Novel enzyme agent and method for producing β-glucobiose-containing carbohydrate

Abstract

To provide novel enzymatic agents capable of selectively producing gentiobiose.SOLUTION: Provided is an enzymatic agent comprising a protein selected from the group consisting of (a), (b), and (c) below: (a) a protein comprising a specific amino acid sequence; (b) a protein consisting of an amino acid sequence having 70% or more identity to a specific amino acid sequence and having β-glucosidase activity; and (c) a protein consisting of an amino acid sequence in which one or more amino acids are modified, in a specific amino acid sequence and having β-glucosidase activity.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a novel enzyme agent and a method for producing a β-glucosidic bond-containing carbohydrate using the enzyme agent.

Background Art

[0002] β-Glucosidase is an enzyme that acts on β-glucosidic bonds such as cellobiose and catalyzes the hydrolysis reaction of the β-glycosidic bond. On the other hand, in the presence of a high concentration of glucose, it is known that β-glucooligosaccharides are produced by a condensation reaction, which is the reverse reaction of the hydrolysis reaction.

[0003] Patent Document 1 describes a method for producing β-glucooligosaccharides by allowing β-glucosidase of microbial origin to act on glucose. Patent Document 2 describes a novel enzyme characterized by having specific physicochemical properties and a method for producing a gentiobiose-rich syrup by allowing the enzyme to act on a high-concentration glucose solution.

[0004] In addition to gentiobiose, the β-glucosidic bonds obtained by the above methods contain a certain amount of β-glucosidic bonds other than gentiobiose, such as laminaribiose and sophorose, and the content of gentiobiose was not sufficient. On the other hand, Non-Patent Document 1 reports that gentiobiose can be synthesized by subjecting glucose to a condensation reaction using β-glucosidase derived from Halobacillus halophilus ( Halobacillus halophilus ).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0006] [Non-Patent Document 1] Abstracts of the Annual Meeting of the Chemical Society of Japan 2018, Presentation Number: 2A26p05, "Functional Analysis of β-1,6-Glucosidase Derived from Halobacillus halophilus", Maki Terada, Naoto Iso [Summary of the Invention] [Problems to be Solved by the Invention]

[0007] An object of the present invention is to provide a novel enzyme agent capable of selectively producing gentiobiose and a novel method for producing a β-glucosidic disaccharide-containing carbohydrate using the enzyme agent. [Means for Solving the Problems]

[0008] The present inventors have now found that seven selected proteins have β-glucosidase activity, and have also found that β-glucosidic disaccharides with particularly high gentiobiose content can be produced using the enzyme. The present invention is based on these findings.

[0009] According to the present invention, the following inventions are provided. [1] An enzyme agent comprising a protein selected from the group consisting of the following (a), (b) and (c): (a) A protein comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 7, (b) A protein consisting of an amino acid sequence having 70% or more identity to any one of the amino acid sequences of SEQ ID NOs: 1 to 7 and having β-glucosidase activity, and (c) A protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in any one of the amino acid sequences of SEQ ID NOs: 1 to 7 and having β-glucosidase activity. [2] The enzyme agent according to [1] above for use in the production of β-glucosidic disaccharides. [3] The enzyme agent according to [2] above, wherein the β-glucosidic disaccharide contains at least gentiobiose. [4] A method for producing a β-glucosaccharide or a saccharide composition containing the same, comprising a step of allowing the enzyme agent according to any one of [1] to [3] above to act on glucose. [5] The production method according to [4] above, wherein the β-glucosaccharide contains at least gentiobiose. [6] A method for producing the enzyme agent according to any one of [1] to [3] above, comprising a step of culturing a host microorganism transformed with a polynucleotide consisting of a base sequence selected from the group consisting of the following (i), (ii), (iii) and (iv) or an expression vector in which the polynucleotide is operably linked: (i) A base sequence encoding any one of the amino acid sequences of SEQ ID NOs: 1 to 7, (ii) A base sequence having 70% or more identity with the base sequence encoding any one of the amino acid sequences of SEQ ID NOs: 1 to 7 and encoding a protein having β-glucosidase activity, (iii) A base sequence consisting of a base sequence in which one or more bases are deleted, substituted, inserted and / or added in the base sequence encoding any one of the amino acid sequences of SEQ ID NOs: 1 to 7 and encoding a protein having β-glucosidase activity, and (iv) A base sequence that hybridizes under stringent conditions to a polynucleotide consisting of a complementary sequence of the base sequence encoding any one of the amino acid sequences of SEQ ID NOs: 1 to 7 and encodes a protein having β-glucosidase activity. [7] The production method according to [6] above, wherein the base sequence encoding any one of the amino acid sequences of SEQ ID NOs: 1 to 7 is any one of the base sequences of SEQ ID NOs: 8 to 14.

[0010] According to the present invention, an enzyme agent useful for producing a β-glucosaccharide having a high gentiobiose content is provided. By allowing the enzyme agent of the present invention to act on glucose, a β-glucosaccharide having a particularly high gentiobiose content can be produced. Detailed description of the invention

[0011] <<The enzyme agent of the present invention>> The enzyme agent of the present invention comprises at least one protein selected from the group consisting of the above (a), (b) and (c) (hereinafter sometimes referred to as "the protein of the present invention"). The enzyme agent of the present invention is an enzyme agent having β-glucosidase activity, and particularly shows glucosidase activity specific to β-1,6-glycosidic bond, and thus is an enzyme agent useful for the selective synthesis and selective decomposition of gentiobiose.

[0012] In the present invention, "β-glucosidase activity" is an enzyme activity that catalyzes a reaction of hydrolyzing β-glucosaccharide to produce glucose. Further, this enzyme also catalyzes a condensation reaction and a sugar transfer reaction. In this condensation reaction and sugar transfer reaction, β-glucosaccharide is produced using glucose as a substrate. Examples of β-glucosaccharide include gentiobiose (β-1,6-glucosaccharide), sophorose (β-1,2-glucosaccharide), laminaribiose (β-1,3-glucosaccharide), cellobiose (β-1,4-glucosaccharide), and isotrehalose (β-1,1-glucosaccharide).

[0013] When the enzyme agent of the present invention contains the protein of (a) above, the protein of the present invention may comprise an amino acid sequence of any one of SEQ ID NOs: 1 to 7, or may consist of an amino acid sequence of any one of SEQ ID NOs: 1 to 7.

[0014] In (b) above, "identity" is used in a sense that includes "homology". Here, "identity" is, for example, the degree of identity when the sequences to be compared are properly aligned, and means the occurrence rate (%) of exact matches of amino acids between the sequences. In calculating identity, for example, the presence of gaps in the sequences and the properties of amino acids are considered (Wilbur, Natl. Acad. Sci. U.S.A. 80:726-730 (1983)). The alignment can be performed, for example, by using any algorithm. Specifically, publicly available homology search software such as BLAST (Basic local alignment search tool) (Altschul et al., J. Mol. Biol. 215:403-410 (1990)), FASTA (Peasron et al., Methods in Enzymology 183:63-69 (1990)), Smith-Waterman (Meth. Enzym., 164, 765 (1988)) can be used. Also, the calculation of identity can be performed, for example, using publicly available homology search programs as described above. For example, in the homology algorithm BLAST of the National Center for Biotechnology Information (NCBI) in the United States (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), it can be calculated by using the default parameters.

[0015] The identity in (b) above is, for example, 70% or more, preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, even more preferably 95% or more, particularly preferably 96% or more, 97% or more, 98% or more, 99% or more, or 99.5%.

[0016] The protein of (c) may have one or more modifications selected from the group consisting of deletion, substitution, insertion, and addition in any of the amino acid sequences of SEQ ID NOs: 1 to 7. The number of amino acids to be modified may be, for example, 1 to 100, preferably 1 to 50, more preferably 1 to 20, still more preferably 1 to 10, even more preferably 1 to 6, particularly preferably 1 to several, 1 to 4, 1 to 3, 1 to 2, or 1. The number of amino acids to be modified can also be the number of mutations generated by known methods such as site-directed mutagenesis, or the number of mutations occurring naturally. Therefore, "modification" is used in a meaning including mutations. In the amino acid sequence, the modifications may occur continuously or discontinuously. The modifications may also be a plurality of the same type of modifications (for example, a plurality of substitutions), or a plurality of different types of modifications (for example, a combination of one or more deletions and one or more substitutions).

[0017] In addition, examples of the insertion of amino acids in (c) include insertion into the interior of the amino acid sequence. Further, the addition of amino acids may be, for example, addition to either the N-terminus or the C-terminus of the amino acid sequence, or addition to both the N-terminus and the C-terminus.

[0018] The modification of the amino acid may be a conservative modification (e.g., conservative mutation). "Conservative modification" or "conservative mutation" means modifying or mutating one or more amino acids so as not to substantially modify the function of the protein. The substitution of the amino acid may also be a conservative substitution. "Conservative substitution" means substituting one or more amino acids with another amino acid and / or amino acid derivative so as not to substantially modify the function of the protein. In a conservative substitution, it is preferable that the amino acid to be substituted and the amino acid after substitution are similar in properties and / or functions, for example. Specifically, it is preferable that chemical properties such as hydrophobicity and hydrophilicity indices, polarity, charge, etc., or physical properties such as secondary structure are similar. Thus, amino acids or amino acid derivatives similar in properties and / or functions are known in the art. For example, non-polar amino acids (hydrophobic amino acids) include, for example, alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, etc. Polar amino acids (neutral amino acids) include glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine, etc. Amino acids having a positive charge (basic amino acids) include arginine, histidine, lysine, etc., and amino acids having a negative charge (acidic amino acids) include aspartic acid, glutamic acid, etc.

[0019] In the above (b) and (c), whether it is a "protein having β-glucosidase activity" can be evaluated using the hydrolysis activity of the test protein as an index. For example, it can be evaluated by allowing the test protein to act on β-glucosidic disaccharide as a substrate and measuring the amount of glucose generated by the decomposition. The strength of β-glucosidase activity can be evaluated using the amount of glucose generated by the decomposition as an index. β-Glucosidase activity can be measured, for example, by allowing the protein (enzyme) of the present invention to act on β-glucosidic disaccharide as a substrate and measuring the amount of glucose generated by the enzyme reaction by a color reaction using Glucose C-II Test Wako (manufactured by Fujifilm Wako Pure Chemical Corporation), and can be evaluated based on the value obtained here and the added amount of the enzyme used in the reaction.

[0020] When the protein of the present invention is the protein of the above (b) or (c) defined based on the amino acid sequence of SEQ ID NO: 1, the protein can be considered to have an activity of about 70% or more, about 80% or more, about 90% or more, about 95% or more, or about 100% or more of the protein consisting of the amino acid sequence of SEQ ID NO: 1 under the conditions of a temperature of 30 to 40°C and a pH of 6 to 8. Similarly, when the protein of the present invention is the protein of the above (b) or (c) defined based on any of SEQ ID NOs: 2 to 7, the strength of its activity can be determined by comparison with the protein consisting of any of the amino acid sequences of SEQ ID NOs: 2 to 7.

[0021] The enzyme agent of the present invention contains a protein having β-glucosidase activity as an active ingredient. As described above, β-glucosidic disaccharide having a high gentiobiose content is generated from glucose by the condensation reaction or glycosyl transfer reaction of β-glucosidase. The production of β-glucosidic disaccharide using the enzyme agent of the present invention will be described later.

[0022] <<Method for Producing β-Glucosidic Disaccharide-Containing Carbohydrate>> According to the present invention, there is provided a method for producing a β-glucosidic disaccharide or a carbohydrate composition containing the same, which comprises a step of allowing the enzyme agent of the present invention to act on glucose.

[0023] When the enzyme agent of the present invention acts on glucose, two molecules of glucose condense to form β-glucobiose. The saccharide composition containing β-glucobiose obtained by the production method of the present invention is, specifically, a mixture that may contain raw material saccharides such as glucose and saccharides having a degree of polymerization of 3 or more produced in the condensation reaction, in addition to β-glucobiose produced in the condensation reaction.

[0024] In the condensation reaction of glucose by β-glucosidase, the produced glucose condensate is one in which glucose molecules are β-glucosidically bonded (β-glucooligosaccharide), and it is known that those with various bonding modes (β-1,6-bond, β-1,3-bond, etc.) are mixed. Therefore, the β-glucobiose obtained by the production method of the present invention may contain β-glucobiose other than gentiobiose (β-1,6-bond). The sugar composition of the β-glucobiose obtained by the production method of the present invention is not particularly limited, but the content of gentiobiose in the β-glucobiose can be 60% by mass or more, preferably 70% by mass or more, 80% by mass or more, 85% by mass or more, 90% by mass or more, 91% by mass or more, 92% by mass or more, 93% by mass or more, 94% by mass or more, or 95% by mass or more. In this specification, when referring to the ratio of the sugar composition, it means the content (% by mass) of each sugar component per solid content.

[0025] In the production method of the present invention, an aqueous glucose solution (glucose syrup) can be used as the raw material glucose. Considering the reaction efficiency, it is preferably an aqueous solution with a solid content concentration of 40% by mass or more, more preferably an aqueous solution with a solid content concentration of 50% by mass or more. The raw material glucose may be a pure product, but considering cost and availability, a saccharide composition mixed with other saccharides may also be used. For example, a starch hydrolyzate with a glucose content of 20 to 100% by mass, a starch hydrolyzate with a glucose content of 50 to 100% by mass, a starch hydrolyzate with a glucose content of 60 to 100% by mass, or a starch hydrolyzate with a glucose content of 70 to 100% by mass can be used as the raw material.

[0026] In the production method of the present invention, the conditions for allowing the enzyme agent to act on the substrate are not particularly limited and may be appropriately adjusted according to the characteristics of the enzyme. For example, the reaction can be carried out for 12 to 144 hours under the conditions of pH 6 to 8 and temperature 30 to 60°C.

[0027] In the production method of the present invention, after the desired β-glucosyl disaccharide is obtained, if necessary, deactivation treatment of the enzyme, purification treatment such as filtration, decolorization, deodorization, and desalting, and / or concentration treatment can be carried out. These treatments can be carried out according to conventional methods. Further, the obtained β-glucosyl disaccharide may be appropriately concentrated to obtain a liquid product, or may be made into a powder product by spray drying or the like. Furthermore, by removing residual raw materials and by-products by fractionation treatment such as membrane fractionation and resin fractionation, or assimilation treatment by microorganisms, and by fractionating those with a specific degree of polymerization, the content of β-glucosyl disaccharide, particularly the content of gentiobiose, can be further increased.

[0028] <Method for Producing the Enzyme Agent of the Present Invention> The enzyme agent of the present invention can be produced by culturing a natural microorganism having the ability to produce the protein of the present invention or a recombinant microorganism described below.

[0029] That is, according to another aspect of the present invention, there is provided a method for producing the enzyme agent of the present invention, which includes a step of culturing a host microorganism transformed with a polynucleotide having a base sequence selected from the group consisting of the above (i), (ii), (iii), and (iv) (hereinafter sometimes referred to as "the polynucleotide of the present invention") or an expression vector in which the polynucleotide is operably linked. The production method of the present invention may further include a step of collecting the expression product from the cultured host microorganism and / or the culture obtained by culturing, and optionally isolating or purifying the enzyme agent.

[0030] In the present invention, the "polynucleotide" includes DNA and RNA, and further includes modified forms and artificial nucleic acids thereof, preferably DNA. Further, DNA includes cDNA, genomic DNA, and chemically synthesized DNA.

[0031] In the above (i), (ii), (iii) and (iv), the "base sequence encoding an amino acid sequence" can be specified based on the genetic code (i.e., codon) by providing a specific amino acid sequence. For example, the base sequence can be specified by replacing the amino acid sequences of SEQ ID NOs: 1 to 7 with the corresponding codons, and specific examples include the base sequences of SEQ ID NOs: 8 to 14.

[0032] In the above (ii), "identity" is used in a sense that includes "homology". Here, "identity" is, as described for the above (b), the degree of identity when the sequences to be compared are appropriately aligned, and means the occurrence rate (%) of exact matches of bases between the sequences. In calculating the identity, for example, the presence of gaps in the sequences and the nature of the bases are considered (Wilbur, Natl. Acad. Sci. U.S.A. 80:726-730 (1983)). Alignment and calculation of identity can be performed according to the description for the above (b).

[0033] The identity in the above (ii) is, for example, 70% or more, preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, even more preferably 95% or more, particularly preferably 96% or more, 97% or more, 98% or more, 99% or more or 99.5% or more.

[0034] The nucleotide sequence of (iii) may have one or more modifications selected from the group consisting of deletion, substitution, insertion, and addition in the nucleotide sequence encoding any of the amino acid sequences of SEQ ID NOs: 1 to 7. The number of nucleotides to be modified may be, for example, 1 to 100, preferably 1 to 50, more preferably 1 to 20, still more preferably 1 to 10, even more preferably 1 to 6, particularly preferably 1 to several, 1 to 4, 1 to 3, 1 to 2, or 1. The number of nucleotides to be modified can also be the number of mutations generated by known methods such as site-directed mutagenesis, or the number of mutations that occur naturally. Therefore, "modification" is used in a meaning that includes mutations. The deleted, inserted, and / or added nucleotide sequences are not particularly limited, but are, for example, nucleotide sequences in the same reading frame as the nucleotide sequence encoding the amino acid sequences of SEQ ID NOs: 1 to 7. The nucleotides to be deleted, inserted, and / or added are preferably, for example, codons consisting of three consecutive nucleotides, and the number of such codons is, for example, 1 to 30, preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 6, particularly preferably 1 to several, 1 to 3, 1 to 2, or 1. In the nucleotide sequence, the modifications may occur continuously or discontinuously. The modifications may also be a plurality of modifications of the same type (for example, a plurality of substitutions) or a plurality of modifications of different types (for example, a combination of one or more deletions and one or more substitutions).

[0035] In addition, examples of the insertion of nucleotides in (iii) include insertion into the interior of the nucleotide sequence. Furthermore, the addition of nucleotides may be, for example, addition to either the 5'-end or the 3'-end of the nucleotide sequence, or addition to both the 5'-end and the 3'-end.

[0036] In the above (iv), "hybridize" means that a polynucleotide forms a double strand by hydrogen bonding between each complementary base and the polynucleotide targeted. "Hybridize" can be detected by various hybridization assays. Examples of hybridization assays include known methods such as Southern hybridization assay and colony hybridization assay.

[0037] In the above (iv), "hybridize" or "hybridization" can be carried out under stringent conditions. For example, it can be carried out by performing a hybridization reaction in a hybridization buffer and then washing with a washing buffer. Here, "stringent conditions" refer to conditions under which specific hybrids are formed between polynucleotides and non-specific hybrids are not formed, and can be determined depending on the Tm (°C) of the double strand of a certain base sequence and its complementary strand and the required salt concentration, etc. For example, after selecting a base sequence encoding any of the amino acid sequences of SEQ ID NOs: 1 to 7 (for example, any of the base sequences of SEQ ID NOs: 8 to 14), setting the corresponding stringent conditions is a technique well known to those skilled in the art (see, for example, J. Sambrook, E. F. Frisch, T. Maniatis; Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory (1989)). Examples of stringent conditions include performing a hybridization reaction at a temperature slightly lower than the Tm determined by the base sequence (for example, a temperature 0 to 10 °C lower than Tm, a temperature 0 to 5 °C lower than Tm, or a temperature 0 to 2 °C lower than Tm) in an appropriate buffer (for example, SSC solution) commonly used for hybridization. Examples of stringent conditions also include performing the washing after the hybridization reaction under severe conditions (for example, a high-temperature and low-salt concentration solution) for the double-stranded polynucleotide that has formed a non-specific hybrid.

[0038] In the above (ii), (iii) and (iv), whether it is a "protein having β-glucosidase activity" can be evaluated using the hydrolysis activity of the test protein as an index. For example, it can be evaluated by allowing the test protein to act on β-glucosyl disaccharide as a substrate and measuring the amount of glucose generated by the decomposition. The strength of β-glucosidase activity can be evaluated using the amount of glucose generated by the decomposition as an index.

[0039] When the polynucleotide of the present invention consists of the base sequence of (ii), (iii) or (iv) determined based on the base sequence encoding the amino acid sequence of SEQ ID NO: 1, the protein encoded by the polynucleotide has an activity of about 70% or more, about 80% or more, about 90% or more, about 95% or more, or about 100% or more of the protein consisting of the amino acid sequence of SEQ ID NO: 1 under the conditions of a temperature of 30 to 40 °C and a pH of 6 to 8. When the polynucleotide of the present invention consists of the base sequence of (ii), (iii) or (iv) determined based on the base sequence encoding any one of SEQ ID NOs: 2 to 7, the strength of its activity can be determined in comparison with the protein consisting of any one of the amino acid sequences of SEQ ID NOs: 2 to 7 in the same manner as above.

[0040] When the enzyme agent of the present invention is produced using a recombinant microorganism having the ability to produce the protein of the present invention, the recombinant microorganism includes a polynucleotide consisting of a nucleotide sequence selected from the above (i), (ii), (iii) and (iv), or a host microorganism transformed with an expression vector formed by operably linking the polynucleotide. As the expression vector, for example, a vector suitable for introduction into a host microorganism such as Escherichia coli or Bacillus subtilis can be selected, and plasmid vectors such as pET21b, pUC18, pET15b, pET32a, pColdI, pGEX-4T, pJEXOPT2 (see JP-A-2009-17841), pHT01, and pHT43 can be mentioned. Among these, when expressing the protein of the present invention in Escherichia coli, pET21b, pUC18, pET15b, pET32a, pColdI, and pGEX-4T are preferred, and when expressing in Bacillus subtilis, pJEXOPT2, pHT01, and pHT43 are preferred. In addition, the ligation of the polynucleotide to the expression vector can be carried out according to a conventional method.

[0041] The expression vector thus obtained can be introduced into host microorganisms such as Escherichia coli and Bacillus subtilis to obtain recombinant microorganisms (i.e., transformants). The introduction into the host microorganism can be carried out according to a conventional method. In addition, the polynucleotide consisting of the nucleotide sequence selected from the above (i), (ii), (iii) and (iv) can also be introduced into the host microorganism without using a vector by methods such as electroporation and lipofection to obtain recombinant microorganisms.

[0042] The recombinant microorganisms and natural microorganisms that produce the protein which is the active ingredient of the enzyme agent of the present invention can be cultured in a medium suitable for culturing the microorganisms. The medium to be used is not particularly limited as long as it is a nutrient medium in which the microorganisms can grow and can produce the protein which is the active ingredient of the enzyme agent of the present invention, and either a synthetic medium or a natural medium may be used. In addition, culture conditions such as time and temperature can be appropriately selected according to the microorganisms to be cultured.

[0043] In the method for producing the enzyme agent of the present invention, after culturing recombinant microorganisms or natural microorganisms, a step of collecting the protein that is the active ingredient of the enzyme agent of the present invention may be included. In the method for producing the enzyme agent of the present invention, for example, after culturing natural microorganisms or recombinant microorganisms, cell lysing agents, surfactants, ultrasonic disruption, bead shockers, etc. are used to disrupt the cells of the bacterial cells, and the crude enzyme solution can be obtained as it is or after separating the insoluble fraction. In the present invention, the crude enzyme solution may be used as it is or after concentration. As the concentration method, ammonium sulfate salting-out method, acetone and alcohol precipitation method, flat membrane, hollow membrane method, etc. can be adopted.

[0044] As described above, the enzyme agent of the present invention can use the crude enzyme solution as it is or after concentration, but if necessary, the one separated and purified by column chromatography or the like can be used. For example, by purifying the supernatant of the culture solution or the disrupted product using affinity chromatography, the protein that is the active ingredient of the enzyme agent of the present invention can be obtained as a purified enzyme showing a single band electrophoretically.

[0045] When the protein that is the active ingredient of the enzyme agent of the present invention is a recombinant protein, depending on the type of recombinant microorganism, the enzyme may accumulate in the bacterial cells or in the culture solution. Even in such a case, as described above, the bacterial cells or their culture may be used as it is, but if necessary, the disrupted cells may be used.

Example

[0046] The present invention will be described more specifically based on the following examples, but the present invention is not limited to these examples.

[0047] Example 1: Protein expression using Bacillus subtilis as a host (1) Construction of expression plasmid New enzyme searches were carried out for various microorganisms. Specifically, Halobacillus halophilus described in Non-Patent Document 1 ( Halobacillus halophilus)A homology search was performed with the amino acid sequence of the original β-1,6-glucosidase, and virtual proteins (SEQ ID NOs: 1 to 7) with an amino acid sequence identity of about 60 to 70% with the amino acid sequence of the enzyme were selected and their functions were analyzed. For each selected virtual protein, the sequence identity with the SEQ ID NO, the originating microorganism, the NCBI registration information (NCBI Reference Sequence), and the amino acid sequence of the enzyme described in Non-Patent Document 1 was as shown in Table 1.

[0048]

Table 1

[0049] An expression plasmid for expressing a protein (hereinafter sometimes referred to as "target protein") consisting of the amino acid sequences shown in SEQ ID NOs: 1 to 7 (hereinafter sometimes referred to as "target amino acid sequence") in Bacillus subtilis (Bacillus subtilis: Bacillus subtilis , the same applies hereinafter) was constructed. First, a His tag sequence was ligated to the C-terminal side of the target amino acid sequence. Next, a plasmid (hereinafter sometimes referred to as "linear plasmid") obtained by modifying pJEXOPT2, a vector for Bacillus subtilis expression (see JP-A-2009-17841 and JP-A-2009-17842), for optimization in this test was prepared. Using the homologous region with the modified plasmid as an additional sequence for cloning, the N-terminal side and the C-terminal side of the target amino acid sequence to which the His tag sequence was added were provided, and a sequence for artificial gene synthesis was designed. A plasmid in which the designed sequence was inserted into the pUC57 vector was designed, and artificial gene synthesis was performed (artificial synthetic plasmid). In addition, codon correction was performed on the base sequence corresponding to the target protein portion for optimization of expression in Bacillus subtilis. The base sequences encoding the amino acid sequences of SEQ ID NOs: 1 to 7 (hereinafter referred to as "target genes") were as shown in SEQ ID NOs: 8 to 14, respectively.

[0050] Using the obtained synthetic plasmid and linear plasmid as templates, the target gene fragment and linear plasmid to be used in the In-Fusion HD Cloning Kit (Takara Bio Inc.) were PCR amplified. The primers shown in Tables 2 and 3 were used for the PCR amplification of the target gene fragment and linear plasmid, respectively.

[0051]

Table 2

[0052]

Table 3

[0053] The composition of the reaction solution used for the PCR reaction was prepared according to the manual of Primestar max Premix (Takara Bio Inc.), and the total volume of the reaction solution was 50 μL. The program for the PCR amplification reaction was to hold at 96°C for 1 minute, then perform 35 cycles with 1 cycle being 10 seconds at 98°C → 5 seconds at 55°C → 35 seconds at 72°C, and then hold at 72°C for 5 minutes. The obtained PCR product was subjected to agarose gel electrophoresis, and the band corresponding to the amplified fragment was cut out from the gel and extracted and purified using illustra TM GFX TM PCR DNA and Gel Band Purification Kit (GE Healthcare). The amplified fragments of the obtained target gene and linear plasmid were ligated using the In-Fusion HD Cloning Kit (Takara Bio Inc.). The ligation reaction was carried out by holding at 50°C for 15 minutes. Then, using 1.5 μL of the ligation reaction solution, E. coli DH5α was transformed, and plasmid DNA was prepared from the culture solution in which the E. coli was cultured using illustra TM plasmidPrep Mini Spin Kit (GE Healthcare) (hereinafter sometimes referred to as "plasmid for Bacillus subtilis expression").

[0054] (2) Expression of Recombinant Protein in Bacillus subtilis and Preparation of Enzyme Samples The plasmid DNA for Bacillus subtilis expression prepared in (1) above was introduced into protoplasted Bacillus subtilis ISW1214 (Takara Bio Inc.) and cultured at 30 °C for 2 days in a regeneration agar medium containing 7.5 μg / mL tetracycline (composition: 8.1% sodium succinate, 1% agar, 0.5% casamino acids, 0.5% yeast extract, 0.15% potassium dihydrogen phosphate, 0.35% dipotassium hydrogen phosphate, 0.5% glucose, 0.4% magnesium chloride, 0.01% bovine serum albumin, 0.001% methionine, and 0.001% leucine). The obtained colonies were cultured in a preculture medium and then in a main culture medium. The culture was carried out as described in JP-A-2009-17841 and JP-A-2009-17842 except for the composition of the medium. Subsequently, the culture was centrifuged (1500×g, 4 °C, 15 minutes), and the supernatant filtered through a 0.45 μm filter (Merck & Co., Inc.) was used as the culture supernatant. The obtained culture supernatant was subjected to ultrafiltration using Amicon Ultra-0.5, 30 kDa (Merck & Co., Inc.) to replace it with 50 mM sodium phosphate buffer (pH 7.0) to remove the medium components, and the resulting solutions were used as enzyme samples 1 to 7, respectively. Enzyme samples 1 to 7 are proteins consisting of the amino acid sequences of SEQ ID NOs: 1 to 7, respectively.

[0055] (3) Confirmation of β-Glucosidase Activity For the enzyme samples 1 to 7 prepared in (2) above, the hydrolysis activity (β-glucosidase activity) using β-glucooligosaccharides as substrates was measured by the following procedure. 10 μL of each appropriately diluted enzyme sample was mixed with 10 μL of various β-glucooligosaccharide (sophorose, laminaribiose, cellobiose, gentiobiose) solutions, and the reaction was carried out at 37 °C and pH 7 for 5 minutes, and then the reaction was stopped by holding at 99.9 °C for 10 minutes. This was used as the sample reaction solution. Also, inactivated enzyme samples 1 to 7 in which enzyme samples 1 to 7 were inactivated by heat treatment at 99.9 °C for 10 minutes in advance were reacted with each β-glucooligosaccharide solution by the same procedure as above, and then the reaction was stopped. These were used as blank reaction solutions 1 to 7. Next, 180 μL of glucose CII-test wako (FUJIFILM Wako Pure Chemical Corporation) was added to each reaction solution (sample reaction solution and blank reaction solution), and after holding at 37 °C for 5 minutes, the absorbance (A492) at a wavelength of 492 nm was measured. Using the blank reaction solution as a blank, the measured value of A492 derived from glucose generated by the reaction of enzyme samples 1 to 7 in the sample reaction solution was determined. The results were as shown in Table 4. For any of the enzyme samples, the generation of glucose was confirmed only when gentiobiose was used as a substrate. From these results, it was shown that enzyme samples 1 to 7 have β-glucosidase activity specific to β-1,6-glycosidic bonds.

[0056]

Table 4

[0057] Example 2: Evaluation of the characteristics of an enzyme sample (1) Condensation reaction using enzyme samples For the enzyme samples 1 to 7 prepared in Example 1(2), a condensation reaction using glucose as a substrate was carried out. Specifically, a mixed solution was prepared by adding 75 μL of each of the enzyme samples 1 to 7 to 100 mg of glucose, and the condensation reaction was carried out by holding it at 37 °C and pH 7 for 72 hours. After the reaction, each reaction solution was held in boiling water for 10 minutes to inactivate the enzyme. Then, after each sample was cooled and appropriately diluted, Amberlite MB-4 was added for desalting. After desalting, each sample was filtered through a 0.45 μm filter, and the obtained reaction products were used for sugar composition analysis as reaction products 1 to 7, respectively. Note that reaction products 1 to 7 are the products obtained by reacting with enzyme samples 1 to 7, respectively.

[0058] (2) Sugar composition analysis of reaction products The sugar composition per solid content of the reaction products 1 to 7 prepared in (1) above was analyzed by high performance liquid chromatography (HPLC). The analysis conditions were as described in Analysis Condition 1 and Analysis Condition 2. The ratio of the peak area of a specific peak to the peak area of the reference substance under each analysis condition was defined as the content ratio (% of solid content) of the substance derived from that specific peak in the reference substance.

[0059] The conditions for analyzing the sugar composition of saccharides (monosaccharides, disaccharides, and trisaccharides) with each degree of polymerization in the reaction product were as in Analysis Condition 1. Note that the sugar composition (% of solid content) of saccharides with each degree of polymerization was calculated by the ratio (%) of the peak area corresponding to the saccharides with each degree of polymerization to the total area of the detected peaks.

[0060] <Analysis Condition 1> Column: Ultron PS80N·L (Showa Chemical Co., Ltd.) Eluent: Ultrapure water Flow rate: 0.9 mL / min Column temperature: 50 °C Detector: Differential refractometer Sample injection volume: 10 μL Analysis time: 20 minutes

[0061] The conditions for analyzing the content ratio of gentiobiose in the disaccharide fraction in the reaction product were as described in Analytical Condition 2. The content ratio of gentiobiose in the disaccharide fraction (solid content %) was calculated based on the ratio (%) of the peak area corresponding to gentiobiose to the total peak area of the peaks corresponding to the disaccharide fraction.

[0062] <Analytical Condition 2> Column: HILICpak VG-50 4E (Shodex) Eluent: 80% acetonitrile Flow rate: 0.6 mL / min Column temperature: 40 °C Detector: Corona charged particle detector Sample injection volume: 1 μL Analysis time: 60 minutes

[0063] The analysis results of the sugar compositions of Reaction Products 1 to 7 were as shown in Tables 5 and 6.

[0064]

Table 5

[0065]

Table 6

[0066] From the results in Table 5, it was confirmed that in the reaction products obtained by the condensation reaction using glucose as a substrate, the content ratio of the disaccharide fraction in the reaction products was 5.57 to 16.05 (solid content %). Also, from the results in Table 6, it was confirmed that in the reaction products obtained by the condensation reaction using glucose as a substrate, the content ratio of gentiobiose in the disaccharide fraction in the reaction products was 95.11 to 96.45 (solid content %). From these results, it was shown that Enzyme Samples 1 to 7 consisting of the amino acid sequences shown in SEQ ID NOs: 1 to 7 selectively produce gentiobiose, which is a β-glucosaccharide, as a disaccharide product in the condensation reaction using glucose as a substrate.

Claims

1. An enzyme agent comprising a protein selected from the group consisting of the following (a), (b), and (c) for use in the production of β-glucobiose, wherein the β-glucobiose contains at least gentiobiose: (a) A protein comprising an amino acid sequence of any one of SEQ ID NOs: 1 and 3 to 7; (b) A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence of any one of SEQ ID NOs: 1 and 3 to 7 and having β-glucosidase activity; and (c) A protein comprising an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of any one of SEQ ID NOs: 1 and 3 to 7 and having β-glucosidase activity.

2. A method for producing a β-glucobiose or a saccharide composition containing the same, comprising the step of allowing the enzyme agent according to Claim 1 to act on glucose.

3. The production method according to Claim 2, wherein the β-glucobiose contains at least gentiobiose.

4. A method for producing the enzyme agent according to Claim 1, comprising the step of culturing a host microorganism transformed with a polynucleotide comprising a base sequence selected from the group consisting of the following (i), (ii), (iii), and (iv) or an expression vector in which the polynucleotide is operably linked: (i) A base sequence encoding an amino acid sequence of any one of SEQ ID NOs: 1 and 3 to 7; (ii) A base sequence having 90% or more identity to the base sequence encoding the amino acid sequence of any one of SEQ ID NOs: 1 and 3 to 7 and encoding a protein having β-glucosidase activity; (iii) A base sequence consisting of a base sequence in which 1 to 20 bases are deleted, substituted, inserted, and / or added in the base sequence encoding the amino acid sequence of any one of SEQ ID NOs: 1 and 3 to 7 and encoding a protein having β-glucosidase activity; and (iv) A base sequence that hybridizes under stringent conditions to a polynucleotide consisting of a complementary sequence of the base sequence encoding the amino acid sequence of any one of SEQ ID NOs: 1 and 3 to 7 and encodes a protein having β-glucosidase activity.

5. The production method according to Claim 4, wherein the base sequence encoding the amino acid sequence of any one of SEQ ID NOs: 1 and 3 to 7 is any one of SEQ ID NOs: 8 and 10 to 14.

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