD3, and mR6 have the same 2-epimerase activity as CE-NE1. Was suggested.

c. Identification by Nuclear Magnetic Resonance (NMR) Method Regarding the product obtained by reacting mR6 with lactose, the solvent was replaced with heavy water using a rotary evaporator, and then a high-resolution nuclear magnetic resonance (NMR) apparatus (BRUKER AMX-500 spectrometer) was used. (500 MHz); Bruker), and the spectrum of ¹³ C was measured ( ¹³ C-NMR). Note that TSP ([2,2,3,3-D ₄ ] sodium 3-3- (trimethylsilyl) propanoate) was used as an external standard. The spectrum of ¹³ C obtained was completely consistent with the commercially available epilactose (Sigma) spectrum. Furthermore, as summarized in chemical shifts in the ¹³ C-NMR spectrum of Table 2, the obtained epilactose is an anomer of α and β, and the product is epilactose from the chemical shift of the peak of the ¹³ C-NMR spectrum. Identified.

The spectrum obtained from the cellotriose reaction product completely coincided with the previously reported spectrum of Glc-Glc-Man derived from cell wall polysaccharide (R. Goldberg et al. Carbohydr. Res., 1991, 210th). (Page 263-276) (data not shown), it was identified as Glc-Glc-Man.

(9) Optimum pH
The optimum pH was measured for mD1, mD2, mD3, and mR6. 100 mM sodium phosphate buffer (pH 6.5, 7.0, 7.5, 8.0) or 100 mM glycylglycine-NaOH buffer (pH 7.5, 8.0, 8.5, 9.0) The activity was measured according to the conditions of (6) above except for the buffer solution. As a result, mD2 was about pH 7.5 in phosphate buffer (FIG. 18A), mD2 was pH 7-8 in phosphate buffer (FIG. 18B), and mD3 was in phosphate buffer and glycylglycine buffer. Over pH 7-8.5 (FIG. 18C), mR6 showed maximum activity at pH 7-8 in phosphate buffer and glycylglycine buffer (FIG. 18D). The maximum activity of CE-NE1 was observed around pH 7.5 in phosphate buffer.

(10) pH stability The pH stability test of mD1, mD2, mD3, and mR6 was conducted. Each enzyme was maintained at 4 ° C. for 20 hours under the condition of pH 2.5 to 10.0, and then the residual enzyme activity was measured according to the condition (6). As a result, mD1 maintained a pH of 8-8, mD2 maintained a pH of 4-8, mD3 maintained a pH of 4-8, and mR6 maintained a pH of 8-8 or more after the retention treatment (FIGS. 19A to 19D). CE-NE1 maintained an activity of 80% or more even after the holding treatment at pH 5-9. mD1, mD2, mD3, and mR6 all had pH stability equivalent to CE-NE1.

(11) Optimum temperature The enzyme activities of mD1, mD2, mD3, and mR6 were measured at each temperature (20, 30, 40, 50, 60, 70 ° C.). Except for the reaction temperature, the activity was measured in the phosphate buffer according to the conditions of (6). As a result, mD1, mD2, mD3, and mR6 are maximum at about 25 ° C. (FIG. 20A), 20-40 ° C. (FIG. 20B), 25-35 ° C. (FIG. 20C), and around 30-45 ° C. (FIG. 20D), respectively. Showed activity. The maximum activity of CE-NE1 was observed around 30 ° C. Compared with CE-NE1, mD2 was an enzyme having a relative activity of 50% even at 0 ° C. and a remarkable activity even in the low temperature region. Further, mR6 showed a relative activity of 60% or more even at 50 ° C., and was an enzyme that acts even in a high temperature region.

(12) Temperature stability Temperature stability tests of mD1, mD2, mD3, and mR6 were performed. After incubation at 30, 40, 50, and 60 ° C. for 1 hour, the enzyme activity was measured after 10 minutes, 30 minutes, and 1 hour according to the condition (6). As a result, mD1 and mR6 maintained an activity of 80% or more after the retention treatment at 50 ° C. and mD2 and mD3 at 40 ° C. (FIGS. 21A to 21D). CE-NE1 maintained an activity of 80% or more even after a holding treatment at 40 ° C. mD1 and mR6 have better temperature stability than CE-NE1, especially mR6 maintains almost 100% activity even when treated at 50 ° C. for 1 hour, which is extremely temperature stable. It was a high enzyme. mD2 and mD3 had the same temperature stability as CE-NE1.

(13) Maximum UV absorption value The absorption of each polypeptide at 230 to 500 nm was measured. As a result, all of mD1, mD2, mD3, and mR6 were proteins having a maximum absorption at 278 to 280 nm.

<Example 4> Prebiotic production test using commercially available milk Using mR6, a prebiotic production test containing epilactose was conducted. Enzymatic reaction is performed by adding 10 μl (about 1 μg) of mR6 to 490 μl of commercially available milk, and the reaction is performed at 30 ° C. for 1 hour, 2 hours, 3 hours, 4 hours, and room temperature for 24 hours. It was. The reaction solution was diluted 10-fold with water and boiled for 10 minutes, and then the supernatant obtained by centrifugation at 15000 × g for 10 minutes was analyzed by TLC. Except for the above, the conditions of (7) of Example 3 were all followed.

In the reaction solution after 3 hours, 4 hours, and 24 hours at room temperature at 30 ° C., formation of epilactose was observed (FIG. 22), and by using the polypeptide of the present invention, it was directly from commercial milk. It has been shown that it is possible to produce prebiotics containing epilactose.

By using the present invention, not only 2-epimerized cellooligosaccharides and epilactose but also epimerized oligosaccharides having a desired β-1,4 bond can be synthesized easily and in large quantities. For example, providing prebiotics, synbiotics, functional foods containing these (for example, non-caloric or low-calorie foods, or foods excellent in mineral (especially Ca ²⁺ ) absorption) at a low price. it can. Further, since foods or food additives having physiological functions can be developed, they are also useful in the field of medicine / pharmaceuticals.

Claims (12)

(1) A polypeptide comprising the amino acid sequence represented by any of SEQ ID NOs: 1 to 4, or (2) one or more amino acids in the amino acid sequence represented by any of SEQ ID NOs: 1 to 4 are deleted. Changes the conformation of the hydroxyl group at the 2-position of the reducing end sugar residue of an oligosaccharide consisting of a substituted or added amino acid sequence and the reducing end sugar residue being linked by a β-1,4 bond. A polypeptide that catalyzes the reaction. Polypeptides having the physicochemical properties shown in (A) to (G) below;
(A) The apparent molecular weight by SDS-PAGE is 42 to 44 kDa,
(B) having the amino acid sequence shown in SEQ ID NO: 9 at the N-terminus,
(C) specifically catalyzing a reaction that changes the conformation of the hydroxyl group at the 2-position of the reducing end sugar residue of the oligosaccharide to which the reducing end sugar residue is bonded by a β-1,4 bond;
(D) When the enzyme activity at pH 7.5 is 100%, the enzyme activity is 80% or more at pH 7.0 to 7.5,
(E) After holding at 4 ° C. for 20 hours at pH 5.0 to 8.0, the enzyme activity remains 80% or more,
(F) Optimum temperature: Shows an enzyme activity of 80% or more at 15-30 ° C., assuming that the enzyme activity at 25 ° C. is 100%, and (G) Temperature stability: after holding at 50 ° C. for 1 hour, The enzyme activity at 30 ° C. remains at 80% or more. Polypeptides having the physicochemical properties shown in (A) to (G) below;
(A) The apparent molecular weight by SDS-PAGE is 43 to 45 kDa,
(B) having the amino acid sequence shown in SEQ ID NO: 10 at the N-terminus,
(C) specifically catalyzing a reaction that changes the conformation of the hydroxyl group at the 2-position of the reducing end sugar residue of the oligosaccharide to which the reducing end sugar residue is bonded by a β-1,4 bond;
(D) When the enzyme activity at pH 7.5 is 100%, the enzyme activity is 80% or more at pH 7.0 to 8.0,
(E) After holding at 4 ° C. for 20 hours at pH 4.0 to 8.0, the enzyme activity remains at 80% or more,
(F) When the enzyme activity at 25 ° C. is 100%, the enzyme activity is 80% or more at 10 to 45 ° C., and (G) the enzyme activity at 30 ° C. is 80 after holding at 40 ° C. for 1 hour. % Or more remain. Polypeptides having the physicochemical properties shown in (A) to (G) below;
(A) The apparent molecular weight by SDS-PAGE is 46 to 48 kDa,
(B) having the amino acid sequence shown in SEQ ID NO: 11 at the N-terminus,
(C) specifically catalyzing a reaction that changes the conformation of the hydroxyl group at the 2-position of the reducing end sugar residue of the oligosaccharide to which the reducing end sugar residue is bonded by a β-1,4 bond;
(D) When the enzyme activity at pH 7.5 is 100%, the enzyme activity is 80% or more at pH 7.0 to 9.0,
(E) After holding at 4 ° C. for 20 hours at pH 4.0 to 8.0, the enzyme activity remains at 80% or more,
(F) When the enzyme activity at 25 ° C. is 100%, the enzyme activity is 80% or more at 15 to 35 ° C., and (G) the enzyme activity at 30 ° C. is 80 after holding at 40 ° C. for 1 hour. % Or more remain. Polypeptides having the physicochemical properties shown in (A) to (G) below;
(A) The apparent molecular weight by SDS-PAGE is 44 to 46 kDa,
(B) having the amino acid sequence shown in SEQ ID NO: 12 at the N-terminus,
(C) specifically catalyzing a reaction that changes the conformation of the hydroxyl group at the 2-position of the reducing end sugar residue of the oligosaccharide to which the reducing end sugar residue is bonded by a β-1,4 bond;
(D) When the enzyme activity at pH 7.5 is 100%, the enzyme activity is 80% or more at pH 7.0 to 7.5,
(E) After holding at 4 ° C. for 20 hours at pH 5.0 to 8.0, the enzyme activity remains 80% or more,
(F) When the enzyme activity at 25 ° C. is 100%, the enzyme activity is 80% or more at 20 to 45 ° C., and (G) the enzyme activity at 30 ° C. is 80 after holding at 50 ° C. for 1 hour. % Or more remain. (1) a polynucleotide encoding a polypeptide comprising the amino acid sequence represented by any of SEQ ID NOs: 1 to 4, or (2) one or more of the amino acid sequences represented by any of SEQ ID NOs: 1 to 4 Of the hydroxyl group at the 2-position of the reducing end sugar residue of an oligosaccharide having an amino acid sequence in which the amino acid is deleted, substituted, or added, and the reducing end sugar residue is linked by a β-1,4 bond. A polynucleotide encoding a polypeptide that catalyzes a reaction that changes conformation. (1) Hybridization under stringent conditions with a polynucleotide comprising the nucleotide sequence represented by any of SEQ ID NOs: 5 to 8 or (2) a complementary sequence of the nucleotide sequence represented by any of SEQ ID NOs: 5 to 8 A polypeptide that catalyzes a reaction that changes the conformation of the hydroxyl group at the 2-position of a reducing end sugar residue of an oligosaccharide having a reducing terminal sugar residue bonded by a β-1,4 bond. The polynucleotide according to claim 6, wherein the polynucleotide encodes any one of the above. A recombinant vector comprising the polynucleotide according to claim 6 or 7. A transformant into which the recombinant vector according to claim 8 has been introduced. A method for producing a polypeptide that catalyzes a reaction that changes the conformation of the hydroxyl group at the 2-position of a reducing terminal sugar residue of an oligosaccharide in which the reducing terminal sugar residue is linked by a β-1,4 bond, A step of culturing the transformant according to claim 9, a step of causing the transformant to produce the polypeptide, a step of recovering the polypeptide from the transformant or a culture supernatant of the transformant, A method comprising the steps of: A method for producing an oligosaccharide in which the reducing end sugar residue in which the conformation of the hydroxyl group at the 2-position of the reducing end sugar residue is changed is bound by a β-1,4 bond, A step of incubating the polypeptide of claim 1 with an oligosaccharide in which a reducing terminal sugar residue is linked by a β-1,4 bond; and a hydroxyl group at the 2-position of the reducing terminal sugar residue of the oligosaccharide on the polypeptide. A method comprising catalyzing a reaction that changes the conformation of said oligosaccharide to produce said oligosaccharide. A method for producing a prebiotic comprising an oligosaccharide in which the conformation of the hydroxyl group at the 2-position of the reducing end sugar residue is changed, wherein the reducing end sugar residue is bound by a β-1,4 bond, A step of incubating the polypeptide according to any one of 1 to 5 with a material containing an oligosaccharide in which a reducing terminal sugar residue is bonded by a β-1,4 bond; and the reduction of the oligosaccharide to the polypeptide A method comprising producing a prebiotic by catalyzing a reaction that changes the conformation of the 2-position hydroxyl group of a terminal sugar residue.

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