WO2008062555A1

WO2008062555A1 - Novel polypeptide having epimerase activity and use thereof

Info

Publication number: WO2008062555A1
Application number: PCT/JP2007/001253
Authority: WO
Inventors: Susumu Ito; Shigeki Hamada; Shigeaki Ito; Hirokazu Matsui; Hidenori Taguchi; Jun Watanabe; Megumi Nishimukai
Original assignee: National University Corporation Hokkaido University
Priority date: 2006-11-20
Filing date: 2007-11-19
Publication date: 2008-05-29
Also published as: JPWO2008062555A1

Abstract

Disclosed are: a enzyme for use in the production of an oligosaccharide having a β-1,4 bond; a process for producing the oligosaccharide; and a functional food relying on a novel function of the oligosaccharide. Specifically disclosed are: a polypeptide comprising an amino acid sequence selected from the items (A) to (C) and having an epimerase activity for at least an oligosaccharide having a β-1,4 bond: (A) the amino acid sequence depicted in SEQ ID NO:1; (B) an amino acid sequence having the substitution, addition or deletion of one or several amino acid residues in the amino acid sequence depicted in SEQ ID NO:1; and (C) an aminoacid sequence having a 51% or more homology to the amino acid sequence depicted in SEQ ID NO:1; and a functional food containing 4-O-β-D-galactopyranosyl-D-mannose.

Description

Specification

Novel polypeptide having epimerase activity and use thereof

[0001] The present invention relates to a novel polypeptide and use thereof. More specifically, the present invention relates to a polypeptide having 2_epimerase activity for oligosaccharides having β-, 4-links, a method for producing the same, Hetero-oligosaccharides synthesized using peptides, methods for their synthesis, and their use

Background art

[0002] Carbohydrates are the main energy source of living organisms and are precursors in the biosynthesis of other compounds such as fatty acids, triglycerides, and several amino acids. Carbohydrates also play an important role as structural components of connective tissue, neural tissue, bacterial cell walls, and nucleic acids. In addition to such roles as nutrients and / or constituents in living organisms, it has recently been clarified that carbohydrates play an important role in the transmission of various information in vivo as sugar chains possessed by glycoproteins. In addition to genetic engineering and / or protein engineering, glycoengineering is attracting attention.

[0003] Oligosaccharide, which is a carbohydrate bound with about 2 to 10 monosaccharides, is contained in, for example, root nodules and / or mammalian milk, and is widely distributed in animal and plant tissues. . In addition, many oligosaccharides having a pharmacological action have been discovered, and pharmaceuticals and / or health foods using them have been actively developed (see, for example, Patent Documents 1 and 2).

[0004] In recent years, attention has been focused on oligosaccharides having various physiological functions. Much research and development has been conducted for the purpose of synthesizing functional oligosaccharides, and various oligosaccharides have been produced on an industrial scale as research and development of functional oligosaccharides. At present, more than half of foods for specified health use contain oligosaccharide as a functional ingredient.

[0005] Hetero-oligosaccharides are formed by combining two or more different monosaccharides. It is a sugar and is known to have various physiological activities. For this reason, hetero-oligosaccharides are used in a wide range of fields. As an example of a method for producing a hetero-oligosaccharide, there is a method for producing a hetero-oligosaccharide from darcos monophosphoric acid and a saccharide using the reverse reaction of cellobiose phosphorylase (see Patent Document 3). Cellobiose phosphorylase is originally an enzyme that phosphorylates cellobiose into glucose 1-phosphate and glucose. By utilizing this reversible reaction, glucose 1-phosphate is used. Various hetero-oligosaccharides have been synthesized from these and various sugars.

[0006] However, the above-described method for producing a hetero-oligosaccharide has the disadvantage that the produced mono-oligosaccharide is expensive because glucose mono-phosphate is expensive. In this method, phosphoric acid generated during the reaction must be removed after the reaction is completed. Furthermore, when this method is used, the yield of heterooligosaccharide is limited. As a method other than the method for producing such hetero-oligosaccharides, sucrose phosphorylase and cholebiose phosphorylase are allowed to act on sucrose, which is inexpensive and readily available, in the presence of glucose-1-phosphate. Patent Document 4 describes a method in which phosphoric acid is not by-produced during the reaction.

[0007] Luminococcus albus (R um inococcusalbus (R. a I bus)) 7 ^ (registered as AT CC 27 2 1 0), an obligate anaerobic rumen bacterium, has 2 cellobioses. —It is known to have an epimerizing activity, and its existence was suggested as cellobiose epimerase (CE) (see Non-Patent Document 1). In order to prove that this activity of being secreted into the culture medium of this fungus is due to CE as a single enzyme, many researchers have attempted to separate and purify the enzyme. However, the enzyme has not yet been purified and identified, and its actual state is unknown.

Patent Document 1: Japanese Patent Application Laid-Open No. 8-256730 (published on 8th October 1996) Patent Document 2: Japanese Patent Application Laid-Open No. 2003-47402 (published on February 18, 1995) Patent Document 3: Japanese Patent Application Laid-Open No. 2-16992 (published on January 19, 1990) Patent Document 4: Special No. 2001-204489 (published July 31, 1991 Non-patent document 1: TR Tyler and JM Leatherwood (1 96 /) Arc h. B ioch em. B iophy s. 1 TR 9, ν3 θ 3. Disclosure of the Invention

Problems to be solved by the invention

[0008] The hetero-oligosaccharide synthesis method using the method as described in Patent Document 4 has the following disadvantages: (1) The raw material is expensive; (2) Multiple substrates and Enzymes are required, resulting in increased costs and complex reaction conditions; (3) In the reaction using sucrose, the reaction process is a single step, but the target hetero-oligosaccharide is in the reaction solution. In addition to the unreacted substrate (sucrose), the fructose produced by sucrose degradation, the unreacted receptor sugar and multiple sugars, the separation of the desired hetero-oligosaccharide is complicated. (4) In the synthesis method using cellobiose phosphorylase, the sugar at the non-reducing end side is limited to glucose.

[0009] The reaction product of the enzyme CE (EC 5.1.3.11), whose existence was suggested in Non-Patent Document 1, is 4_0_S_D_glucoviranosyl 1-D-mannose (glucosyl mannose; GI c -Ma n). However, whether or not this enzyme is a secretory enzyme has not been determined, and detailed properties of the enzyme, substrate specificity, and detailed structure of the product have not yet been determined.

[0010] As described above, CE has a unique enzymatic activity and is a very interesting enzyme from an enzymatic viewpoint. Therefore, many advantages and usefulness can be expected for hetero-oligosaccharide synthesis using this enzyme.

[0011] The present invention has been made in view of the above problems, and an object of the present invention is to provide a novel oligosaccharide synthesis method by realizing purification of CE and elucidating various enzyme chemical properties. It is in.

Means for solving the problem [0012] In order to solve the above-mentioned problems, the present inventors have sought and searched for CE-producing bacteria in nature. As a result, an excellent CE was found from the enzymes produced by R. a I bus isolated from cattle rumen (see Example 1), and a gene encoding CE produced by this microorganism was obtained. (See Example 2 and the text below) to complete the present invention. The polypeptide according to the present invention is characterized by having 2_epimerase activity for oligosaccharides having β-Λ, 4 bonds, and has overcome the problem to be solved.

[0013] That is, the polypeptide according to the present invention comprises (i) an amino acid sequence represented by SEQ ID NO: 1; (ii) one or several amino acids in the amino acid sequence represented by SEQ ID NO: 1 are substituted, An added or deleted amino acid sequence; or (C) an amino acid sequence having 51% homology or more with the amino acid sequence represented by SEQ ID NO: 1, and a polypeptide comprising any one amino acid sequence It is characterized by having epimerase activity for oligosaccharides having at least S-1, 4 bonds.

[0014] The polypeptide according to the present invention is (i) derived from a bacterium of the genus Ruminococcus; (B) the apparent molecular weight by SDS-PAGE is 40 to 42 kDa. (C) having the amino acid sequence shown in SEQ ID NO: 4 at the N-terminus; and (D) having a biological property of having at least 2 epimerase activity for oligosaccharides having 4 bonds; It is a feature. The polypeptide according to the present invention further has (E) an activity of 2-epomerizing glucose at the reducing end of at least cellobiose, cellotriose, cellotetraose or lactose; (F) UV radiation (G) isoelectric point (p I) is 4.69; (H) activity shown in (D) is a metal salt (eg, Fe 3 +, Co ^{2 +} , Cu ^{2 +} , P b ² +, Z n ^{2 +,} or Ag ⁺ ); (I) The activity shown in (D) is inhibited by a chemical (eg, N-promosuccimi , Odoacetic acid or p-chloromer benzoate); (J) The maximum activity shown in (D) is pH 7.7-8 in Britton-Robinson buffer . Or (K) (D) has the property that the maximum activity shown in (D) is shown in Tris-maleate buffer at 28-32 ° C. preferable. The preferred concentration of the metal salt or chemical is 1 mM, and the preferred concentration of the buffer is 40 to 1 O OmM. Moreover, the activity shown in the above property (E) is reversible, and the polypeptide according to the present invention also catalyzes the reversible reaction.

[0015] The polynucleotide according to the present invention is characterized by encoding the above-mentioned polypeptide.

[0016] The polynucleotide according to the present invention comprises: (A) a nucleotide sequence represented by SEQ ID NO: 2; (B) one or several nucleotides in the nucleotide sequence represented by SEQ ID NO: 2 are substituted, added or missing. Or (C) a polynucleotide comprising a complementary sequence of the base sequence shown in SEQ ID NO: 2 and a base sequence that is hybridized under stringent conditions, and having an epimerase activity for oligosaccharides It is characterized by coding the possessed polypeptide.

[0017] A vector according to the present invention is characterized by containing the above-mentioned polynucleotide.

[0018] A method for producing a polypeptide according to the present invention is characterized by using the above-mentioned vector.

[0019] A kit for producing a polypeptide according to the present invention comprises the above vector

It is characterized by having —.

[0020] The transformant according to the present invention is characterized by containing the above-mentioned polynucleotide. The transformant according to the present invention is preferably a bacterium belonging to the genus Ruminococcus, and more preferably a luminococcus albus (R. a I b us).

[0021] A method for producing a polypeptide according to the present invention is characterized by using the transformant described above.

[0022] A kit for producing a polypeptide according to the present invention is characterized by comprising the above-described transformant. [0023] The antibody according to the present invention is characterized by specifically binding to the above-mentioned polypeptide.

[0024] A method for producing a polypeptide according to the present invention is characterized by using the above-mentioned antibody.

[0025] A kit for producing a polypeptide according to the present invention is characterized by comprising the above-described antibody.

[0026] A method for synthesizing a hetero-oligosaccharide according to the present invention is characterized by including a step of reacting the above-mentioned polypeptide with an oligosaccharide having a β-Λ, 4-bond.

[0027] A reagent kit for synthesizing a hetero-oligosaccharide according to the present invention is characterized by comprising the above-mentioned polypeptide. The reagent kit according to the present invention preferably further comprises an oligosaccharide having β-Λ, 4 bonds.

[0028] A reagent composition for synthesizing a hetero-oligosaccharide according to the present invention is characterized by containing the above-mentioned polypeptide.

[0029] The functional food according to the present invention is characterized in that it contains a compound produced using the above-mentioned polypeptide in order to function as a prebiotic. The functional food according to the present invention is preferably a bifidobacterial growth promoter and a low-strength (indigestible) food. Further, the functional food according to the present invention may contain a further functional substance. The functional food according to the present invention may be provided as a single product in the form of a composition or in the form of a kit.

[0030] The method for producing a functional food according to the present invention comprises the above-described polypeptide,

And a step of reacting with an oligosaccharide having 4 bonds; and a step of adding a reaction product to food. The method for producing a functional food according to the present invention may further include a step of adding a further functional substance to the food.

[0031] The functional food for improving the intestinal environment according to the present invention is characterized by containing 4_0_; S_D_galactopyranosyl_D-mannose. In particular the functionality Food is intended to improve the intestinal environment by promoting the growth of bifidobacteria.

[0032] The functional food for improving lipid metabolism according to the present invention is characterized by containing 4_0_; S_D_galactopyranosyl_D-mannose. In particular, the functional food is intended to improve lipid metabolism by reducing blood cholesterol levels.

[0033] The functional food for promoting mineral absorptivity according to the present invention is characterized by containing 4-0-; 3_D_galactobilanosyl-D-mannose.

[0034] The functional food for diabetic patients according to the present invention is characterized by containing 4_0_; S_D_galactobilanosyl 1-D-mannose.

[0035] The low-strength low-reactivity sweetener according to the present invention is characterized by containing 4_0_; S_D-galactobillanoyl D-mannose.

[0036] The constipation improving agent according to the present invention is characterized by containing 4_0_; S_D_galactopyranosyl_D_mannose.

The invention's effect

[0037] By using the polypeptide according to the present invention, it is possible to easily and abundantly produce 4_0_; S_D_galactobyrosine D-mannose, etc., from a product in which the reducing end glucose of the cellooligosaccharide is 2-epidemerized or lactose. In addition to being able to synthesize, the desired hetero-oligosaccharide can be easily synthesized. Thus, it is possible to provide functionality food material (e.g., non-caloric or low-calorie foods, or Mi Neraru (particularly, Ca ² +, etc.) excellent food absorption) at low cost. In addition, if the present invention is used, foods or food additives that cause a reduction in blood cholesterol can be developed. Furthermore, it is possible to promote the growth of bifidobacteria with anti-enteric action. However, this does not deny that other physiological activities can be recognized.

[0038] 4_0_ S_D_Garactopyranosyl-D-mannose, also known as epilactose, is known to exist in trace amounts in milk (TR I. Cataldi et al., (1 999) A nal. C he m. 7 1, 491 9). Following this compound epilac! Abbreviated as “su”. Brief Description of Drawings

[FIG. 1] FIG. 1 is a scheme showing a reaction catalyzed by a polypeptide according to the present invention to isomerize the hydroxyl group at the 2nd-position glucose of cellobiose and convert it to GIc-Man.

FIG. 2 is a scheme showing a reaction catalyzed by a polypeptide according to the present invention to isomerize the 2-position hydroxyl group of glucose at the reducing end of lactose and convert it to epilactose.

FIG. 3 is a CBB-stained photograph of an SDS-PAGE gel showing a polypeptide according to the present invention purified using various chromatographic methods. M, molecular weight marker; C, crude extract; P, CE purified enzyme preparation.

[FIG. 4] FIG. 4 shows a polypeptide according to the present invention comprising N-acetyl-D-glucosamine (A), UDP-glucose (B), glucose_6_phosphate (C), glucose (D), Mannose (E), Fluke I (F), Galax I (G), Xylose (H), Arabinose (I), Sophorose (J), Lamina Revios (K), Gentiobiose (L), Rak TLC was used for the reaction products of Tols (M), Maltose (N), Sucrose (O), Cellobiose (P), Cellotriose (Q) and Cellotetraose (R) as substrates. It is a figure which shows the result of praying.

[FIG. 5] FIG. 5 shows the ¹ H-NMR (a) and ¹³ C-NMR (b) spectra of the product (epitaxial!) From lactose produced by the polypeptide of the present invention. is there.

[FIG. 6] FIG. 6 shows ¹ H-NMR (a) and ¹³ C-NMR (b) of the product (G lc -GI c-Man) from cellotriose produced by the polypeptide of the present invention. This is the Spectra.

FIG. 7 is a scheme showing a reaction catalyzed by a polypeptide according to the present invention to isomerize the hydroxyl group at the 2-position of the reducing terminal glucose of cellotriose and convert it to GI cGIc-Man. [Fig.8] Fig.8 is epirac! This is a result showing that (a) or DFA III (b) has a mineral absorption promoting effect.

[FIG. 9] FIG. 9 shows the results showing that epilactose has a mineral absorption promoting effect. Panel A ⁺ C a ^2, Panel B Mg ² +, Panel C respectively the absorption of Z n ² +.

[FIG. 10] FIG. 10 is a graph showing the Ca ² + absorption-promoting effect of epicrust measured by the inverted sack method. The vertical axis represents the amount of C a ² + absorbed per length (cm) of the small intestine used for the sac.

[FIG. 11] FIG. 11 is a graph showing the change in the total cholesterol level in the rats ingesting the dietary supplement with epilactose. Panel A represents fasting and Panel B represents feeding.

[FIG. 12] FIG. 12 is a graph showing changes in blood neutral lipid, phospholipid, and cholesterol levels in rats fed an epilactose supplemented diet.

[FIG. 13] FIG. 13 is a graph showing changes in blood glucose level after feeding epilactose.

BEST MODE FOR CARRYING OUT THE INVENTION

[0040] Up to now, 32 types of epimerases classified as one of isomerases have been reported. Many of them use sugars modified with nucleotides, phosphates, acyl groups, etc. as substrates, and those with unmodified sugars as substrates are aldos 1-epimeraise (EC 5.1. 3. 3) There are only three types: Malus 1-epimelase (EC 5.1.3.2 1) and CE (EC 5. 1. 3. 1 1).

[0041] The present inventors have refined CE derived from Rumicoccus albus (R. a I bu s) N E 1 found by themselves and attempted to elucidate various enzyme chemical properties. This enzyme isomerized the 2-position hydroxyl group of cellobiose at the reducing end side glucose and catalyzed the interconversion to G I c — Man (Fig. 1). Of the three types of epimerases described above, CE is the only epimerase that acts at position 2 on the reducing end side.

[0042] Thus, since the reaction catalyzed by CE was very unique, the present inventors Thought that a new product could be obtained by making this enzyme act on a substrate other than cellobiose or modifying the substrate specificity of this enzyme, and further attempted synthesis of a new oligosaccharide by CE.

[0043] [1] Polypeptide and polynucleotide

[1_1] Polypeptide

The present invention provides a polypeptide having 2-epimerase activity for oligosaccharides having a β-4 bond.

As used herein, a “polypeptide according to the present invention” has 2-epimerase activity against oligosaccharides derived from bacteria of the genus Ruminococcus and having at least β-Λ, 4 bonds. Polypeptides or variants thereof are contemplated, more particularly CE or variants thereof. The bacterium is preferably R. a I bus, more preferably R. a I bus N E 1 strain. R. albus N E 1 described in the present specification can be provided by the accession number F ERM P-2 1 03 6 (National Institute of Advanced Industrial Science and Technology Patent Organism Depositary).

[0044] As used herein, "CE from Ruminococcusa I bus (R. a I bus) NE 1" has an apparent molecular weight of about 41 k D according to SDS-PAGE. a (for example, 40 to 42 kDa) and having the amino acid sequence shown in SEQ ID NO: 4 at the N-terminus. This apparent molecular weight is calculated from the actual amino acid sequence because it varies slightly depending on the degree of post-translational modification in the polypeptide (protein) used and the concentration of the gel used for SDS_PAGE. One skilled in the art will readily appreciate that the molecular weight can be from about 35 kDa to about 46 kDa. The molecular weight calculated based on the CE sequence information identified by the present inventors is 452 17 Da. In addition, if the position of the single band shown in lane P in Fig. 3 is accurately represented, the apparent molecular weight by SDS-PAGE is 43.1 kDa. [0045] The activity of the polypeptide according to the present invention is preferably an activity of isomerizing the 2-position hydroxyl group of the sugar on the reducing end side of the oligosaccharide. As used herein, “oligosaccharide” is intended to be a sugar composed of 2 to 10 monosaccharides, and the number of monosaccharides is preferably 2 to 6, cellobiose, cellotrio More preferably, it is monos, cellotetraose or lactose.

[0046] As shown in the Examples below, the polypeptide according to the present invention has a maximum absorption of ultraviolet rays of 2 78 to 28 80 nm and an isoelectric point (p I) of 4.69. is there. In addition, in the polypeptide according to the present invention, the 2-epepimerase activity for the oligosaccharide is a metal salt (for example, Fe ^{3 +} , Co ^{2 +} , Cu ^{2 +} , P b ² +, Z n 2 ₊ or A g +) and is inhibited by chemicals (eg N_prosuccimide, codoacetic acid or p-chloromercurybenzoate). The concentration of the metal salt or chemical substance preferable for inhibiting the activity of the polypeptide according to the present invention is 1 mM. Further, in the polypeptide according to the present invention, 2_epepimerase activity with respect to the above oligosaccharide has a maximum activity at pH 7.7 to 8.2 in Priton-Robinson buffer, and T Its maximum activity is shown at 28-32 ° C in ris_maleic acid buffer. The preferred concentration of the buffer is 40-1OOmM.

[0047] As used herein, the term "polypeptide" is used interchangeably with "peptide" or "protein". In addition, “fragment” of a polypeptide is intended to be a partial fragment of the polypeptide. Polypeptides according to the present invention may also be isolated from natural sources or chemically synthesized.

[0048] The term "isolated" polypeptide or protein is intended to be a polypeptide or protein that has been removed from its natural environment. For example, recombinantly produced polypeptides and proteins expressed in host cells can be expressed as simple as natural or recombinant polypeptides and proteins that have been substantially purified by any suitable technique. It is thought that they are separated.

[0049] The polypeptide according to the present invention was purified from R. a I bus NE 1. However, the polypeptide according to the present invention is not limited to this, and other natural purified products. Products, products of chemical synthesis procedures, or products produced by recombinant techniques from prokaryotic hosts.

[0050] Based on the description of the present specification, a person skilled in the art will know the entire amino acid sequence of the polypeptide according to the present invention and the entire nucleotide sequence (or open reading sequence) of the polynucleotide encoding the polypeptide. Frame or part of it). For example, a polynucleotide encoding a polypeptide according to the present invention can be obtained as follows.

[0051] The method for isolating a polynucleotide encoding the polypeptide according to the present invention includes a step of preparing a genomic library prepared from cells expressing the polypeptide according to the present invention. In order to isolate a polynucleotide encoding the polypeptide according to the present invention, it is preferable to use a genomic library. A gene library suitable for isolating a nucleotide sequence encoding a polypeptide according to the present invention or a portion thereof using a wide range of cloning vectors such as plasmids, cosmids, phages, YACs, etc. One can be created.

[0052] A method of screening a gene library for confirming the presence of a polynucleotide encoding a polypeptide according to the present invention is an oligonucleotide based on amino acid sequence information shown in SEQ ID NO: 1. Includes the process of preparing the probe. Using the standard triplet genetic code, oligonucleotide sequences of about 17 base pairs or longer can be prepared by conventional in vitro synthesis techniques. This oligonucleotide sequence is synthesized to correspond to the full length or a part of the amino acid sequence shown in SEQ ID NO: 1. The obtained oligonucleotide is then labeled with a radionuclide, an enzyme, biotin, a fluorescent reagent, etc., and used as a probe for screening a gene library. Alternatively, a polymerase chain reaction (PCR) using a degenerate primer designed based on the amino acid sequence shown in SEQ ID NO: 1 may be performed.

[0053] The polynucleotide encoding the polypeptide according to the present invention is the above gene. It can be obtained from recombinant DNA recovered from library isolates. Polynucleotides encoding the polypeptides according to the invention can be obtained by sequencing the non-vector single nucleotide sequences of these recombinant molecules. Nucleotide sequence information is available from widely used DNA sequencing protocols (eg, S. L. Berger and AR Kimmel (1 987) M ethods E nz ymo to 1 52, 307, A cad em ic P ress, NY) (incorporated herein by reference)) can be obtained using, for example, sequencing methods that can be found in. By combining nucleotide sequence information from several recombinant DNA isolates (including both isolates from cDNA and isolates from a single genomic library), the polypeptides of the present invention Of all amino acids, as well as upstream and downstream nucleotide sequences.

[0054] Nucleotide sequences obtained from gene library isolates specific for the polypeptides according to the present invention are subjected to analysis in order to identify important regions of the polypeptide genes according to the present invention. The These important areas include open reading frames, promoter sequences, and termination sequences. The analysis of nucleotide sequence information is preferably performed on a computer. Software suitable for analyzing nucleotide sequences for important regions is commercially available and includes, for example, forces such as D NAS IS ^TM (Pharmacia LKBT echnology, Piscataway, NJ). The When analyzing polynucleotide sequence information according to the present invention, the amino acid sequence information obtained from N-terminal sequencing of the purified polypeptide according to the present invention is used to improve the accuracy of the nucleotide sequence analysis. It is also important.

[0055] The polypeptide according to the present invention can be expressed by a recombinant technique when the nucleotide sequence of the polynucleotide encoding the polypeptide according to the present invention is functionally inserted into a vector. As used herein, “functionally inserted” is intended to be inserted into an expression vector according to the appropriate open reading frame and orientation. [0056] As described above, those skilled in the art who have read this specification, according to the present invention, not only a polypeptide having 2-epimerase activity for an oligosaccharide having a β-, 4-bond, but also the polypeptide. It will be readily appreciated that polynucleotides that code for are also provided. The polynucleotide encoding the polypeptide according to the present invention will be described in detail later in this specification. When producing a gene construct comprising the complete open reading frame of a polypeptide according to the present invention, the preferred starting material is a genomic library isolate encoding the polypeptide according to the present invention. More preferably, it is a polynucleotide obtained by the method described above.

[0057] Typically, the polynucleotide encoding the polypeptide according to the present invention is inserted downstream of the promoter, followed by a stop codon and, if desired, produced as a hybrid protein and Subsequent cleavage can be used. In general, sequences specific to the host cell that improve the production yield of the polypeptide of the present invention are used, and appropriate control sequences (such as an enhancer sequence, polyadenylation sequence, and ribosome binding site). Is added to the expression vector. Once the appropriate coding sequence has been isolated, it can be expressed using a variety of different expression systems; eg, bacteria, yeast, baculovirus or mammalian cells. The polypeptide according to the present invention may be any polypeptide in which amino acids are peptide-bonded, but is not limited thereto, and may be a complex polypeptide including a structure other than the polypeptide.

[0058] In addition, the polypeptide according to the present invention may include an additional polypeptide. Additional polypeptides include, for example, epitope labeled polypeptides such as H i s, My c, F I ag and the like.

[0059] For example, a polypeptide according to the present invention is an extract from a natural bacterium, or a transformant genetically modified to produce a polypeptide according to the present invention (for example, prokaryotic cells and Eukaryotic cells) can be easily purified by affinity chromatography using an antibody capable of specifically binding to the polypeptide of the present invention. Antibody affinity chromatography In addition to use, the polypeptides according to the present invention and the polypeptides derived from the polypeptides according to the present invention may be produced using a wide variety of other known protein purification techniques (eg, ammonium sulfate fractionation, solvent precipitation, immunoprecipitation, Gel filtration, ion exchange chromatography, hydrophobic chromatography, isoelectric precipitation, electrofocusing, electrophoresis, etc.) may be used alone or in combination.

[0060] The fraction isolated in the course of the purification may be immunoassay using the polypeptide-specific antibody according to the present invention, or the polypeptide-specific bioassay according to the present invention (for example, lactose or The presence of the polypeptide according to the present invention can be analyzed by analysis using an enzymatic reaction using cellooligosaccharide as a substrate and analysis of its product.

[0061] The antibody specific for the polypeptide of the present invention immunizes an appropriate vertebrate host (eg, rabbit) with the purified polypeptide of the present invention alone or in combination with a normal adjuvant. It is produced by. Usually, two or more immunizations are included, and blood or spleen is taken several days after the last injection. For polyclonal antisera, immunoglobulins can be precipitated, isolated, and purified by a variety of standard techniques. This includes affinity purification using a polypeptide according to the invention bound to a solid surface such as a gel or bead in an affinity ram. For monoclonal antibodies, spleen cells are usually fused with immortalized lymphocytes, such as bone marrow cell lines, under selective conditions for hyperidoma formation. The hybridomas can then be cloned under limited dilution conditions and their supernatants can be screened for antibodies with the desired specificity. Techniques for producing antibodies are known in the literature, for example, “Antibibodies: AL aboratry Manual, E. Harbor and Ed. Lane, Ed., Old Spring Harbor Laboratories Press, NY (1 988 ) "(Incorporated herein by reference).

[0062] As described above, those skilled in the art who have read this specification can make β-—, It is easy to provide not only a polypeptide having a 2-epimerase activity against an oligosaccharide having a combination and a polynucleotide encoding the polypeptide, but also an antibody that specifically binds to the polypeptide. To understand. The antibody that specifically binds to the polypeptide of the present invention will be described in detail later in this specification.

[0063] As used herein with respect to a protein or polypeptide, the term "variant" has the polypeptide according to the present invention; 2-epimerase activity for oligosaccharides having S_1,4 bonds Polypeptides that hold are intended.

[0064] As used herein, as an example of a mutant, one or several amino acids are substituted, added, or deleted in the amino acid sequence of CE derived from R. a I bus NE 1. Examples include mutants containing lost, inverted, translocated, repetitive and type-substituted amino acid sequences.

[0065] 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. . Furthermore, it is also well known that there are mutants in natural proteins that do not change artificially, but do not significantly alter the structure or function of the protein.

[0066] One skilled in the art can readily mutate one or several amino acids in the amino acid sequence of a polypeptide using well-known techniques. For example, according to a known point mutation introduction method, any base group of a polynucleotide that codes for a polypeptide can be mutated. In addition, a deletion mutant or an addition mutant can be prepared by designing a primer corresponding to an arbitrary site of a polynucleotide encoding a polypeptide. In addition, the objective can be achieved by random mutation. Furthermore, by using the activity measurement method described in the present specification, it can be easily determined whether or not the produced mutant has a 2-epimerase activity for an oligosaccharide having a desired _ 1,4 bond. obtain.

[0067] Preferred variants are conservative or non-conservative amino acid substitutions, deletions, or With additions. Silent substitution, addition, and deletion are preferred, and conservative substitution is particularly preferred. These do not change the 2_epimerase activity of the polypeptide according to the present invention for oligosaccharides having β-Λ, 4 bonds.

[0068] Representative conservative substitutions include substitution of one amino acid for another in the hydrophobic amino acids AI a, V a I, Leu, and II e; hydroxyl amino acids Ser and T exchange of hr, exchange of acidic residues A sp and GI u, substitution between amido amino acids A sn and GI n, exchange of basic amino acids L ys and A rg, and aromatic amino acids P he, Examples include substitution between Tyr.

[0069] As used herein, other examples of mutants include deletion of one or several bases in the base sequence encoding CE from R. a I bus NE 1. It is preferably coded by a polynucleotide comprising a base sequence inserted, substituted, or added.

[0070] As used herein, another example of a variant is one or several bases of a complementary sequence of a base sequence encoding CE from R. a I bus NE 1. Is preferably encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a substituted, added, deleted or inserted nucleotide sequence.

[0071] The hybridization is “M o l e c u l a r C l o n i n g: A

Laboratory Manual fed reversal, edited by J. Sambrook and DW Russll, C. Old Spring Harbor, NY (200 1) (incorporated herein by reference) It can be performed by a known method such as a method. Usually, the higher the temperature and the lower the salt concentration, the higher the stringency (which makes it difficult to hybridize), and a more homologous polynucleotide can be obtained. The appropriate hybridization temperature depends on the base sequence and the length of the base sequence. For example, DNA consisting of 18 bases encoding 6 amino acids. When fragments are used as probes, temperatures below 50 ° C are preferred.

[0072] As used herein, the term “stringent hybridization conditions” refers to hybridization solutions (50% formamide, 5 XSSC (1 50 1 \ 1 a CI, 1 5 in 5 mM sodium phosphate (pH 7.6), 5 X Denhardt's solution, 10% dextran sulfate, and 20 g / mL denatured sheared salmon sperm DN A) It is intended to wash the filter in 0.1 X SSC at approximately 65 ° C after a total incubation of 42 ° C. Depending on the polynucleotide that hybridizes to a “portion” of the polynucleotide, at least about 15 nucleotides (nt) of the reference polynucleotide, and more preferably at least about 20 nt, even more preferably at least about 3 O nt, and Even more preferably, a polynucleotide (either DNA or RNA) that hybridizes to a polynucleotide longer than about 30 nt is contemplated. Such a polynucleotide (oligonucleotide) that hybridizes to a “part” of the polynucleotide is also useful as a detection probe as discussed in more detail herein.

[0073] As used herein, another example of a variant is at least 50% identical to the base sequence encoding CE from R. a I bus NE 1, more preferably at least 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 72%, 75%, 80%, 82%, 85%, It is preferably encoded by a polynucleotide comprising a base sequence that is 90%, 92%, 95%, 96%, 97%, 98% or 99% identical. In addition, a natural sequence that is homologous at the amino acid level with CE from the NE 1 strain shows only 51% homology. This sequence is the N-vasizoleg, Norecosamin 2_epimerase (G lc NA c_2_e pi protein (registration number Q 1 FKV 5) derived from C. ostridium phytofermentans. is there. [0074] For example, the reference nucleotide sequence of the polynucleotide according to the present invention is based on the reference (QU ERY) polynucleotide comprising at least 95 <½ identical nucleotide sequence to the nucleotide sequence. Identical to the reference sequence except that it can contain up to 5 mismatches per 100 nucleotides (bases) of the reference base sequence of the polynucleotide encoding the polypeptide of the invention. It is intended to be. In other words, up to 5% of the bases in the reference sequence can be deleted or replaced with another base to obtain a polynucleotide comprising a base sequence at least 95 <½ identical to the reference base sequence, Alternatively, as many bases as 5 <½ of all bases in the reference sequence can be inserted into the reference sequence. These discrepancies in the reference sequence are dispersed either individually at the 5 ′ or 3 ′ end position of the reference base sequence or at the base in the reference sequence or in one or more adjacent groups within the reference sequence, Can occur anywhere between the end of

[0075] Any specific nucleic acid molecule is, for example, at least 50%, 51%, 52%, 53%, 54%, 55% of the nucleotide sequence of the polynucleotide encoding the polypeptide of the present invention. %, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 72%, 75%, 80%, 82%, 85%, 90%, 92%, 95%, 96%, Whether 97%, 98% or 99% are identical is determined by a known computer program (for example, Bestfitprogr am (WI sconsinsequence Analysis package, Version 8 for Unix), G enetics and om puter G roup, U niversity R esearch Park, 5/5 Science Drive, Madison, WI 537 1 1) B estfit is the locality of Sm ith and Wa terman Using homology algorithm (TF Smith and MS Watterman (1 98 1) A d v. A pp Mat h. 2, 482 (incorporated herein by reference)) Find the best homologous segment between two sequences Highest homology (B es tfit) or any other sequence alignment program, to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present invention, the percent identity Is calculated over the entire length of the reference sequence, and the parameters are set to allow a gap in homology of up to 5% of the total number of nucleotides in the reference sequence.

In a specific embodiment, the identity (also referred to as global sequence alignment) between a reference (QU ERY) sequence (sequence according to the invention) and a target sequence is DL B rut I ag et al. (1 990) Is determined using the FAS TDB computer program based on the azorego rhythm (Comp. A pp Biosci. 6, 237 (incorporated herein by reference)). To calculate% identity, the preferred parameters used in FASTDB alignment of DNA sequences are: Matrix = Unit, k—tup I e = 4, M is ma tch Penalty = 1, Joining , P enalty = 30, Randomization G roup L ength = 0, Cutoff S core = 1, G ap P enalty = 5, G ap S ize P ena I ty = 0. 05, W ind ow S ize = 500 or the length of the target nucleotide sequence (whichever is shorter). According to this embodiment, if the target sequence is shorter than the QUERY sequence (due to an internal deletion) due to a 5 'or 3' deletion, the F AS TDB program When calculating, manual corrections are made to the results, taking into account the fact that 5 'and 3' shortenings of the target sequence are not taken into account. For subject sequences that are truncated at the 5 'or 3' end compared to the QU ERY sequence, the percent identity is 5 'and 3' of the subject sequence that are not matched / aligned QU ERY sequences Is corrected by calculating the number of bases as a percentage of the total bases in the QU ERY sequence. The determination of whether a nucleotide is matched / aligned is determined by the results of the FAS TDB sequence alignment. This percentage is then subtracted from the identity percentage calculated by the above FAS TDB program using the specified parameter, resulting in a final identity. —Reach gender percent score. This corrected score is used for the purpose of this embodiment. Only the 5 'and 3' bases outside the target sequence that do not match / align with the QU ERY sequence are calculated to manually adjust the percent identity score, as shown in the FASTDB alignment. Is done. For example, a 90 base subject sequence is aligned with a 100 base QU ERY sequence to determine percent identity. The deletion occurs at the 5 ′ end of the subject sequence, so the FAS TDB alignment does not show a match / alignment of the first 10 bases at the 5 ′ end. 10 unpaired bases represent 10% of the sequence (number of unmatched bases at 5 'and 3' ends / total number of bases in QU ERY sequence), so 10% Is subtracted from the identity percent score calculated by the F AS TDB program. If the remaining 90 residues are perfectly matched, the final percent identity is 90%. In another example, a 90 residue subject sequence is compared to a 100 base QU ERY sequence. In this case, the deletion is an internal deletion, so there is no base at the 5 'end or 3' end of the subject sequence that is not aligned / aligned with the QUERY sequence. In this case, the percent identity calculated by FAS TDB is not manually corrected. Again, only the 5 'and 3' end bases of the target sequence that do not match / align with the QU ER Y sequence are manually corrected. No other manual correction is made for the purposes of this embodiment.

[0077] As described above, the present invention provides a polypeptide having 2_ epimerase activity for oligosaccharides having S_1, 4 bonds. The polypeptide according to the present invention has 2-epimerase activity for cellobiose, but also has 2-epimerase activity for cellooligosaccharides other than lactose or cholebiose. The polypeptide according to the present invention having such properties is very useful for the synthesis of a novel oligosaccharide.

[0078] The method of synthesizing a hetero-oligosaccharide using the polypeptide according to the present invention has the following advantages compared with the synthesis method described in Patent Document 4: (1) Because it is a reaction of 1 substrate-1 enzyme, the reaction system is simple and inexpensive. Yes; (2) Since there is no reaction by-product, purification of the target product is easy and suitable for mass production; (3) Since the substrate specificity for non-reducing end sugars is not strict, Hetero-oligosaccharides that cannot be synthesized by the synthesis method can be synthesized.

[0079] [1-2] Polynucleotide

As described above, the present invention also provides a polynucleotide that codes for a polypeptide having 2_epimemerase activity to an oligosaccharide having a β-, 4 bond.

[0080] As used herein, the term "polynucleotide" is used interchangeably with "gene", "nucleic acid" or "nucleic acid molecule" and is intended to be a polymer of nucleotides. As used herein, the term “base sequence” is used interchangeably with “nucleic acid sequence” or “nucleotide sequence” and refers to deoxyribonucleotides (abbreviated as A, G, C, and T). Shown as an array. In addition, “a polynucleotide containing the base sequence shown in SEQ ID NO: 2 or a fragment thereof” is a polynucleotide containing a sequence shown by each of the deoxynucleotides A, G, C and / or T of SEQ ID NO: 2. Or a fragment of it is intended.

[0081] The polynucleotide according to the present invention may exist in the form of R N A (eg, m R N A) or D N A (eg, c D N A or genomic D N A). DNA may be double stranded or single stranded. Single stranded DNA or RNA can be the coding strand (also known as the sense strand) or can be the non-coding strand (also known as the antisense strand).

[0082] As used herein, the term "oligonucleotide" is intended to be a combination of several to several tens of nucleotides, and is used interchangeably with "polynucleotide". Oligonucleotides are represented by the number of nucleotides polymerized, such as dinucleotides and trinucleotides for short ones and 30 or 10 for long ones. Oligonucleotides are chemically synthesized, even if they are produced as longer polynucleotide fragments. May be.

[0083] The polynucleotide according to the present invention can also be fused to the polynucleotide encoding the tag tag (tag sequence or marker sequence) described above on its 5 'side or 3' side.

[0084] The polynucleotide according to the present invention comprises an untranslated region sequence or a vector sequence.

It may contain a sequence such as (including an expression vector sequence).

[0085] It should be noted that since the signal sequence and signal peptide do not exist in the entire base sequence (SEQ ID NO: 2) and amino acid sequence (SEQ ID NO: 1) of CE of the present invention, CE is an intracellular enzyme. It is obvious. However, it should be noted that this does not completely deny that an undiscovered secretory mechanism exists in CE.

[0086] The object of the present invention is to: a polypeptide having 2_epimerase activity for oligosaccharides having S_1,4 bonds; and 2_epimerase activity for oligosaccharides having S-1,4 bonds The present invention is to provide a polynucleotide that encodes a polypeptide having a nucleotide, and does not exist in a method for preparing a polypeptide, a method for preparing a polynucleotide, or the like specifically described in the present specification. Therefore, obtained by a method other than the above methods; a polypeptide having 2_epimerase activity for oligosaccharides having S_l, 4 bonds; and 2-epimerase activity for oligosaccharides having S_l, 4 bonds; It should be noted that a polynucleotide encoding a polypeptide having a phenotype also belongs to the technical scope of the present invention.

[0087] [2] Antibody

As described above, the present invention also provides an antibody that specifically binds to a polypeptide having 2_epimerase activity against an oligosaccharide having a β-, 4 bond. As used herein, the term “antibody” refers to an immunoglobulin (

IgA, IgD, IgE, IgG, IgM and their Fab fragments, F (ab ') ₂ fragment, Fc fragment), for example, polyclonal antibodies, Monoclonal antibody, single chain antibody, anti-idiotype Examples include but are not limited to antibodies and humanized antibodies.

[0088] Peptide antibodies are produced by methods well known in the art. For example, M. Chow et al., (1985) Proc. Natl. A cad. Sc, USA 82, 910, and F. J. Bittle et al., (1985) J. Ge. n. See V iro 66, 2347, both of which are incorporated herein by reference. In general, animals can be immunized with free peptide; however, anti-peptide antibody titers couple the peptide to a macromolecular carrier (eg, mosianin or tetanus toxoid to keyhole limpet). Can be boosted. For example, peptides containing cysteine are coupled to a carrier using a linker such as m-maleimidobenzoyl-N-hydroxysuccinimide ester, while other peptides such as glutaraldehyde. More common linking agents can be used to couple to the carrier. Animals such as rabbits, rats, and mice are either free or carrier-coupled peptides, such as intraperitoneal and intraperitoneal in emeraldions containing about 100 g of peptide or carrier protein and Freund's adjuvant. Immunized by intradermal injection. In order to provide useful titers of anti-peptide antibodies that can be detected by ELISA assay using, for example, free peptides adsorbed on a solid surface, for example, at intervals of about 2 weeks Can be needed. The titer of anti-peptide antibodies in serum from immunized animals can be determined by selection of anti-peptide antibodies, e.g., adsorption to peptides on solid supports and elution of selected antibodies by methods well known in the art. Can be increased.

[0089] As used herein, an "antibody" according to the present invention refers to a complete antibody molecule and an antibody fragment (eg, "313") that can specifically bind to a polypeptide according to the present invention. And “(ab ′) ₂ fragments”. F ab and F (ab ′) ₂ fragments lack the Fc portion of the complete antibody and are removed more rapidly by circulation and Rarely have non-specific tissue binding of antibodies (RL Wa h I et al., (1 983) J. Nu c to Me d. 24, 3 1 6 (incorporated herein by reference)). Therefore, these fragments are preferred.

[0090] Furthermore, additional antibodies that can bind to the peptide antigens of the polypeptides of the invention can be produced in a two-step procedure through the use of anti-idiotype antibodies. Such a method uses the fact that the antibody itself is an antigen, and thus it is possible to obtain an antibody that binds to a secondary antibody. According to this method, an antibody that specifically binds to a polypeptide according to the present invention is used to immunize an animal (preferably a mouse). The splenocytes of such animals are then used to produce hybridoma cells, and the hybridoma cells have the ability to bind to antibodies that specifically bind to the polypeptides of the invention. Screened to identify clones that produce antibodies that can be blocked by the polypeptide antigen. Such an antibody includes an anti-idiotype antibody against an antibody that specifically binds to the polypeptide of the present invention, and induces the formation of an antibody that specifically binds to the polypeptide of the present invention. Can be used to immunize animals to

[0091] It is clear that F ab and F (ab ′) ₂ and other fragments of the antibodies according to the invention can be used according to the methods disclosed herein. Such fragments are typically produced by proteolytic cleavage using enzymes such as papain (resulting in Fab fragments) or pepsin (resulting in F (ab ') ₂ fragments). Alternatively, the polypeptide binding fragments according to the invention can be produced by the application of recombinant DNA technology or by synthetic chemistry.

[0092] The antibody according to the present invention may be a chimeric monoclonal antibody. Such antibodies can be generated using genetic constructs derived from hybridoma cells that produce the monoclonal antibodies described above. Methods for producing chimeric antibodies are known in the art. For previous reports, see SL Morrison, (1 98 5) Science, 229, 1202; VT O i et al., (1 986) B io T echniques 4, 2 1 4; S. C abi I ly and H L. Heyneker, US Pat. No. 4, 8 1 6,567; M. T aniguchi and M. Kurosawa, EP 1 7 1 496; SL Morrison and L. Herzenberg, EP 1 73494; M. S N euberger and TH Rabbitts, WO 860 1 533; RR R obinson and AY Liu, WO 870 267 1; GL Boulianne et al., (1 984) Nature, 3 1 2, 643; MS N euberger et al., ( 1 985) Nature, 3 1 4, 268, both of which are incorporated herein by reference.

[0093] Thus, antibodies of the present invention, at least, have a Poribe petit de antibodies that recognize fragments of the present invention (e.g., _"3 13 Oyobi" (ab ') ₂ fragment) That is, it should be noted that an immunoglobulin comprising an antibody fragment that recognizes the polypeptide of the present invention and an Fc fragment of a different antibody molecule is also included in the present invention.

[0094] Thus, when the antibody according to the present invention is used, it is a polypeptide according to the present invention; a polypeptide having 2-epimerase activity with respect to an oligosaccharide having S_1,4 bonds can be easily obtained. The polypeptide according to the present invention can be produced by combining with affinity chromatography and the like. That is, the present invention also provides a method for producing a polypeptide using the antibody, a kit for producing a polypeptide comprising the antibody, and a method for producing the polypeptide and a specific kit for producing the polypeptide. Those skilled in the art who have read this specification will readily understand these embodiments.

[0095] [3] Vector

The present invention provides a vector that is used to produce a polypeptide having 2_epimerase activity for oligosaccharides having β_4 linkages. The vector according to the present invention may be a vector used for in vitro translation or a vector used for recombinant expression.

[0096] The vector according to the present invention includes the above-described polynucleotide according to the present invention. If it is a thing, it will not specifically limit. For example, a recombinant expression vector into which a polynucleotide c D Ν coding for a polypeptide having 2_epimelase activity for an oligosaccharide having a β-, 4 bond is inserted. Examples of the method for producing the recombinant expression vector include, but are not limited to, a method using plasmid, phage, or cosmid.

[0097] 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 host cell, a promoter sequence is appropriately selected in order to reliably express the polynucleotide according to the present invention, and a vector in which this and the polynucleotide according to the present invention are incorporated into various plasmids, etc. What is necessary is just to use as an expression vector. In addition, transformation of the host with the expression vector can also be performed according to conventional methods.

[0098] After culturing, cultivating or raising a host transformed with the above expression vector, conventional methods (for example, filtration, centrifugation, cell disruption, gel filtration chromatography, ion The target protein can be recovered and purified according to exchange chromatography.

[0099] The expression vector preferably includes at least one selection marker.

Such best practices include tetracycline resistance genes or ampicillin resistance genes for culturing in E. coli (E s c h e r i c h i a c o l i), Bacillus subtilis (B a c i l l u s su b t i i i s), and other bacteria.

[0100] The method of introducing the expression vector into a host cell, that is, the transformation method is not particularly limited, and a conventionally known method such as an electroporation method, a calcium phosphate method, a ribosome method, or a DEA E dextran method is preferable. Can be used for

[0101] When the vector according to the present invention is used, if the polynucleotide is introduced into an organism or a cell, the polypeptide having 2_epepimerase activity for an oligosaccharide having an S_1,4 bond is introduced into the organism or cell. Can be expressed. Furthermore, the vector according to the present invention can be used in a cell-free protein synthesis system. For example, it is possible to synthesize a polypeptide having 2-epimerase activity for oligosaccharide having 4 bonds.

[0102] Thus, it can be said that the vector according to the present invention should include at least a polynucleotide encoding the polypeptide according to the present invention. That is, it should be noted that vectors other than the expression vector are also included in the technical scope of the present invention.

[0103] As described above, when the vector according to the present invention is used, the polypeptide according to the present invention can be easily produced. That is, the present invention also provides a method for producing a polypeptide using the vector, a kit for producing a polypeptide comprising the vector, and a method for producing a polypeptide and a kit for producing a polypeptide. Specific embodiments are readily understood by those of ordinary skill in the art who have read this specification.

[4] Transformant

The present invention provides a transformant in which a polynucleotide that codes for a polypeptide having 2_epimerase activity for an oligosaccharide having a β_4 bond is introduced. If the transformant according to the present invention is used, the polypeptide according to the present invention can be produced easily and in large quantities. As used herein, the term “transformant” is intended to include not only cells, tissues or organs, but also living organisms, but cells (especially prokaryotic cells, fungi (eg, And filamentous fungi).

[0105] The transformant according to the present invention is characterized in that a polypeptide having a 2-epimerase activity for an oligosaccharide having a β_4 bond is expressed. In the transformant according to the present invention, it is preferable that a polypeptide having 2-epimerase activity with respect to an oligosaccharide having β-, 4 bonds is stably expressed, but it may be expressed transiently.

[0106] In one embodiment, the transformant according to the present invention comprises a recombinant vector comprising a polynucleotide that codes for a polypeptide having 2_epimerase activity for oligosaccharide having 4 linkages. Polypeptides with 2_epepimerase activity against oligosaccharides with β- and 4-bonds can be expressed. It is obtained by introducing it into living organisms.

[0107] As described above, when the transformant according to the present invention is used, the polypeptide according to the present invention can be easily produced. That is, the present invention also provides a method for producing a polypeptide using the transformant, a kit for producing a polypeptide comprising the transformant, and a method for producing the polypeptide and polypeptide production. Those skilled in the art who have read this specification will readily understand the specific embodiment of the kit.

[5] Kits, reagent compositions and methods for synthesizing hetero-oligosaccharides The present invention provides kits, reagent compositions and methods for synthesizing hetero-oligosaccharides. By using the present invention, it is possible not only to synthesize GIc_Man much much more easily than the conventional method, but also to easily obtain a new oligosaccharide.

[0109] The kit for synthesizing the hetero-oligosaccharide according to the present invention comprises the polypeptide according to the present invention. In addition to the polypeptide according to the present invention, the kit according to the present invention may be provided with a reagent for promoting the enzymatic reaction of the polypeptide or a reagent for appropriately performing the enzymatic reaction. These reagents can be appropriately selected depending on the enzyme reaction. The carbon source used when applying the kit according to the present invention is not particularly limited, and examples thereof include cellooligosaccharides (disaccharide, trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide, etc.) and lactose. . Bulk materials for obtaining these carbon sources include, for cello-oligosaccharides, unused resources including cellulose or low-utilized resources (for example, pulp), but are not limited to these. Β-, 4 Hemicellulose containing conjugated polysaccharides can also be mentioned as a bulk material. For lactose, examples of bulk materials include raw milk such as sushi and goats, powdered milk, and whey. These bulk materials may be used directly or may be decomposed in advance by adding a degrading enzyme (for example, cellulase or xylanase).

[0110] The reagent composition for synthesizing the hetero-oligosaccharide according to the present invention includes the polypeptide according to the present invention. The reagent composition according to the present invention comprises a polypeptide according to the present invention. In addition to tide, a reagent for promoting the enzymatic reaction of the polypeptide described above, or a reagent for appropriately performing the enzymatic reaction may be included together. The carbon source used when applying the reagent composition according to the present invention is not particularly limited. Power cellooligosaccharides (disaccharide, trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide, etc.) and lactose are included. Can be mentioned. Bulk materials for obtaining these carbon sources are as described above.

[0111] The method of synthesizing the hetero-oligosaccharide according to the present invention uses the polypeptide according to the present invention. In one embodiment, the method according to the present invention includes a step of incubating the polypeptide according to the present invention together with an oligosaccharide having a β-, 4 bond. The oligosaccharide is preferably lactose or cellooligosaccharide. The carbon source used when applying the method according to the present invention is not particularly limited, and examples include cellooligosaccharides (disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, etc.) and lactose. . Bulk materials for obtaining these carbon sources are as described above. If HPLC, silica gel, activated charcoal column chromatography, etc. are used, the synthesized hetero-oligosaccharide can be purified at a large production level.

[0112] [6] Functional food

“Prebiotics” is defined by GR Gibson and MB Roberfroid (1 995) (J. Nut r. 1 25, 1 401) as “intestinal flora (group of microorganisms inhabiting the digestive tract). (Mainly anaerobic bacteria)), which has a positive impact on the health of the host by improving the balance of the food. "As used herein," of animals (including humans) A substance that promotes improvement of the intestinal environment by growing microorganisms useful in the intestinal environment (for example, probiotics such as lactic acid bacteria and bifidobacteria) is also referred to as a useful microbial growth factor. Typical prebiotics include indigestible substances such as oligosaccharides, fermented whey products from propionic acid bacteria, and food fiber. Oligosaccharides serve as food for probiotics, The fiber stores enteric bacteria and assists in their growth. [01 13] The effects of prebiotics include absorption of minerals, blood cholesterol and neutral fat levels, prevention of arteriosclerosis, blood sugar levels, diabetes improvement, obesity, Activation of exercise, improvement of constipation, activation of immunity, prevention of infectious diseases, prevention of cancer, suppression of blood ammonia level, improvement of hepatic encephalopathy due to decreased liver function, promotion of vitamin synthesis by enteric bacteria Examples include, but are not limited to, promoting absorption of various minerals and improving symptoms of ulcerative colitis. In addition, recent research has shown that prebiotic intake improves the absorption of minerals (eg, C a ^{2 +} , Mg ² +, Z n ² +, F e ³ +, etc.). It is, in particular attention is C a ² + absorption promoting effect. Oligosaccharides 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 tit junction (TJ)) between cells inside the small or large intestine. It is thought to be because of this.

[0114] As shown in the following Examples, by using the polypeptide according to the present invention; a method of synthesizing epilactose, which is a hetero-oligosaccharide synthesized from lactose having an S_1,4 bond, Those of ordinary skill in the art who have read this specification will readily appreciate that functional foods as prebiotics can be provided.

[0115] For example, as shown in Example 5, Bifidobacteria can assimilate epilactose (Table 6). In addition, as shown in Example 9, epilactose promotes the growth of cecal bifidobacteria in rats, reducing pH (Table 9), and reducing the amount of short-chain fatty acids and organic acids in the cecum. Since it can be increased (Table 10), the intestinal environment can be improved and can be used as a functional food for improving the intestinal environment.

[01 16] In addition, as shown in Examples 7 and 8, Epilactos has the ability to promote mineral absorption capacity (Figs. 8, 9 and 10), and therefore, the mineral absorption capacity promoter or mineral It can be used as a functional food for promoting absorption.

[0117] Furthermore, as shown in Example 9, since Epi lactose increases the content of the cecum (Table 9), it can be used as an agent for improving constipation. [01 18] Furthermore, as shown in Example 10, Epilactos can reduce the amount of cholesterol in the blood (Figures 11 and 12), and therefore, lipid metabolism improving agents or lipids can be reduced. It can be used as a functional food for improving metabolism.

[01 1 9] In addition, epilactose is difficult to digest and absorb in the stomach and intestine (Table 7), and is low in calories, as shown in Example 11 and also increased in blood glucose after eating. Therefore, it can be used as a functional food for diabetics who need to avoid an increase in blood glucose level (Fig. 13).

[0120] Epilactose is weak in sweetness and can be used as a low-strength, low-sweetening agent.

[0121] That is, the present invention provides a functional food and a method for producing the same. In one embodiment, the functional food according to the present invention is characterized in that it contains epilactose, which is an oligosaccharide having a β_4 bond, a growth promoter for bifidobacteria, a constipation improving agent, A lipid metabolism improving agent, a low-calorie food or a mineral absorption promoter is preferred. The functional food according to the present invention may contain a further functional substance other than epilactose.

[0122] In another embodiment, the functional food according to the present invention is characterized by comprising a compound produced using a polypeptide having epimerase activity for oligosaccharides having Π4 bonds.

[0123] In order to produce the functional food according to the present embodiment, the above-described polypeptide may be reacted with an oligosaccharide having β-Λ, 4 bonds, and the reaction product may be added to the food. For example, epilactose can be produced as a reaction product by reacting the above polypeptide with lactose. As described above, this reaction product can improve the intestinal environment, promote mineral absorption, act as a lipid. Since it has functions such as metabolism improving action, low caloric properties, and also does not cause an increase in blood glucose level, the function of the reaction product can be obtained by adding the reaction product to other foods. Added functional foods can be produced. In addition, the reaction product, epilactose, has a lower sweetness than sucrose, so it can be used as a low-strength, low-sweetness sweetener to produce low-strength, low-sweetness foods while suppressing sweetness. Can.

[0124] The reaction product, epilactose, is a natural raw material containing lactose, especially livestock milk such as ushi, goat or hidge as raw or degreased. It can be produced by reacting with a polypeptide. It is also possible to react whey (milk) fraction collected from livestock milk and processed milk such as low-fat milk, low-protein milk, and non-fat ■ deproteinized milk or low-lactose milk with the above-mentioned polypeptides. Epilactose can be produced.

[0125] The present invention may be provided as a symbiotic that combines probiotics and prebiotics and a method for producing the same.

[0126] Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples, and various modifications may be made within the scope of the claims and the above embodiments. Embodiments that can be obtained by appropriately combining technical means disclosed in different embodiments are also included in the technical scope of the present invention.

Example

[Example 1: Isolation method and mycological properties of N E 1 strain]

[1. Strains isolation method]

Collected rumen contents from the force Nyure with bovine anaerobic diluent (M. P. B ryant and LA B urkey (1 953) J . D in airy S c 36, 205) 1 0 5 -fold with Dilute to The diluted solution was used as an inoculum. RGCA (described in American Type Culture Collection (ATCC))) A roll tube of medium was prepared. Colonies that appeared on the surface of the agar in the roll tube were inoculated into the RM_filter paper medium, and strains that disrupted the filter paper were selected. The composition of RM medium is shown in Table 1.

[0128] 1) Mineral solution I 75 ml

Mineral solution 11 ^b ) 75 ml

Park 卜 Peptone 2.0 g

Yeast extract 1.2 g

Lumen solution 300 ml

■ L-cystine hydrochloride. 0 0.5 g

Segubiose 10 g

0.1 ml resazurin solution 1 ml

8% Na ₂ C0 ₃ 50 ml

(Adjust to H 6.8) 1

a ⁾ Composition of mineral solution 1 4.5 g KH ₂ P0 ₄ / L

b ⁾ Mineral solution [Composition of I 4.5 g NaCI, 4.5 g (NH ₄ ) ₂ S0 ₄ , 0.1 g MnS0 ₄ -H ₂ 0,

_{_{0.33g CaCI 2 -2H 2 0, 0.512g}} MgS0 4 .7H,

0.1g FeS0 ₄ -7H _z 0, 0.1g ZnS0 ₄ -7H ₂ 0,

0.01 g CoCI ₂ -7H ₂ 0 / L

e ⁾ Carbon source is optional

[0129] [2. CE activity measurement]

As a qualitative assay, it is thought to coexist; Tris buffer (1 O OmM Tris—maleic acid buffer (pH 7. 8) ^ 0 UL 1 0 OmM cellobiose for the purpose of inhibiting S-glucosidase activity 5 L, enzyme solution 10 L) was used in the purification process. The reaction was performed at 30 ° C, the reaction time was adjusted according to the enzyme activity, and the reaction was stopped by boiling for 5 minutes. Reaction products were identified using thin-layer chromatography (TLC). After spotting the reaction solution 2.5 / and 0.5 mm thick glass plate Silicagel 60 F ₂₅₄ (Merck), the developing solution [2-propanol-butanol / H ₂ 0 = 7: 1: 2 , v / v]. After drying, the p_anisaldehyde solution was sprayed and heated in a hot plate or oven to develop color.

[0130] Quantitative measurement was performed according to the following method. The composition of the reaction solution is 100m MT ris _ maleate buffer (pH 7.0) 3.2mL, 10OmM lac! 6.2 mL (2 1 2 mg) of the enzyme and 1.6 mL (10.9 JI g) of enzyme solution were allowed to react at 30 ° C for 20 or 40 minutes. After stopping the reaction by boiling the sample for 5 minutes, ion exchange resin (AG 50 1 —X 8 resin, Bio_Rad) column, and a non-adsorbable fraction was obtained by centrifugation. 10 μL of this was analyzed by high performance liquid chromatography (column: Sh o dex Sugar SP 08 10 (Shodex), eluent: water, flow rate: 0.6 mL / min, column temperature: 80 ° C, detector: vaporized light scattering detector (Oltech)), and calculated the epilactose concentration in the reaction solution from the area of the peak derived from epilactose eluted with a retention time of approximately 17 minutes . The calibration curve was prepared using the reagent epilactose (Sigma). Enzyme reaction rate is calculated from the epicose generated between 20 min and 40 min after the start of the reaction, where the reaction proceeds linearly, 1 per minute; The reaction rate produced was defined as 1 unit (U). The specific activity of the enzyme was calculated using bovine serum albumin as a standard protein and expressed as U / mg.

[0131] [3. Mycological properties of N E 1 strain]

Among the obtained isolates, a bacterium having a high CE activity was selected and named N E 1 strain. This bacterium is an anaerobic bacterium, depending on the growth conditions, present alone, in double or short chain, and spherical or oval (0.3-1.5 X 0.7-1.8 m) . The bacterium was Gram positive and assimilated cellulose, cellobiose, xylan, lactose and glucose.

[0132] [4. Preparation of £ 1 strain chromosome 0-8]

After culturing R. a I bus N E 1 using RM-C medium, the culture supernatant was removed by centrifugation. The resulting precipitate was suspended in 5.67 7 mL of 10 mM Tris_HCI (containing 1 mM EDTA) (TE), 0.3 mL of 10% SDS and 9 L of 2 Omg / m Add L Proteinase K (T a K a R a) and mix 3 7. Incubated at C for 1 hour. Add 1.25 mL of 4M NaCI to the reaction mixture and mix gently to adjust the salt concentration to 0.7 M, then 0.8 1_ 10% cetyltrimethylammonium. Mbromide (cety I trimethy I a mm on ι umbromide / MN a

CI was added and incubated at 65 ° C for 10 minutes. 8 mL of black mouth form was added and mixed gently at room temperature for 30 minutes. 6 minutes at 6,000 rpm, chamber The aqueous layer was recovered by separating it into a water layer, an intermediate layer, and a phenol layer by centrifugal separation with temperature. An equal volume of phenol / black mouth form was added to the recovered aqueous layer and mixed gently at room temperature. Again, the aqueous layer was recovered by separating it into an aqueous layer, an intermediate layer, and a phenol layer by centrifugation at 6,000 rpm for 20 minutes at room temperature. An equal amount of 2_propanol was added to the collected aqueous layer and mixed gently to precipitate chromosome DNA. The precipitate was collected, rinsed with 70% ethanol, and then centrifuged at 12,000 rpm for 5 minutes at 4 ° C. The obtained chromosome DNA was dissolved in an appropriate amount of TE and stored at -20 ° C.

[5. Determination of 1 63 r DNA sequence of £ 1 strain]

Using the chromosomal DNA described above as a template DNA, the region encoding 16 S r DNA was PCR amplified using 27 f primer and 1 492 r primer. PCR was performed using TaKaRa PCR TymalCyCerD ice, and TaKaRa EX Taq was used as the DNA polymerase. The amplified fragments were separated by agarose gel electrophoresis and purified with G FX PCR DNA and Gel Band Purification Kit (Amersh am Biosciences). The purified fragment was ligated to p GEM—T easy (Prome ega) and transformed into E. coli XL 1-BI ue. This was applied to a 1_8 agar medium with an ampicillin concentration of 50 _§ / 1_ to form a single colony. The formed colonies were picked and cultured in a 1_8 liquid medium with an ampicillin concentration of 50 _§ / 1_. The culture was collected by centrifugation, and the plasmid was collected using QIA prep Spin Miniprep Kit (QI AG EN). Using the prepared plasmid as a template, use DTCS Quick Start

Sequence reaction was performed using M ix (B ec kman Co ulter), and C EQ8000 (B ec kman Co ulter) was used for sequence angle analysis. The composition of the reaction solution was in accordance with the manual, and the reaction time and temperature were determined according to the size of the target fragment or the primer used in the reaction. Primers used for sequence reaction and PCR (SEQ ID NOs: 5 to 1 1) Table 2 shows the list. The base sequence of the obtained 16 S rRNA is SEQ ID NO: 3. The nucleotide sequence shown in SEQ ID NO: 3 showed 99% homology with that of the standard strain (typestrain) R. a I bus ATCC 27210. In view of the above bacteriological properties, the £ 1 strain was identified as an aI bus.

[0134] [Table 2]

27f 5 '-agagtttgatcctggctcag-3'

1492r 5 '-ggctaccttgttacgactt-3'

529r 5 '-accgcggckgctggc-3'

786f 5 '-gattagataccctggtag-3'

926 r 5 '-ccgtcaattcctttragttt-3'

T7W 5 '-taatacgactcactatagggc-3'

SP6 5 '-atttaggtgacactatagaatactc-3'

[Example 2: Preparation of CE of R. a I bus N E 1]

[1. Culture method]

The R. a I bus N E 1 strain obtained in Example 1 was used. As a pre-culture liquid medium, 1% cereal biose was added to RM medium. The RGC medium modified from the RGC A medium described in the ATCC manual, the medium reported by Ty Ir et al. (Non-Patent Document 1), etc. can be used, but our medium can be prepared more easily.

In the case of pre-culture, after placing 2 OmL of the above medium in a pressure culture test tube (Sangent Industrial) and replacing the inside of the test tube with carbon dioxide gas using the anaerobic bacteria culture device AG-2 (Sangent Industrial) Sterilized. The cells in the frozen state were thawed on ice, then inoculated with 1 mL and cultured at 35 ° C for 24 hours.

[0136] In the case of main culture, 75 Om L and 375 mL of the above-mentioned medium are placed in 1 L or 50 Om L wide-mouthed medium bin (I WA KI), respectively, and an anaerobic culture apparatus AG_2 is used. Then, the inside of the medium bottle was replaced with carbon dioxide gas, and then sterilized. Inoculate the medium with 2 Om L and 1 Om L of preculture at 35 ° C, respectively. The culture was stationary for 1 week. In addition, carbon dioxide substitution was performed using an anaerobic bacteria culture apparatus A G — 2 at the time of inoculation. Usually, cellobiose or filter paper is appropriately used as the carbon source, so that CE is efficiently expressed in the cells. The culture solution obtained in the pre-culture was stored at −80 ° C. while being placed in a pressure culture test tube.

[0137] [2: Preparation of crude enzyme solution]

After culturing R. a I bus N E 1 for 1 week using the above medium, the cells were collected by centrifugation. 1 5 1_ 1 1 \ 1 Phenylmethanesulfonyl fluoride (PMSF) containing buffer A (10 OmM Tris-maleic acid (pH 7.0), 1 mM EDTA, 1 mM dithiothre!) After suspension, the cells were crushed with a French press. After disrupting the cells, centrifugation was performed at 12,000 rpm for 30 minutes, and the resulting supernatant was used as a crude enzyme solution. Ammonium sulfate was added to the obtained culture supernatant to 70% saturation, and the mixture was stirred gently and then centrifuged at 12,000 rpm for 30 minutes. The resulting precipitate was added to buffer A (2 OmM MES (pH 6.0) containing 1 mM PMS F,

1 mM EDTA, 1 mM dithiothre! The crude enzyme solution was dialyzed with Buffer A after being suspended in All operations after incubation were performed at 4 ° C.

[0138] [3. Purification of CE]

(1) Anion exchange chromatography

In the case of using DtAbSep h a r o s e l_—o B (Ame r s h am B i o s c i enc e s), the sample was dialyzed with buffer A and then applied to a carrier previously equilibrated with the same buffer. Elution was carried out with a linear concentration gradient increasing the Na C I concentration from 0 M to 0.6 M, and 100 mL fractions were collected in 4 mL aliquots.

[0139] Using RESOURCE Q (Ame rsh am Biosciences), FPL was performed, and System (Ame rsh am BIOSCIences) was used, and the same operation as above was performed. However, elution is performed with a linear concentration gradient that increases the Na CI concentration from 0 M to 0.5 M, and 0.5 100 mL fractions were collected in milliliters (flow rate: 0.5 mL / min).

[0140] (2) Hydrophobic interaction chromatography

Ammonium sulfate was added to the active fraction obtained by anion exchange chromatography (DEAE) to make it 50% saturated, and then it was subjected to R E S O U R S E ETH (Am er s s s c e nc es) using FPLC Sy s tem. The carrier was pre-equilibrated with buffer B (2 OmM MES (pH 6.0), 1 mM EDTA, 1 mM dithiothreyl I (DTT), 50% saturated ammonium sulfate). Elution was performed with a linear concentration gradient that decreased the ammonium sulfate concentration from 50% saturation to 0% saturation, and 100 mL fractions were collected in 1.5 mL portions (flow rate: 1.5 mL / min). The elution fraction was desalted using Microcon (MiiIipore) and then subjected to activity measurement and SDS_PAGE.

[0141] (3) Adsorption chromatography

The active fraction obtained by hydrophobic interaction chromatography was dialyzed against buffer C (5 mM phosphate buffer (pH 6.0), 1 mM EDTA, 1 mM dithiothreitol), and then the same buffer. The solution was applied to hydroxyapatite (Wako Pure Chemical Industries, Ltd.) that had been equilibrated with the solution. Washing after adsorption was performed using buffer D (5 mM phosphate buffer (pH 6.0), 1 mM EDTA, 1 mM dithiothre !, 0.5 M KCI). Elution was performed with a linear concentration gradient that increased the phosphate concentration from 5 mM to 20 OmM, and 100 mL each of 4 mL was collected.

[0142] (4) Gel filtration chromatography

The active fraction (4.5 mL) obtained by anion exchange chromatography (RESOURCE Q) was fractionated into five 0.9 mL portions. Each fraction was pre-equilibrated with buffer E (2 OmM MES (pH 6.0), 1 mM EDTA, 1 mM dithiosyl I, 0.1 MN a CI) using FP LC syst em. Superdex 200 HR 1 0/30 (Amersham Biosciences) was used. Elution with the same buffer 50 fractions of 250 L were collected (flow rate: 0.5 ml / min, collected 20 minutes after the start of elution).

[0143] [4. CE activity measurement]

CE activity was measured qualitatively and quantitatively according to the procedure described in Example 1.

[0144] [5. Protein quantification]

Proteins were quantified according to the Bradford method (M. M. Bradford, (1976) AnaI. Biochem. 72, 248). A calibration curve was prepared using bovine serum albumin.

[0145] [6. S DS— PAG E]

S DS—PAG E was performed according to the method of Laemmli (U. K. LaemmIi, (1970) Natre, 227, 680). 4. An 8% concentrated gel and a 10% separation gel were used, and electrophoresis was carried out at a constant current of 20 mA using MiniProteanIII (Bio-Rad) as the electrophoresis apparatus. The gel after electrophoresis was stained with S i m p l y B l e e s a f e S t a i n (I n v i t r o g e n) or 7 n i s i l — B e S t Sta i n f o r "r o te n / PAGE (Nacalai Tesque).

[0146] [7. Analysis of N-terminal amino acid sequence and nucleotide sequence]

The protein contained in the gel after SDS—PAGE was transferred to a P VDF membrane using T RAN S—BLOT SDS EM I—DRY T RAN SFERCELL (Bio-Rad). Blotted buffer to the transfer (2 5 mM T ris, 1 92 mM glycine, 0. 1% SDS, 20% methanol) using a 1 hour at a constant current of membrane 1 cm ² per 0. 8 m A I went. After staining the transferred membrane with Ponso_S, the target band was cut out and the N-terminal amino acid sequence was analyzed using a protein sequencer.

[0147] Submarine electrophoresis Mupid-2 (C o smo B io) is used for the agarose gel electrophoresis apparatus, and A garose L 03 (T a K a R a) is used for the agarose for electrophoresis. used. Sample solution contains dye for electrophoresis (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glyceride) is added 1/10 volume, and 1 XTAE (4 OmM Tris_HC I, 1 mM EDTA (pH 8.0)) was used. The gel after electrophoresis was immersed and stained in 1 X TAE to which ethimub mouth amide was added. A 1 Kb Plus DNA L adder (Invitrogen) was used for DNA molecular weight control. Recovery of various DNA fragments separated by electrophoresis from an agarose gel was performed using GEN EC LEAN III kit (BIO 1101) according to the manufacturer's instructions.

[8148] [8. Homology analysis of clone base sequence and amino acid sequence]

Genomic information of R. a l b u s 8 strain is stored in T IG R (h t p p

1 g r .o r g /). This gene is described as an unidentified gene. Analysis of nucleotide sequence using D RAW32 (A ca Clonesoft wa re) and homology using B LAST (SF Altschul et al., (1 990) J. Mo and B iol. 21 5, 403) I did a search. For the multiple alignment (alignment) of the base sequence and amino acid sequence, C I u s t a I W (J. D. T h omp s o n et al., (1 994) N c c and A c i ds R e s. 22, 4673) was used.

[0149] Cloning of the R. albus N E 1 gene and determination of the nucleotide sequence were performed according to the following method. Note that the method described below is an example, and does not limit the present invention.

[0150] First, based on the sequence of the unidentified gene from the chromosome of R. albus 8 strain, Primer_ (5, 1 GATGATGAGAACGGCGGCT TT— 3 '(SEQ ID NO: 1 2) and 5' -GCTTCCGCCTGCACCCACCAT A-3 '( SEQ ID NO: 1 3)) was constructed, and PCR was performed using the chromosomal DNA of R. albus NE 1 as a saddle. 95 ° C for 1 minute, 60 ° C for 30 seconds, 7

Thirty cycles of reaction at 2 ° C for 2 minutes were performed. Based on the reaction products obtained, the perfectly matched primers (5'-CGCT T AT ATGACACTGG GCG-3 '(SEQ ID NO: 1 4) and 5'-TCAGCCTGTCAGCT ACCTT C-3 '(SEQ ID NO: 15)) was constructed, and PCR was performed in the same cycle using PCRDIGL abeling MIX (Roche) to produce a probe labeled with DIG.

[0151] Chromosomal DNA of isolated R. albus N E 1 was digested with various restriction enzymes (Pst I, Pvu I I, E co R I, E co RV, B g I I I). Using 10 U of restriction enzyme per 3 g of chromosome DNA 3 7. After reacting with C for 12 hours, 10 U restriction enzyme was further added and allowed to react for 12 hours. The reaction solution was subjected to electrophoresis using a 0.8% agarose gel. The DNA was separated by electrophoresis at a constant voltage of 30 V for 4 hours. The isolated DNA was transferred to Hybond_N + (Amersham Biosciences), which is a Nai-Kan-Mu, using capillary action according to a conventional method, and the DNA was fixed to the Nai-Cann membrane by UV irradiation. .

[0152] For the hybridization and detection, A n t i — D i g o x i g e n i n -A P (R o c h e) was used. Hybridization was performed at 60 ° C for 16 hours using the labeled probe prepared by the above procedure and a nylon membrane. The probe was used at 10 ng / mL of the hybridization solution. The membrane was washed by shaking in 0.1% S DS, 0.1 X S S C solution at 68 ° C for 15 minutes, and the same operation was performed three times in total. Detection was performed according to the manufacturer's instructions, and C D P—S tar (R o c h e) was used as the luminescent substrate. The X-ray film was exposed to FUJIMEDICALLX_RAYFILM (FUJIFILM) for 1 minute.

[0153] 3 g of chromosomal DNA of R. albus NE 1 was extinguished with 10 U Eco RI, and the DNA was purified by phenol / chloroform extraction. After purification, ethanol precipitation was performed and dissolved in 4 L of TE, and self-ligation was performed at 4 ° C. The reaction solution after self-ligation is a vertical type and is designed based on the probe sequence (5 '-CGAGAAG GT AG CT GA CA GGCT GAAG TTC C-3' (SEQ ID NO: 1 6) and 5 '— C CA TCATC CAG TATACTC CA C CG TATT C- 3 '(SEQ ID NO: 1 7) ) Was used to perform inverse PCR. Thirty cycles of reaction were performed at 95 ° O for 1 minute, 55 ° C for 30 seconds, and 72 ° C for 4 minutes. By performing a sequencing reaction using a DNA fragment amplified by inverse PCR as a saddle shape, information on the base sequence in the vicinity including the CE gene was obtained. Based on the obtained sequence information, primers (5 '-CT CT TATAG T TGCACATATATAAT TGAGG-3' (SEQ ID NO: 1 8) and 5 '-TT CCATG TAG TT CT CCT TT CG G-3 ′ (SEQ ID NO: 19)) was prepared, and a DNA fragment obtained by amplifying the chromosomal DNA of R. albus NE 1 into a cage was subcloned into pT 7 Blue vector (N ovagen).

[0154] The base sequence was determined by the method of d angelo Xy of Sang ger et al. (F. Sang ger et al., (1 977) P ro c. Nat l. A c a d. S c i. U SA, 74, 5463). The DNA fragment amplified by PCR or the amplified fragment was subcloned into the multicloning site of pT7Bluevector, and the purified plasmid was used as a saddle to perform sequencing. For the anti-sequence J, we used B i g D y e T e r m i n a t o r v .1 C y c l e S e q u e n c i n g k i t ^ A p p I ι e d B i o s y s t em s). For the analysis, A B I P R I SM 3 10 e n e t i c A n a y z e r f o r W i indow s (registered trademark) (A p p I i e d B i o ys y s t em s) was used.

[0155] [9. Substrate specificity]

The substrate specificity of CE was analyzed qualitatively using TLC. The composition of the reaction solution was 100 mM Tris-maleic acid buffer (pH 7.0) 1 5 1_, 1 O OmM substrate 5 L, purified enzyme 5 L (34 ng) Reacted. After stopping the reaction by boiling the sample for 5 minutes, it was subjected to TLC (developing solvent system; 2_propanol-butanol / H ₂ 0 = 7: 1: 2).

[0156] [1 0. Analysis of reaction products] (1) When lactose is used as a substrate

The composition of the reaction mixture was 100 mM Tris _ maleate buffer (pH 7.0), 3.2 mL, 100 mM Lac! The reaction volume was 6.2 mL (2 12 mg) and the enzyme solution was 1.6 mL (10.9 IJ g). The reaction was stopped by boiling the sample for 5 minutes and the entire volume was subjected to TLC. The product was isolated by coloring a part of the TLC plate and measuring the mobility of the reaction product, and then scraping the silica gel. The obtained product was extracted with twice the amount of silica gel demineralized water, and then concentrated with a rotary evaporator (Tokyo Rika Kikai). Since this product contains a trace amount of lactose as a contaminant, the entire product was again subjected to TL C, and the reaction product was purified by the same operation. To the reaction product after purification, 50 M (2 62 g) was added with 8 M trifluoroacetic acid and then left to stand at 100 ° C. for 3 hours for complete hydrolysis. After replacing the solvent of this reaction solution with demineralized water using a centrifugal concentrator, the degradation product was qualitatively identified by subjecting the reactive product to TL C.

[0157] (2) When cellotriose is used as substrate

The composition of the reaction mixture was 4.1 mL of 10 O mM Tris_maleic acid buffer (pH 7.0) and 2.7 mL (1 36 mg) of 10 O mM cellotriose. After the reaction solution was separated and purified by the same procedure as above, the enzyme solution was adjusted to 1.8 mL (12.2 μg), and then the high resolution nuclear magnetic resonance (NMR) apparatus (BRUKER AM X ^-1 000 spectrometer (5 000 MHz)), ¹ H and ¹³ C spectra were measured ( ¹ H _ NMR and ¹³ C NMR).

[0158] [B. Results]

[1. Purification of CE]

A single protein was obtained electrophoretically by five-step kumatography (Fig. 3). The yield was about 0.2% (10.2 μg). When CE activity was measured with various buffer solutions (0.1 M, pH 7.8), the highest activity was found in glycylglycine mono-NaOH. The specific activity of the enzyme in this buffer solution is 3 6 U / mg.

[0159] The molecular weight of the purified enzyme estimated from S DS_P AG E was about 43.1 kDa. The molecular extinction coefficient of the purified enzyme (E value, absorbance at 280 nm with 1% enzyme solution) was about 18 (when bovine serum albumin was used as the standard protein). The maximum enzyme activity is at pH 7.7 to 8.2 (in 40 mM Britton-Robinson buffer), and the maximum enzyme activity is 28 to 32 ° C (4 OmM Tris_malein). Acid buffer). Enzyme activity is Fe ^{3 +} , Co ^{2 +} , Cu ^{2 +} , Z n ^{2 +} , P b ^{2 +} , Ag +, N-bromosuccinide, thioacetic acid, p-chloropolymer benzoate ( 1 mM each) (when the enzyme was pretreated at 30 ° C for 30 minutes).

[0160] (Table 3: CE purification process)

[0161] [Table 3]

[0162] [2. Analysis of amino acid sequence at the heel terminal and substrate specificity]

The purified protein was subjected to SDS-PAGE, transferred to a PVDF membrane, and the target band was cut out. The Ν terminal sequence of the target protein was decoded. The obtained heel end sequence was MMISEIRQE LTDHIPIFPFWNKRD (SEQ ID NO: 4). Although amino acid sequences having similarities were searched by BLAST, amino acid sequences showing high homology were not obtained (data not shown).

[0163] Therefore, a search for an amino acid sequence having similarity to this sequence in the genomic database TIGR of R. a I bus 8 strain, whose genome sequence was partially clarified, revealed that out of 23 residues. 1 A sequence MMKEEVKQE LTS HIIP FWN KLRD (SEQ ID NO: 20) was found that matched 8 residues. This protein listed in the genome database of R. a I bus 8 strain is Bacteroide Bacteroidesfragi I is YCH 4 6, GI c NA c _ 2 _ epi from Microburbiferdegradans 2-40 and amino acid sequences at 40% and 37 o / o, respectively Was consistent. GI c NA c-2-epi is an enzyme that isomerizes the hydroxyl group at the 2-position of acetyl glucosamine to produce N-acetyl methyl nosamine.

[0164] Therefore, the enzyme of the present invention was measured by thin layer chromatography (TLC) for GI c NA c-2-epi activity using acetyl glucosamine, which is a substrate of GI c NA c _2 epi. As a result of confirmation, no reaction product was obtained. In addition, in order to examine whether or not the epimerase activity has been reported so far, the enzyme of the present invention is used in reactions using UDP-glucose, glucose_6_phosphate and various monosaccharides as substrates. However, no reaction product was obtained in any reaction.

[0165] Enzymes of the present invention can be converted into typical disaccharides such as maltose (1-1, 4), sucrose, sophorose (β_1, 2), laminobiosis (S_1, 3). ) And gentiobiose (β-, 6) as a substrate, no reaction product was obtained. Surprisingly, however, when the enzyme of the present invention was subjected to a reaction using lactose (yS — 1, 4) as a substrate, spots other than the substrate were detected as reaction products. To date, no epimerase has been known that produces a terminal oligosaccharide using lactose as a substrate. Further, when the enzyme of the present invention was subjected to the reaction with at least cellobiose, cellotriose and cellotetraose, spots other than the substrate were detected as reaction products (FIG. 4).

[0166] Table 4 summarizes the above results, and the results of using cellopentaose and cepoxahexaose as substrates. From these results, it was clarified that this enzyme is a novel epimerase enzyme having a substrate specificity completely different from the epimerase reported so far. In addition, the reactivity of various sugars suggested that this enzyme may act only on oligosaccharides having S-1, 4 bonds. [01 67] Also, TLC confirmed that the products of epilactose or GI c _ Man as a substrate were lactose or cellobiose, respectively (not shown) . This result shows that the CE according to the present invention performs a reversible catalytic reaction.

[01 68] (Table 4: Substrate specificity of purified protein)

[01 69] [Table 4]

Substrate Product

/ V-acetyl-D-Darkosamine ND

UDP-glucose ND

Glucose-6-phosphate ND

Glucose ND

Mannose ND

Fluk I ND

Garaku 卜 ND

Xylose ND

Arabinose ND

Sophorose ND

Laminaribiose ND

Gentiobiose ND

Rakutosu D

Marutous ND

Sucrose ND

Isomare! Su ND

Cellobiose D

Cerro Triose D

Cellotetraose D

Cellopentaose D

Seguchi Hexaose D

ND: Not detected

D: Detected [0170] [3. Identification of reaction products]

From the analysis of the substrate specificity described above, it has been clarified that, in addition to cellobiose, at least cello-oligosaccharide and lactose can be substrates for the above-mentioned novel enzyme. This suggests that a novel oligosaccharide that has never been reported has been synthesized.

[0171] Therefore, the structure of the resulting oligosaccharide was analyzed. Specifically, an enzymatic reaction was performed using lactose as a substrate, the reaction products were separated by TL C, and the product was isolated by scraping the silica gel. This product was hydrolyzed with trifluoroacetic acid, and the degradation product was detected by TLC. As a result, it was observed that the product was galactose and mannose. From this, this enzyme is similar to cellobiose; by using lactose having 4 bonds as a substrate and converting reduced terminal glucose to mannose, a novel oligosaccharide; S-4 galactosylmannose It was thought that it was generated.

[Example 3: Structural analysis of reaction product by N MR]

The product from lactose was purified according to the procedure described in Example 2. The sample after extraction was concentrated, and the solvent was replaced with heavy water using a rotary evaporator, followed by ¹ H-NMR and ¹³ C-NMR, and ¹ H and ¹³ C spectra were measured. In addition, TSP ([2, 2, 3, 3-D 4] sod I um 3—i — (trimethy I s I I I propanoate) was used as an external standard. (Fig. 5).

[0173] The composition of the reaction mixture when cellotriose was used as a substrate was 4.1 mL of 100 mM Tris-maleic acid buffer (pH 7.0) and 2 of 10 OmM cell triotriose. 7 mL (1 36 mg) and the enzyme solution were 1.8 mL (1 2 g), and reacted at 25 ° C for a while. The reaction was stopped by boiling for 5 minutes, and the entire amount was subjected to TLC. After confirming the formation of a reaction product by coloring a part of the TLC plate, the silica gel was scraped off and the product was isolated. After extraction with deionized water twice the amount of silica gel, the mixture was concentrated on a rotary evaporator. This sample is Since it contained a small amount of cellotriose, the whole amount was again subjected to TLC and purified by the same operation. The sample after extraction was concentrated, and the solvent was replaced with heavy water using a single evaporator. The sample was then subjected to ¹ H NMR and ¹³ C NMR. TSP was used as an external standard. The obtained spectrum is a cell wall polysaccharide-derived glucopyranosyl 1, 4 _0_; S _ D_ glucopyranosyl 1, 4— 0— S— D— mannose (GI c -GI c— Man (R. G oldberg et al., (1 9 9 1) Carbohydr. Res. 2 1 0, 2 6 3) (Fig. 6, Fig. 7)).

[Example 4: Preparation of recombinant enzyme]

The amino acid sequence and base sequence of the mature CE enzyme of the N E 1 strain were found to be the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2. The N-terminal amino acid sequence (SEQ ID NO: 4) obtained by a previous experiment was completely identical to that of the amino acid sequence shown in SEQ ID NO: 1. Based on the amino acid sequence shown in SEQ ID NO: 1, the molecular weight of CE was 45, 2 17 Da.

[0175] A polynucleotide encoding the CE enzyme (polypeptide) (having the base sequence shown in SEQ ID NO: 2) was expressed by the following method.

[0176] [1. Construction of expression plasmid]

Genomic DNA was used as a cage, and the entire CE gene was amplified by PCR. 9 5 C for 1 minute, 55. C for 30 seconds, 7 2. The reaction for 2 minutes at C was performed 30 cycles. The DNA fragment was recovered from the reaction solution, and after confirming that no mutation was introduced into the subcloned amplified fragment, it was inserted into the E. coli expression vector pET-23a (+) vector.

[0177] [2. Induction of enzyme]

The host Escherichiacoli BL2 1 (DE 3) was transformed with the expression plasmid and then selected on LB agar medium supplemented with 100 g / mL ampicillin at the final concentration. Incubated at 37 ° C for 1 hour. Inoculate 200 mL of the same liquid medium in a 50 Om L Erlenmeyer flask with 4 mL of the pre-culture solution (total 400 mL) and shake culture at 200 rpm at 37 ° C. Nourished. When the absorbance at 600 nm reached 0.6, IPTG was added to a final concentration of 1 mM to induce expression of the target protein. All operations after incubation were performed at 4 ° C.

[0178] [3. Disruption of bacterial cells]

6, Bacteria were recovered from 4 O OmL of E. coli culture solution by centrifugation at O O O rpm for 20 minutes. After suspending the cells in buffer solution A (10 OmM Tris-maleic acid (pH 7.0), 1 mM EDTA 1 mM dithiothre!) Containing 15 mL of 1 mM PMSF, French It was crushed by a press. After disruption of the cells, the mixture was centrifuged at 12, O O O rpm for 30 minutes, and the resulting supernatant was used as a crude enzyme solution.

[0179] [4. Purification of recombinant enzyme]

1) Anion exchange chromatography

The crude enzyme solution obtained in the above-described Examples was subjected to Q—Sep h a ros e Fa s t ι- I ow (Ame r s h am B i os c i nc e s) previously equilibrated with buffer A. Elution was performed with an a CI linear concentration gradient of OM 0.51 \! And collected in 4 1_100 fractions.

[0180] 2) Adsorption chromatography

The active fraction obtained by anion exchange chromatography was dialyzed against buffer B (5 mM phosphate buffer (pH 6.0), 1 mM EDTA, 1 mM dithiothreitol), and equilibrated with the same buffer in advance. Washed after adsorption, buffer C (5 mM phosphate buffer (pH 6.0), 1 mM EDTA 1 mM dithiothresyl I, 0.5 M KC) I) was used. Elution was performed with a linear phosphate gradient of 5 mM 20 OmM and collected in 41 1 to 100 fractions.

[0181] 3) Purity of recombinant enzyme

The recombinant enzyme obtained by purification gave a single band with SDS_PAGE. 20 mg of a purified sample with a yield of about 70% and a specific activity of about 10 Ug was obtained. [0182] 4) Enzymatic properties of recombinant enzymes

When tested using the above-mentioned experimental method of enzymatic chemistry, this recombinant CE was almost the same as CE derived from R. albus N E1.

[Example 5: Utilization of epilactose by bifidobacteria]

According to the following procedure, Bifidobacteria were cultured in a medium containing epilactose (Table 5), and a growth test was conducted. The Bifidobacteria strains tested were Bifidobacterium bifidum J CM 1 255, B. breve J CM 1 1 92, Bifidobacteria B. longum J CM 1 2 1 7 B. adolescentis J CM 1 275, B. infantis JCM 1 222, Bifidobacteria catenatum (B catenulat um) J CM 1 1 94.

1. Considering that the medium is diluted by adding the test sugar solution, the concentration of the medium was adjusted to several times.

2. The test sugar solution was sterilized by filtration and then added to the medium (final concentration 1%).

3. 1% of the medium was inoculated with the bacterial solution of the test strain pre-cultured in a medium containing 1% glucose. 4. Cultivation was carried out by the gas pack method using Aneropackenki (Anaeropackekenki; Mitsubishi Gas Chemical). The cells were cultured at 37 ° C for 1 to 3 days under anaerobic conditions, and the assimilation of the test strains was examined.

[0184] (Table 5. Composition of epilactose assimilation test medium)

[0185] 5) Pactever leachate '* 1000 ml

Proteo 1 Spepton No.3 10 g

Tripty case (BBL) 5 g

Yeast extract 3 g

Tween 80 1 g

Saline solution ² * 5 ml

L-Sistine 'HGI'H ₂ 0 0.2 g

¹ * 1050 g of purified water added to 5.5 g of pactever leachate, leached for about 1 and a half hours with occasional stirring in a 50-60 ° C warm bath, and then filtered through filter paper

² * MgS0 ₄ -7H ₂ 0 10g, FeS0 ₄ -7H ₂ 0 0.5g, NaCI 0.5g, nS0 ₄ 0.337g

Dissolved in 250m I purified water

[0186] Table 6 shows the growth of epilactose. It was found that any bifidobacteria can use epilactose.

[0187] (Table 6. Epilactose assimilation ability of bifidobacteria)

[0188] [Table 6]

Co-test B. bifidum B. breve B. longum B. adolescentis B. inTantis B. eaten ulatum Test ffi ^^ JCM1255 JC 1192 JCM1217 JCM1275 JCM1222 JCM1194 Epilactosis + + + + + + + + + + +

[Example 6: Indigestibility of epilactose]

The digestive tolerance of epilactose in the stomach and intestine was examined by the following method.

[0190] The Japanese Pharmacopoeia Disintegration Test 1st liquid (artificial gastric juice) was used to examine digestive tolerance in the stomach. That is, artificial gastric juice (0.4% sodium chloride, 1.4% concentrated hydrochloric acid) 25 1_ of 2 times concentration is added to 25 L of 1 O OmM epilactose aqueous solution, and then left at 37 ° C for 2 hours. did. After neutralizing the above solution by adding 100 mM sodium hydroxide, the solution was applied to AG501_XB resin, and a non-adsorbing fraction was obtained by centrifugation. Remaining epilactose in the same way as quantitative measurement of CE activity Quantitatively. Digestion resistance was examined for lactose and sucrose as well as for epilactose.

[0191] Intestinal resistance to digestion was examined using rat small intestine crude enzyme solution prepared according to the method of T. Mishima et al. (丄 Agri c. Food Chem. 53, 7257 (2005)). It was. Specifically, 25 L of rat small intestine crude enzyme solution was added to 25 L of 1 O OmM epilactose aqueous solution, and then allowed to stand at 37 ° C for 3 hours. The solution was boiled for 5 minutes to inactivate the contained enzyme, and then subjected to AG 50 1 —X B resin, and a non-adsorbing fraction was obtained by centrifugation. Remaining epilactose was quantified in the same manner as the quantitative measurement of CE activity. The digestibility of lactose and sucrose as well as epilactose was examined.

[0192] Table 7 shows the digestive resistance of epilactose, lactose and sucrose in the stomach and intestine. Epilactose was confirmed to have high stability against digestive juices in the stomach and intestine.

[0193] [Table 7]

Survival rate ")

Stomach Intestine Epilactose 104 82

Raku Shinichi 101 54

Sucrose 102 3

[Example 7: Promotion of mineral absorption by epilactose]

Caco_2 cells are epithelial cell lines established from human colon cancer tissue and cultured on flasks and permeable membranes to form a polar monolayer. Caco_2 cells can be separated into small intestinal epithelial cells without the use of differentiation-inducing agents by culturing for about 2 to 3 weeks in a medium supplemented with 10 to 15% urine fetal serum. Turn into. TJ is formed between cells, and microvilli are formed on the brush border membrane side of the cells. And some minerals are absorbed through this TJ. Rats were used; the addition of indigestible sugars, which had been shown to promote Ca ²⁺ absorption in n V ivo experiments, increased C a ²⁺ absorption. It has been suggested that this Ca absorption promoting action is mediated by TJ (T. Suzuki and H. Hara, (2004) J. Nutr. 1 34, 1 935).

[0195] Therefore, difract, a kind of indigestible saccharide having C a ²⁺ absorption promoting action.

The effect of the epilactice on T J was examined using the triad anhydride (d i _D_f r u c t o_ f u r a n o s e 1, 2 ': 2, 3' d i a n h y d r i d e; DFA I I I) as a control.

[0196] A human colon cancer-derived cell line C aco_2 (HTB 37, passage number 19) was purchased from AT CC. 10 OmL / L urine fetal serum (inactivated, T emo T race), 44 mM Na ₂ C0 ₃ , 1 mM Na—hyzorenoic acid, 50,000 IU / L penicillin, and 5 Dulbecco's modified Eag Ieme dium (Invitrogen) supplemented with Omg / L streptomycin sulfate is used as the medium. The aco_2 cells were cultured at 37 ° C. in the presence of 5% carbon dioxide and 95% air in a 75 cm ² cell culture flask (Corning). In this experiment, Caco_2 cells with passage number 39 were used.

[0197] The evaluation of the effect on the intercellular permeability by the indigestible saccharide added to the brush border membrane side is as follows: (Π) Hanksbalancedsaltsolut ion (HBSS, 1 37 mM Na CI, 5.4 mM KC I, 4. 2 mM N a HC0 3, 3. 4 mM N a 2 H P0 4, 4. 4 mM KH 2 P 0 4, 1. 25 mM Ca CI 2, 0. 41 mM Mg S0 4, 0. _{49 mM M g CI 2, 1} OmM H EP ES, 1 OmM D- glucose, 4 mM L - glutamine (p H 7. 4)), and (2) the D FA III or Epiraku preparative Ichisu (HBSS for 1 M Dissolution) The solution was used.

[0198] Transwell System (T ranswe II; diameter 12 mm, Poasa 0.4, culture area 1.0 cm ² (Corning) was used. Intercellular permeability was evaluated by measuring transepithelial electrical resistance (T ER) according to the following method.

1. C aco — 2 cells seeded in T ran swel I at a density of 0.63 × 10 ⁴ eel I s / cm ² were cultured for 25-26 days.

2. After rinsing the brush border membrane side and basement membrane side of Tran swel I cultured with C aco-2 cells twice with HBSS, each 0.5 1_powder 1. OmL of H Filled with BS S.

3. C0 ₂ incubator within one (5% carbon dioxide and 95% hands in the presence of air 37 ° C) for 30 minutes after standing was measured TER as a measure of 0 min.

4. When evaluating the effects of the addition of indigestible saccharides from the brush border side, the final concentration should be 0, 20, 40, and 80 mM for each indigestible saccharide on the brush border side. Added to the solution.

[0199] As a result, as shown in Figure 8, Epirak! Depending on the concentration of TER or D FA III added to Ca co-2, TER decreases, and after 3 hours of 80 mM addition, epilac! Even when either DFA III or DFA III was added, the amount dropped to around 5000 cm ² .

[0200] Further, after measuring TER 3 hours after addition of the hardly digestible saccharide, each difficultly digested saccharide was removed and replaced with a culture medium. The TER at 24 hours after the medium change was restored to the initial value of 1 200-1400 Ωcm ² for both Epilac I and D FA III.

[0201] Thus, Epilactose showed the same action as DFA III, which has a mineral absorption promoting action on TJ. These results indicate the possibility that Epiraku bets one scan has a mineral absorption promoting effects, including C a ² +.

[0202] [Example 8: Promotion of mineral absorption to rattle]

The effect of epilactose on mineral absorption in rats was examined.

[0203] (1) Four-week-old Wistar-ST male rats (Japan SLC) were raised individually in stainless steel cages and freely fed food and water (ion exchange water) I let you. Body weight and food intake were measured every morning at the same time. The state of the animal individual was determined using hair loss and / or diarrhea as an index. The test feed was changed every day and the drinking water was changed every 3 days. The breeding room was set to room temperature 23 ± 1 ° C, temperature around 60%, and light / dark cycle 12 hours (light period 8:00 to 20:00, dark period 20:00 to 8:00:00). After 5 days of breeding with AIN 93 G standard purified feed (basic diet), the animals were reared for 15 days with the test diet shown in Table 8 (foods supplemented with epilactose, lactose supplemented diet) or control diet.

[0204] [Table 8] Feed

Composition

Control Epi-Lacose Lac I ^ I added

Food additive food

g / kg

Casein 200.000 200.000 200.000 Sucrose 644.486 599.486 599.486 Epilac I -Ice 50.000

Lactose 5.000 50.000 Corn oil 50.000 50.000 50.000 Crystalline cellulose 50.000 50.000 50.000 Mineral mix (AIN93G-MX) 35.000 35.000 35.000 Vitamin mix (AIN93G-VMX) 10.000 10.000 10.000 Cystine 3.000 3.000 3.000 Choline bitartrate 2.500 2.500 2.500 t -Ptylhydroquinone 0.014 0.014 0.014

[0205] Breeding 1 Feces collected for 4 days from the second day, freeze-dried, pulverized, about 1 g dry ashed (550 ° C, 18 hours), dissolved in 3% hydrochloric acid solution Minerals were measured by the atomic absorption method (polarized Zeman atomic absorption spectrometer, HI TACH I). The same operation was performed on 1 g of feed. From the amount of minerals obtained, the absorption rate of minerals for 4 days was determined.

[0206] As shown in Fig. 9, it was confirmed that the absorption rate of C a ²⁺ , Mg ^ +, and Z n ²⁺ increased with the intake of epilactose.

[0207] (2) 1 After 5 days of breeding, dissect the rat and remove the small intestine. After rinsing out the contents of the small intestine with water, three segments of about 3 cm were made from the jejunum and ileum, and the front and back sides were reversed. One end of the inverted segment was ligated with suture silk (special suture silk No. 4, Hashimoto). The other end is the internal solution (125 mM NaCI, 4 mM KCI, 1.25 mM CaCI2 2 H20, 3 OmM Tris, 1 OmM D_glucose, pH 7.4) After injecting 0.7 mL with a syringe, it was similarly purpled to create an inverted small intestine sac.

[0208] The prepared reverse sac contains Ca 2 ⁺ and epilactose 0, 50, and 100 mM, respectively (1 25 mM NaCI, 4 mM KCI, 10 mM CaCI 2 2H 2 O 3 OmM Tris, 10 mM D—glucose, pH 7.4, osmotic pressure adjusted by Na CI) 1 Transfer to a 5 Om L centrifuge tube containing 5 mL Shake on a water bath at 37 ° C, 1 10 osci II ation / min. After shaking for 30 minutes, the internal solution in the inverted sac was collected, and the Ca ²⁺ concentration was measured with a commercially available measurement kit (Calcium C Test Koichi, Wako Pure Chemical Industries). In addition, the length of the small intestine reversal sack was measured with a ruler. The results are shown in FIG.

[0209] The vertical axis in Fig. 10 represents the amount of C a ²⁺ absorbed per length (cm) of the small intestine used for the sac. Depending on the amount of epilactose added, the Ca ²⁺ concentration in the internal fluid collected from both the jejunum and ileum increased. From this, it was confirmed that epilactose has a function of promoting C a ²⁺ absorption not only in the large intestine but also in the jejunum and ileum.

[0210] [Example 9: Epilactose improves intestinal environment]

[0211] Rats were raised for 15 days under the conditions of Example 8, and then laparotomized under ether anesthesia. Both ends of the cecum were ligated with sutures, the cecum containing the contents was removed, and the total cecal weight was measured. After removing the cecal contents, the cecal wall weight was measured. The value obtained by subtracting the cecal wall weight from the total cecal weight was taken as the cecal content weight. A 4-fold amount of deionized water was added to a portion of the cecum contents (diluted 5 times) and homogenized under ice-cooling. A portion of the homogenizer was centrifuged, and the pH of the supernatant was measured. In addition, add crotonic acid as an internal standard to the whole homogen, centrifuge and collect the upper layer. Add the black mouth form, mix, centrifuge, collect the upper layer again, and pass the filter to make the sample for organic acid analysis. Using the Shimadzu high performance liquid chromatograph organic acid analysis system, the following separation and detection conditions The analysis was performed.

[0212] Separation conditions

Column: S h i m p a k S CR— 1 02 H (8 mm I. D. x 300 mm)

Mobile phase: 5mM p_Toluenesulfonic acid aqueous solution

Flow Mi: 0.8 m L / m i n

Temperature: 40 ° C

Detection condition

Reagent: 20 mM Bis-Tris aqueous solution containing 5 mM p-toluenesulfonic acid aqueous solution and 100 M EDTA

Flow Mi: 0.8 mL / m i n

Detector: CD D-6A

Temperature: 45 ° C

[0213] In addition, a portion of the cecum content was anaerobic dilution buffer (20 g / LB ufferedpeptonewater (D ι fco), 0.5 g / L-Sing Inn (Wako Pure Chemical Industries), 1 mL / L Tw e _en 80 (Wako Pure Chemical Industries) was suspended in 1 g / LB actoagar (D ifco )), it was further diluted it with dilution buffer scratch. To count the total number of anaerobic bacteria, BL agar medium (Eiken Chemical) supplemented with 50 ml L / L horse defibrinated blood (Nippon Biotest) was used, and the number of lactic acid bacteria was 8 g / L L. LBS agar (Difco) supplemented with abl em cop owd er (O xoid) and 1.3 ml / L acetic acid was used. After culturing the plate coated with the cecal content dilution in Aneropackenki (Mitsubishi Gas Chemical) at 37 ° C for 24 hours, the number of colonies was counted.

[0214] DNA was extracted from a part of the cecal contents using Fecal DNA I solation Kit (MO Bio). Using Sma rt Cycler II (C epheid), the number of bifidobacteria was determined by realtime PCR. The counting was performed under the following conditions. The composition of the reaction solution is 200 n Mg B ifif-F, 200 n Mg B ifif _ R, 1 x SYBRP remix E x T aq (Takara Bio), initial denaturation 9 5 ° C, after 30 seconds, 9 6 4 at 5 ° C for 5 seconds. C for 1 5 seconds, 7 2. C for 15 cycles of 15 seconds.

[0215] For statistical analysis, one-way AN OVA was performed, and when the P value was 0.05 or less, a significant difference between the mean values was calculated using Duncans Multiple Range Γ est. Judged.

[0216] Table 9 shows the total cecal weight, cecal wall weight, cecal content weight, and pH of the cecal content, and Table 10 shows the organic acid content in the cecal content.

[0217] [Table 9] Control Epilactose-added lactose P-value

Food Additive Additive

Total cecal weight 1.06 ± 0.09 2.62 ± 0.10 1.27 ± 0.09 <0.0001

(g / 100g body weight)

Cecal wall weight 0.23 Sat 0.01 0.38 ± 0.02 0.24 ± 0.01 + 0.0001

(g / 100g body weight)

Cecal content weight 1.76 ± 0.17 4.82 ± 0.20 2.20 ± 0.19 <0.0001

(g)

Cecal contents pH 7.60 ± 0.04 6.62 ± 0.18 7.52 ± 0.07 0.0002

[0218] [Table 10] Control diet Epilactose Lactose P-value

Additive food additive food

Content (mmol / cecal content)

Acetic acid 88.3 ± 6.9 275 ± 19 99.1 ± 9.3 <0.0001 Propic acid 32.3 ± 3.2 126.6 ± 8.3 41.8 ± 4.6 <0.0001 Butyric acid 17.1 ± 1.3 46.2 ± 7.7 36.6 + 4.0 0.0008 Total short chain fatty acids 138 ± 11 447 ± 31 178 ± 16 <0.0001 Succinic acid 1.52 ± 0.43 185 ± 27 11.51 ± 5.86 <0.0001 Lactic acid 0.742 ± 0,167 44.9 ± 30.6 3.04 ± 1.61 0.1792

[0219] As shown in Table 9, the content of the cecal content was increased by the intake of epilactose-added feed. The amount and cecal wall weight increased significantly compared to the group that received the control diet. Since an increase in the content of the cecum means an increase in the number of defecations, epilactose is expected to have an effect of improving constipation.

[0220] Also, as shown in Table 10, regarding the amount of organic acids in the cecum contents, the amount of short-chain fatty acids significantly increased in the epilactose intake group, and the total amount of short-chain fatty acids in the control group 3.2 times as much. Furthermore, the amount of succinic acid was 120 times that of the control group. As the amount of organic acid increased, pH in the cecal contents decreased in the epilactose intake group.

[0221] The total anaerobic bacterial count in the cecum (I o g 10 C FU / g contents) was compared to the control diet group 9.4 ± 0.2, epilac! In the dietary supplement group, there was an increase of 10.2 ± 0.2. The number of lactic acid bacteria (I o g 10 C FU / g content) is as follows: In the dietary supplement group, it was 9.1 ± 0.1, and increased by about 10 times as a result of intake of the diet supplemented with epilactose. In addition, the number of bifidobacteria (I o g 10 copy number / g content) is epilac against the control diet group 5.6 ± 0.3! It was 7.8 ± 0.3 in the dietary supplement group, and increased by about 150 times due to the intake of epilactos supplemented diet.

[0222] In this way, it was concluded that good bacteria such as bifidobacteria and lactic acid bacteria were increased by the feed containing epilactose, and it was concluded that epicactus fermentation was advanced by these bacteria, The effectiveness as a take was shown.

[0223] [Example 10: Epilactose improves lipid metabolism]

Four-week-old male WIStar-ST rats (Japan SLC) were housed individually in stainless cages and allowed to freely feed and feed (ion exchange water). Body weight and food intake were measured every morning at the same time. The state of the animal individual was judged using hair loss and / or diarrhea as an index. The test feed was changed every day and the drinking water was changed every 3 days. The breeding room was set to room temperature 23 ± 1 ° C, temperature around 60%, and light / dark period to 12 hours (light period 8:00 to 20:00, dark period 20:00 to 8:00). After raising with AIN 93G standard refined feed (basic diet) for 5 days, They were bred for 15 days on the test feeds shown in Table 8 (epi-lactose added diet, lactose-added diet) or control diet.

[0224] Blood was collected from the tail vein at 7 o'clock, 7 days later, 14 days later at 9 o'clock (at the time of feeding) and 20:00 (at the time of fasting), and centrifuged at 4 ° C, 3000 rpm for 10 minutes. Plasma, neutral lipid, total cholesterol, HD L cholesterol, choline-type phospholipid, TG-EN Kainos (Kainos Co., Ltd.), cholesterol E-test ヮ koichi (Wako Pure Chemical Industries), HD L, Cholesterol Test Measured using KOKOI (Wako Pure Chemical Industries, PL-EN Kainos Co., Ltd., Kainos Co., Ltd.) (Figure 1 1) LDL + VLDL cholesterol is more HDL cholesterol than total cholesterol In addition, after 15 days of breeding, the rats were dissected and abdominal aortic blood was collected and centrifuged at 4 ° C, 3000 rpm for 10 minutes, and plasma was similarly obtained. Medium neutral lipid, total cholesterol, HD L cholesterol (Figure 1 2) In addition, for statistical analysis, one-way ANOVA was performed, and when the P-value power was below 0.05, the Duncans Multiple Range Test was performed. ¾r was used to determine the significant difference between the mean values.

[0225] As a result, changes in the concentrations of neutral finger and phospholipids were not confirmed, but total cholesterol and LD L + V LD L cholesterol levels were lower in the epilac I supplemented food group than in the control group It was confirmed that

[0226] [Example 11: Change in blood glucose level during epilactose consumption]

After 7-week-old female d d Y mice (Japan SLC, 6 mice per group) were fasted, Lax I or Epilac! (0.1 mmo I / animal) were orally administered, blood was collected from the tail vein at 0, 30, 60, 120 minutes after administration, and the blood glucose level was measured using a glucose CI test. The results are shown in Figure 13.

As shown in Fig. 13, the blood glucose level significantly increased after administration in the lactose administration group and reached the highest level 30 minutes after administration, while no increase in blood glucose level was observed in the epilactose administration group. This result, together with the digestibility results shown in Table 7, shows that epilactose has a low caloric character. [0227] CE is an aldose 1-epimemerase in that it rearranges the configuration of the hydroxyl group bonded to the carbon atom at the 2-position of the reducing end glucose of cellobiose and catalyzes the reaction leading to glucosylmannose. (EC 5. 1. 3. 3) and Mal I Spi-Epimelase (EC 5. 1. 3. 2 1) are different. CE is very interesting from an enzymological point of view because it has a very unique enzymatic activity. However, elucidation of various enzyme chemistry properties of CE, acquisition of CE-encoding gene (CE gene), and analysis of amino acid sequence have never been done so far.

[0228] When CE derived from R. albus NE 1 is used, the present inventors produce GI c -Man using cellobiose as a substrate (Fig. 1), but also cellotriose and cellotetraose. Was found to be integrated into 2—epima (Fig. 4, Fig. 7). Furthermore, it has been found that when CE uses lactose as a substrate, it produces epi- lactose (Figs. 2 and 5).

[0229] Since many oligosaccharides have physiologically active functions, their functionality is also expected in epilactose, which is a reaction product of CE. However, at present, its physicochemical properties and physiological activities have not been clarified, and it has not been industrially used. In order to clarify the characteristics of epilactose, it is necessary to establish a practical mass synthesis method.

[0230] So far, it has been reported that lactic acid can be synthesized by inducing isomerization of lactose under high temperature and high pressure conditions, effective due to the presence of many contaminants. (J. F. Moreno et al., (20 03) J. Agri c. Food C he m. 5 1, 1 894).

[0231] In addition, the production of epilactose is possible by enzymatic synthesis using p-nitrophenylgalactose and mannose as substrates; and 3-galactosidase (M. Miy amo to and K. A jisaka (2004) BIOSCI. ι ι otechno I. B ioch em. D 8, 2086). This production method can also produce galactosyl mannose at different binding positions, and the target product can be easily isolated by subsequent enzymatic treatment. Practicality is higher than the manufacturing method described above. However, since p_nitrophenylgalactose is used as a substrate, the cost is high, which is not suitable for an industrial production method.

[0232] Compared to these production methods, the enzymatic synthesis method of epicase by CE is highly practical because of its low cost, simple reaction conditions, and few contaminants. It can be said that it is a synthesis method. However, it is practically difficult to prepare a large amount of this enzyme using R. a I bus which is a difficult-to-culture bacterium. From this point of view, it is very effective to isolate the CE gene and obtain a large amount of recombinant enzyme. Furthermore, since the physiological significance and metabolic mechanism of CE in R. a I b us has not been clarified, it also makes a great academic contribution.

Industrial applicability

[0233] By using the present invention, it is possible to synthesize novel oligosaccharides in addition to G lc _ Man, so in the food field, functional foods (for example, non-caloric or low-calorie foods or minerals) (Especially Ca ²⁺ ) (food with excellent absorption) can be provided at a low price. Since the present invention can develop foods or food additives that cause a decrease in blood cholesterol, it is also useful in the field of medicine / pharmaceuticals.

[0234] Since the present invention synthesizes a new oligosaccharide such as GIc_Man, particularly in the case of epilactose, in the food field, functional foods (for example, foods having an action to improve the intestinal environment, Non-caloric or low-calorie foods or minerals (especially foods with excellent absorption of C a ² +) can be provided at low prices. In addition, if the present invention is used, foods or food additives that cause a reduction in blood cholesterol can be developed, which is also useful in the pharmaceutical / pharmaceutical field.

Claims

The scope of the claims

[1] A polypeptide comprising an amino acid sequence of any one of (A) to (C), and having at least; an epimerase activity for oligosaccharides having 4 bonds:

(A) the amino acid sequence shown in SEQ ID NO: 1;

(B) an amino acid sequence in which one or several amino acids in the amino acid sequence represented by SEQ ID NO: 1 are substituted, added or deleted; or

(C) An amino acid sequence having 51% or more homology with the amino acid sequence represented by SEQ ID NO: 1.

[2] Polypeptide characterized by having the biological properties shown in (A) to (D): (A) It is derived from a bacterium belonging to the genus Ruminococcus;

(B) S DS—the apparent molecular weight by PAGE is 40-42 kDa;

(C) having the amino acid sequence shown in SEQ ID NO: 4 at the N-terminus;

(D) At least; has 2-epimerase activity for oligosaccharides having 4 bonds.

[3] A polynucleotide encoding the polypeptide according to claim 1 or 2.

[4] A polynucleotide comprising a nucleotide sequence of any one of (A) to (C), wherein the polynucleotide has an epimerase activity against oligosaccharides:

(A) the base sequence shown in SEQ ID NO: 2;

(B) a nucleotide sequence in which one or several nucleotides in the nucleotide sequence shown in SEQ ID NO: 2 are substituted, added or deleted; or

(C) A nucleotide sequence that hybridizes with a complementary sequence of the nucleotide sequence shown in SEQ ID NO: 2 under stringent conditions.

[5] A vector comprising the polynucleotide according to claim 3 or 4.

[6] A production method for producing a polypeptide having epimerase activity with respect to an oligosaccharide having a β-Λ, 4 bond, wherein the vector according to claim 5 is used.

[7] A kit for producing a polypeptide having an epimerase activity for an oligosaccharide having a β-Λ, 4 bond, comprising the vector according to claim 5.

[8] A transformant comprising the polynucleotide according to claim 3 or 4.

[9] The transformant according to claim 8, wherein the transformant is a bacterium belonging to the genus Ruminococcus.

[10] The transformant according to claim 9, wherein the transformant is Zoleminococcus azolevus (RuminococcusaIbus).

[11] A production method for producing a polypeptide having epimerase activity with respect to an oligosaccharide having a β-Λ, 4 bond, wherein the transformant according to claim 8 is used.

[12] A kit for producing a polypeptide having an epimerase activity with respect to an oligosaccharide having a β-Λ, 4 bond, comprising the transformant according to claim 8. kit.

[13] A key body that specifically binds to the polypeptide according to claim 1 or 2.

[14] A production method for producing a polypeptide having epimerase activity with respect to an oligosaccharide having a β-Λ, 4 bond, wherein the antibody according to claim 13 is used.

[15] A kit for producing a polypeptide having an epimerase activity for an oligosaccharide having a β-Λ, 4 bond, the kit comprising the antibody according to claim 13.

[16] A hetero-oligosaccharide comprising the step of reacting the polypeptide of claim 1 or 2 with an oligosaccharide having an S_1,4 bond; Method.

[17] A reagent kit for synthesizing a hetero-oligosaccharide comprising the polypeptide according to claim 1 or 2.

[18] The reagent kit according to claim 17, further comprising an oligosaccharide having a β-Λ, 4 bond.

[19] A reagent composition for synthesizing a hetero-oligosaccharide comprising the polypeptide according to claim 1 or 2.

[20] A method for producing a functional food comprising:

Reacting the polypeptide of claim 1 or 2 with an oligosaccharide having a β-Α, 4 bond; and

Adding reaction products to food

A method characterized by comprising.

[21] 4 _0_; S_ D_ Galactopyranosyl- A functional food for improving the intestinal environment, characterized by containing D-mannose.

[22] A claim for improving the intestinal environment by promoting the growth of bifidobacteria.

2 Functional food according to 1.

[23] 4 _0_; S_ D_Galactopyranosyl-Functional food for improving lipid metabolism, characterized by containing D-mannose.

[24] The functional food according to claim 23 for improving lipid metabolism by reducing blood cholesterol level.

[25] 4 _0_ S_ D_ Galactopyranosyl-D Functional food for promoting mineral absorption, characterized by containing D-mannose.

[26] 4 _0_ S_ D_ Galactopyranosyl-D Functional food for diabetic patients, characterized by containing D-mannose.

[27] 4 _0_; S_ D_Galactopyranosyl- A low-strength, low-sweetener characterized by containing D-mannose.

[28] 4 _0_ S_ D_ Galactopyranosyl mono-D-mannose is a constipation-improving agent.