JP2022152998A - NOVEL ENZYMATIC AGENT AND METHOD FOR PRODUCING β-GLUCOSE DISACCHARIDE-CONTAINING CARBOHYDRATE - Google Patents

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

The present invention relates to a novel enzymatic agent and a method for producing a β-glucodisaccharide-containing carbohydrate using the enzymatic agent.

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

Patent Document 1 describes a method for producing β-glucooligosaccharides by reacting microbial β-glucosidase on glucose. Patent Document 2 describes a novel enzyme characterized by having specific physicochemical properties, and a method for producing a gentiooligosaccharide-rich syrup by acting the enzyme on a high-concentration glucose solution.

The β-glucodisaccharide obtained by the above method contained a certain amount of β-glucodisaccharides other than gentiobiose, such as laminaribiose and sophorose, in addition to gentiobiose, and the content of gentiobiose was not sufficient. On the other hand, Non-Patent Document 1 reports that gentiobiose can be synthesized by condensing glucose with β-glucosidase derived from Halobacillus halophilus .

JP-A-2-219584 JP-A-1-222779

2018 Annual Meeting of Japan Society for Bioscience, Biotechnology and Agrochemistry, Lecture number: 2A26p05, "Functional analysis of β-1,6-glucosidase derived from Halobacillus halophilus", Mai Terada, Naoto Isono

An object of the present invention is to provide a novel enzymatic agent capable of selectively producing gentiobiose, and a novel method for producing β-glucodisaccharide-containing carbohydrates using the enzymatic agent.

The present inventors have recently discovered that the selected seven proteins have β-glucosidase activity, and that the enzyme can be used to produce β-glucodisaccharide with a particularly high gentiobiose content. The present invention is based on these findings.

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

INDUSTRIAL APPLICABILITY According to the present invention, an enzymatic agent useful for producing β-glucodisaccharide with a high gentiobiose content is provided. By allowing the enzymatic agent of the present invention to act on glucose, β-glucodisaccharide having a particularly high gentiobiose content can be produced.

Specific description of the invention

<<Enzyme agent of the present invention>>
The enzymatic agent of the present invention comprises at least one protein selected from the group consisting of (a), (b) and (c) (hereinafter sometimes referred to as "the protein of the present invention"). be. The enzymatic agent of the present invention is an enzymatic agent having β-glucosidase activity, and since it exhibits β-1,6-glycoside bond-specific glucosidase activity, it is useful for selective synthesis and selective degradation of gentiobiose. is.

In the present invention, "β-glucosidase activity" is an enzymatic activity that catalyzes a reaction that hydrolyzes β-glucodisaccharide to produce glucose. Furthermore, this enzyme also catalyzes condensation reactions and transglycosylation reactions. In this condensation reaction and glycosyltransferase reaction, β-glucodisaccharide is produced using glucose as a substrate. β-glucodisaccharides include gentiobiose (β-1,6-glucodisaccharide), sophorose (β-1,2-glucodisaccharide), laminaribiose (β-1,3-glucodisaccharide) , cellobiose (β-1,4-glucodisaccharide), and isotrehalose (β-1,1-glucodisaccharide).

When the enzymatic agent of the present invention contains the protein (a), the protein of the present invention may comprise any amino acid sequence of SEQ ID NOS: 1-7, or any of SEQ ID NOS: 1-7. It may consist of any amino acid sequence.

In (b) above, "identity" is used in the sense of including "homology". Here, "identity" is, for example, the degree of identity when the sequences to be compared are properly aligned (aligned), and means the occurrence rate (%) of exact amino acid matches between the sequences. . In calculating identity, for example, the presence of gaps in the sequence and the nature of the amino acids are taken into account (Wilbur, Natl. Acad. Sci. U.S.A. 80:726-730 (1983)). The alignment can be performed, for example, by using any algorithm, specifically 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 do. In addition, the identity can be calculated using, for example, a publicly available homology search program such as those described above, for example, the homology algorithm BLAST (https: http://blast.ncbi.nlm.nih.gov/Blast.cgi) by using the default parameters.

The identity in (b) 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%.

The protein (c) may have one or more modifications selected from the group consisting of deletion, substitution, insertion and addition in the amino acid sequence of any one of SEQ ID NOs: 1 to 7. . The number of amino acids to be modified is, 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 produced by known methods such as site mutagenesis, or the number of mutations produced naturally. Therefore, the term "modification" is used in the sense of including mutation. In the amino acid sequence, the modifications may occur continuously or discontinuously. The modification may also be multiple homologous modifications (e.g., multiple substitutions) or multiple heterologous modifications (e.g., a combination of one or more deletions and one or more substitutions), good too.

In addition, the amino acid insertion in (c) above includes, for example, insertion into the inside of the amino acid sequence. Furthermore, 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.

The amino acid alterations may be conservative alterations (eg, conservative mutations). A "conservative modification" or "conservative mutation" means modifying or mutating one or more amino acids so as not to substantially alter the function of the protein. The amino acid substitutions may also be conservative substitutions. A "conservative substitution" refers to the replacement of one or more amino acids with another amino acid and/or amino acid derivative that does not substantially alter the function of the protein. In conservative substitution, the substituted amino acid and the substituted amino acid are preferably similar in properties and/or functions, for example. Specifically, it is preferred that the indices of hydrophobicity and hydrophilicity, chemical properties such as polarity and charge, or physical properties such as secondary structure are similar. Thus, amino acids or amino acid derivatives with similar properties and/or functions are known in the art. For example, nonpolar amino acids (hydrophobic amino acids) include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, and the like. Polar amino acids (neutral amino acids) include glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine and the like. Examples of positively charged amino acids (basic amino acids) include arginine, histidine, and lysine, and examples of negatively charged amino acids (acidic amino acids) include aspartic acid and glutamic acid.

In (b) and (c) above, whether or not the "protein having β-glucosidase activity" can be evaluated using the hydrolytic activity of the test protein as an index. - It can be evaluated by acting on glucodisaccharide and measuring the amount of glucose produced by decomposition. The strength of β-glucosidase activity can be evaluated using the amount of glucose produced by decomposition as an index. The β-glucosidase activity can be measured, for example, by allowing the protein (enzyme) of the present invention to act on β-glucodisaccharide as a substrate, and measuring the amount of glucose produced by the enzymatic reaction with Glucose C-II Test Wako (Fujifilm Wako Pure Chemical Industries, Ltd.). ), and can be evaluated based on the value obtained here and the added amount of the enzyme used in the reaction.

When the protein of the present invention is the protein (b) or (c) defined based on the amino acid sequence of SEQ ID NO: 1, the protein can be prepared at a temperature of 30 to 40° C. and a pH of 6 to 8. It can have about 70% or more activity, about 80% or more activity, about 90% or more activity, about 95% or more activity, or about 100% or more activity of a protein consisting of one amino acid sequence. . When the protein of the present invention is the protein (b) or (c) defined based on any of SEQ ID NOs: 2 to 7, the amino acid sequence of any one of SEQ ID NOs: 2 to 7, respectively, in the same manner as above The strength of its activity can be determined by contrasting it with a protein consisting of

The enzymatic agent of the present invention contains a protein having β-glucosidase activity as an active ingredient. As described above, β-glucodisaccharide with a high content of gentiobiose is produced from glucose by condensation reaction and transglycosylation reaction of β-glucosidase. Production of β-glucodisaccharide using the enzymatic agent of the present invention will be described later.

<<Method for producing carbohydrate containing β-glucodisaccharide>>
According to the present invention, there is provided a method for producing a β-glucodisaccharide or a carbohydrate composition containing the same, comprising a step of allowing the enzymatic agent of the present invention to act on glucose.

By allowing the enzymatic agent of the present invention to act on glucose, two molecules of glucose are condensed to produce β-glucodisaccharide. Specifically, the saccharide composition containing β-glucodisaccharide obtained by the production method of the present invention includes, in addition to β-glucodisaccharide produced by the condensation reaction, raw saccharides such as glucose, It is a mixture that can contain the produced saccharides having a degree of polymerization of 3 or more.

In the condensation reaction of glucose by β-glucosidase, the produced glucose condensation product is a β-glucoside bond between glucose (β-gluco-oligosaccharide), and various bonding modes (β-1,6-bond, β-1,3-bonds, etc.) are known to be mixed. Therefore, the β-glucodisaccharide obtained by the production method of the present invention may contain β-glucodisaccharide other than gentiobiose (β-1,6-linkage). The sugar composition of the β-glucodisaccharide obtained by the production method of the present invention is not particularly limited. % 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 sugar composition ratio, it means the content (% by mass) of each sugar component per solid content.

In the production method of the present invention, an aqueous glucose solution (glucose syrup) can be used as the raw material glucose, and considering the reaction efficiency, it is preferable to use an aqueous solution having a solid content concentration of 40% by mass or more, more preferably. It is an aqueous solution with a solid content concentration of 50% by mass or more. Glucose as a raw material may be a pure product, but considering cost and availability, it may be a sugar composition in which other sugars are mixed. A starch hydrolyzate with a glucose content of 100% by mass, a starch hydrolyzate with a glucose content of 60-100% by mass, or a starch hydrolyzate with a glucose content of 70-100% by mass can be used as a raw material.

In the production method of the present invention, the conditions under which the enzymatic agent acts on the substrate are not particularly limited, and may be appropriately adjusted according to the characteristics of the enzyme. Allow to react for ~144 hours.

In the production method of the present invention, after the desired β-glucodisaccharide is obtained, enzyme deactivation treatment, purification treatment such as filtration, decolorization, deodorization, and desalting and/or concentration treatment are performed as necessary. be able to. These treatments can be carried out according to conventional methods. Further, the obtained β-glucodisaccharide may be appropriately concentrated into a liquid product, or may be powdered by spray drying or the like. In addition, β-glucolysis can be achieved by removing raw materials and by-products remaining after fractionation such as membrane fractionation and resin fractionation, assimilation by microorganisms, and by separating those with a specific degree of polymerization. Disaccharide content, especially gentiobiose content, can be higher.

<Method for Producing Enzyme Preparation of the Present Invention>
The enzymatic agent of the present invention can be produced by culturing natural microorganisms or recombinant microorganisms described later that are capable of producing the protein of the present invention.

That is, according to another aspect of the present invention, a polynucleotide consisting of a base sequence selected from the group consisting of (i), (ii), (iii) and (iv) (hereinafter referred to as "polynucleotide of the present invention There is provided a method for producing the enzymatic agent of the present invention, which comprises the step of culturing a host microorganism transformed with an expression vector operatively linked with said polynucleotide. After the culturing step, 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, and optionally isolating or purifying the enzymatic agent. good.

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

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

In (ii) above, "identity" is used in the sense of including "homology". Here, "identity" is the degree of identity when the sequences to be compared are properly aligned, as described in (b) above. means the occurrence rate (%) of good matches. In calculating identity, for example, the presence of gaps in the sequence and the nature of the bases are taken into account (Wilbur, Natl. Acad. Sci. U.S.A. 80:726-730 (1983)). Alignment and identity calculation can be performed according to the description of (b) above.

The identity in (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.

The nucleotide sequence of (iii) has one or more alterations selected from the group consisting of deletion, substitution, insertion and addition in the nucleotide sequence encoding the amino acid sequence of any of SEQ ID NOs: 1 to 7. may have. The number of bases to be modified is, 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 modified bases can also be the number of mutations produced by a known method such as site mutagenesis, or the number of mutations produced naturally. Therefore, the term "modification" is used in the sense of including mutation. The deleted, inserted and/or added base sequences are not particularly limited, but are, for example, base sequences in the same reading frame as the base sequences of the polynucleotides encoding the amino acid sequences of SEQ ID NOS: 1-7. The deleted, inserted and/or added bases are, for example, preferably codons consisting of three consecutive bases, and the number of such codons is, for example, 1 to 30, preferably 1 to 20, and more. The number is preferably 1 to 10, more preferably 1 to 6, particularly preferably 1 to several, 1 to 3, 1 to 2 or 1. In the base sequence, the modifications may occur continuously or discontinuously. The modification may also be multiple homologous modifications (e.g., multiple substitutions) or multiple heterologous modifications (e.g., a combination of one or more deletions and one or more substitutions), good too.

In addition, the insertion of bases in (iii) above includes, for example, insertion into the interior of the base sequence. Furthermore, the addition of bases may be, for example, addition to either the 5' end or the 3' end of the base sequence, or addition to both the 5' end and the 3' end.

In (iv) above, "hybridize" means that a certain polynucleotide forms a double strand by hydrogen bonding between bases complementary to the target polynucleotide. "Hybridization" can be detected by various hybridization assays. Hybridization assays include, for example, known methods such as Southern hybridization assays and colony hybridization assays.

In (iv) above, "hybridization" or "hybridization" can be performed under stringent conditions. It can be implemented by Here, "stringent conditions" refer to conditions under which specific hybrids are formed between polynucleotides and non-specific hybrids are not formed, and Tm (° C. ) and the required salt concentration, etc. For example, after selecting a nucleotide sequence encoding any of the amino acid sequences of SEQ ID NOs: 1 to 7 (eg, any of the nucleotide sequences of SEQ ID NOs: 8 to 14), it is appropriate to set stringent conditions accordingly. Techniques well known to those skilled in the art (see, eg, J. Sambrook, E. F. Frisch, T. Maniatis; Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory (1989)). Stringent conditions include, for example, a temperature slightly lower than the Tm determined by the nucleotide sequence (for example, 0 to 10° C. lower than the Tm) in an appropriate buffer (eg, SSC solution) commonly used for hybridization. and performing the hybridization reaction at a low temperature, 0 to 5° C. lower than the Tm or 0 to 2° C. lower than the Tm. Stringent conditions also include post-hybridization washing under stringent conditions (eg, high temperature, low salt solution) for non-specifically hybridized double-stranded polynucleotides.

In (ii), (iii) and (iv) above, whether or not the protein is a "protein having β-glucosidase activity" can be evaluated using the hydrolytic activity of the test protein as an index. It can be evaluated by acting on the substrate β-glucodisaccharide and measuring the amount of glucose produced by decomposition. The strength of β-glucosidase activity can be evaluated using the amount of glucose produced by decomposition as an index.

When the polynucleotide of the present invention consists of the nucleotide sequence (ii), (iii) or (iv) defined based on the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, the protein encoded by the polynucleotide is about 70% or more activity, about 80% or more activity, about 90% or more activity, about 95% or more of the protein consisting of the amino acid sequence of SEQ ID NO: 1 under conditions of temperature 30 to 40 ° C. and pH 6 to 8 It can have activity or about 100% or more activity. When the polynucleotide of the present invention consists of the nucleotide sequence (ii), (iii) or (iv) defined based on the nucleotide sequence encoding any of SEQ ID NOs: 2 to 7, the same sequence as above The strength of the activity can be determined by comparison with a protein consisting of any one of the amino acid sequences of numbers 2-7.

When the enzymatic agent of the present invention is produced using a recombinant microorganism capable of producing the protein of the present invention, the recombinant microorganism includes (i), (ii), (iii) and (iv) Examples include host microorganisms transformed with a polynucleotide consisting of a selected nucleotide sequence or an expression vector operably linked to the polynucleotide. As the expression vector, for example, a vector suitable for introduction into host microorganisms such as Escherichia coli and Bacillus subtilis can be selected. 17841), pHT01 and pHT43. Among these, pET21b, pUC18, pET15b, pET32a, pColdI and pGEX-4T are preferable when the protein of the present invention is expressed in E. coli, and pJEXOPT2, pHT01 and pHT43 are preferable when it is expressed in Bacillus subtilis. In addition, ligation of polynucleotides to expression vectors can be carried out according to conventional methods.

The expression vector thus obtained can be introduced into host microorganisms such as Escherichia coli and Bacillus subtilis to obtain recombinant microorganisms (ie, transformants). Introduction into host microorganisms can be carried out according to conventional methods. Also, a polynucleotide consisting of a base sequence selected from (i), (ii), (iii) and (iv) above can be introduced into a host microorganism without using a vector by electroporation, lipofection or the like. , a recombinant microorganism can also be obtained.

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

The method for producing the enzymatic agent of the present invention may include a step of collecting the protein, which is the active ingredient of the enzymatic agent of the present invention, after culturing the recombinant or natural microorganism. In the method for producing the enzymatic agent of the present invention, for example, after culturing natural microorganisms or recombinant microorganisms, bacterial cells are crushed using a bacteriolytic agent, a surfactant, ultrasonic disruption, a bead shocker, or the like, and The separated fraction can be used as a crude enzyme solution. In the present invention, the crude enzyme solution may be used as it is or after concentrating. As a concentration method, an ammonium sulfate salting-out method, an acetone and alcohol precipitation method, a flat membrane method, a hollow membrane method, or the like can be employed.

As described above, the enzymatic preparation of the present invention can be used as is or after concentration, but can be separated and purified by column chromatography or the like, if necessary. For example, the protein, which is the active ingredient of the enzymatic preparation of the present invention, can be obtained as a purified enzyme that electrophoretically exhibits a single band by purifying the culture supernatant or the homogenate using affinity chromatography. can be done.

When the protein, which is the active ingredient of the enzymatic agent of the present invention, is a recombinant protein, depending on the type of the recombinant microorganism, the enzyme may accumulate in the cells or in the culture medium. In such a case, as described above, the bacterial cells or their culture may be used as they are, but if necessary, disrupted cells may be used.

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

Example 1: Expression of protein using Bacillus subtilis as a host (1) Construction of expression plasmid Various microorganisms were searched for novel enzymes. Specifically, a homology search was performed with the amino acid sequence of β-1,6-glucosidase derived from Halobacillus halophilus described in Non-Patent Document 1, and the identity of the amino acid sequence with the amino acid sequence of the enzyme was confirmed. Hypothetical proteins (SEQ ID NOS: 1 to 7) with about 60 to 70% were selected and their functions were analyzed. For each hypothetical protein selected, the sequence identity with the amino acid sequence of the enzyme described in SEQ ID, originating microorganism, NCBI registration information (NCBI Reference Sequence) and Non-Patent Document 1 is shown in Table 1.

A protein consisting of the amino acid sequences shown in SEQ ID NOs: 1 to 7 (hereinafter sometimes referred to as "target amino acid sequence") (hereinafter sometimes referred to as "target protein") is prepared by Bacillus subtilis , The same applies hereinafter) to construct an expression plasmid for expression. First, a His tag sequence was ligated to the C-terminal side of the target amino acid sequence. Next, pJEXOPT2 (see JP-A-2009-17841 and JP-A-2009-17842), which is a Bacillus subtilis expression vector, was modified to optimize it for this test (hereinafter referred to as "linear plasmid" ) was produced. A sequence for artificial gene synthesis was designed by attaching the region homologous to the modified plasmid as an additional sequence for cloning to the N-terminal side and C-terminal side of the target amino acid sequence to which the above His tag sequence was added. A plasmid was designed by inserting the design sequence into the pUC57 vector, and artificial gene synthesis was performed (artificially synthesized plasmid). The base sequence corresponding to the target protein portion was subjected to codon correction to optimize expression in Bacillus subtilis. The nucleotide sequences encoding the amino acid sequences of SEQ ID NOs: 1-7 (hereinafter referred to as "target genes") were as shown in SEQ ID NOs: 8-14, respectively.

Using the resulting artificially synthesized plasmid and linear plasmid as templates, the target gene fragment and linear plasmid used in the In-Fusion HD Cloning Kit (Takara Bio Inc.) were amplified by PCR. The primers shown in Tables 2 and 3 were used for PCR amplification of the target gene fragment and linear plasmid, respectively.

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

(2) Expression of recombinant protein in Bacillus subtilis and preparation of enzyme sample The Bacillus subtilis expression plasmid DNA prepared in (1) above was introduced into protoplasted Bacillus subtilis ISW1214 (Takara Bio Inc.), and 7.5 μg/ Regeneration agar medium containing 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) for 2 days at 30°C. The colonies obtained were cultured in the pre-culture medium and then in the main culture medium. The culture was performed as described in JP-A-2009-17841 and JP-A-2009-17842, except for the composition of the medium. Then, the culture was centrifuged (1500×g, 4° C., 15 minutes), and the supernatant was filtered through a 0.45 μm filter (Merck) to obtain a culture supernatant. The resulting culture supernatant was subjected to ultrafiltration using Amicon Ultra-0.5, 30 kDa (Merck) to remove medium components by replacing with 50 mM sodium phosphate buffer (pH 7.0). The resulting solutions were designated 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.

(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-glucodisaccharide as a substrate was measured by the following procedure. 10 μL of each appropriately diluted enzyme sample and 10 μL of various β-glucodisaccharide (sophorose, laminaribiose, cellobiose, gentiobiose) solutions are mixed, reacted at 37° C. and pH 7 for 5 minutes, and held at 99.9° C. for 10 minutes. to stop the reaction. This was used as a sample reaction solution. In addition, inactivated enzyme samples 1 to 7, which were previously inactivated by heat treatment at 99.9° C. for 10 minutes, were treated with each β-glucodisaccharide solution in the same manner as above. After reacting, the reaction was stopped. These were used as blank reaction solutions 1-7. Then, 180 μL of glucose CII-Test Wako (Fujifilm Wako Pure Chemical Industries, Ltd.) 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. It was measured. Using the blank reaction solution as a blank, the measured value of A492 derived from glucose produced by the reaction of the enzyme samples 1 to 7 in the sample reaction solution was determined. The results were as shown in Table 4. For all enzyme samples, glucose production was confirmed only when gentiobiose was used as a substrate. These results indicated that enzyme samples 1-7 had β-1,6-glycosidic bond-specific β-glucosidase activity.

Example 2: Evaluation of properties of enzyme samples (1) Condensation reaction using enzyme samples Enzyme samples 1 to 7 prepared in Example 1 (2) were subjected to a condensation reaction using glucose as a substrate. Specifically, mixed solutions were prepared by adding 75 μL of each of the enzyme samples 1 to 7 to 100 mg of glucose, and the condensation reaction was performed by maintaining the mixture at 37° C. and pH 7 for 72 hours. After the reaction, each reaction solution was kept in boiling water for 10 minutes to inactivate the enzyme. After cooling and diluting each sample appropriately, 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. Reaction products 1 to 7 are products obtained by reacting enzyme samples 1 to 7, respectively.

(2) Sugar Composition Analysis of Reaction Products The sugar compositions per solid content of the reaction products 1 to 7 prepared in (1) above were analyzed by high performance liquid chromatography (HPLC). The analytical conditions were as described in the analytical conditions 1 and 2. The ratio of the specific peak area to the peak area of the reference substance under each analysis condition was defined as the content ratio (solid content %) of the specific peak-derived substance in the reference substance.

Analysis conditions 1 were used to analyze the sugar composition of sugars (monosaccharides, disaccharides and trisaccharides) of each degree of polymerization in the reaction product. The sugar composition (solid content %) of the sugar with each degree of polymerization was calculated from the ratio (%) of the peak area corresponding to the sugar with each degree of polymerization to the total area of the detected peaks.

<Analysis conditions 1>
Column: Ultron PS80N L (Shinwa Kako 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

Analysis condition 2 was used as the condition for analyzing the content ratio of gentiobiose in the disaccharide fraction in the reaction product. The content ratio (solid content %) of gentiobiose in the disaccharide fraction was calculated from the ratio (%) of the peak area corresponding to gentiobiose to the total area of the peak corresponding to the disaccharide fraction.

<Analysis conditions 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

The sugar composition analysis results of reaction products 1-7 were as shown in Tables 5 and 6.

From the results in Table 5, the content ratio of the disaccharide fraction in the reaction product obtained by the condensation reaction using glucose as a substrate is 5.57 to 16.05 (solid content %). was confirmed. Further, from the results in Table 6, in the reaction product obtained by the condensation reaction using glucose as a substrate, the content ratio of gentiobiose in the disaccharide fraction in the reaction product was 95.11 to 96.45 (solid minutes %). From these results, enzyme samples 1 to 7 consisting of the amino acid sequences shown in SEQ ID NOS: 1 to 7 selectively produced gentiobiose, which is a β-glucodisaccharide, as a disaccharide product in the condensation reaction using glucose as a substrate. It was shown to generate

Claims (7)

An enzymatic agent comprising a protein selected from the group consisting of (a), (b) and (c) below:
(a) a protein comprising the amino acid sequence of any of SEQ ID NOS: 1-7;
(b) a protein consisting of an amino acid sequence having 70% or more identity to any of the amino acid sequences of SEQ ID NOS: 1-7 and having β-glucosidase activity, and (c) a protein of SEQ ID NOS: 1-7 A protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and/or added to any amino acid sequence and having β-glucosidase activity. The enzymatic agent according to claim 1, which is used for producing β-glucodisaccharide. 3. The enzymatic agent according to claim 2, wherein the β-glucodisaccharide contains at least gentiobiose. A method for producing a β-glucodisaccharide or a carbohydrate composition containing the same, comprising a step of allowing the enzymatic agent according to any one of claims 1 to 3 to act on glucose. The production method according to claim 4, wherein the β-glucodisaccharide contains at least gentiobiose. 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 operably linked to the polynucleotide: The method for producing the enzymatic preparation according to any one of claims 1 to 3, comprising the step of culturing:
(i) a nucleotide sequence encoding the amino acid sequence of any one of SEQ ID NOS: 1-7;
(ii) a nucleotide sequence that has 70% or more identity with the nucleotide sequence encoding the amino acid sequence of any one of SEQ ID NOs: 1 to 7 and encodes a protein having β-glucosidase activity;
(iii) a nucleotide sequence encoding the amino acid sequence of any one of SEQ ID NOs: 1 to 7, consisting of a nucleotide sequence in which one or more nucleotides are deleted, substituted, inserted and/or added, and β-glucosidase hybridizes under stringent conditions to a polynucleotide consisting of a nucleotide sequence encoding a protein having activity and (iv) a sequence complementary to the nucleotide sequence encoding the amino acid sequence of any one of SEQ ID NOs: 1 to 7, and , a nucleotide sequence encoding a protein having β-glucosidase activity. 7. The production method according to claim 6, wherein the nucleotide sequence encoding any of the amino acid sequences of SEQ ID NOs: 1-7 is any of the nucleotide sequences of SEQ ID NOs: 8-14.