JP2016158570A - Mutant glycoside hydrolases - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide mutant glycoside hydrolases with high heat resistance and high stability in liquid detergents, in particular, excellent stability to surfactants.SOLUTION: The mutant glycoside hydrolase comprises a specified amino acid sequence or an amino acid sequence having 80% or more sequence identity to the specified amino acid sequence, where the amino acid residues at the positions 138, 301 and 310 or at positions corresponding to these positions are: (a) at position 138 or corresponding position: histidine; (b) at position 301 or corresponding position: histidine; and (c) at position 310 or at corresponding position: glutamic acid.SELECTED DRAWING: None

Description

  The present invention relates to mutant glycoside hydrolases.

  Mixing a plurality of enzymes such as protease, lipase, cellulase, amylase, xylanase, mannanase, pectate lyase, cutinase, and lichenase into a detergent as a cleaning aid has been studied or practiced. Cellulase, which is one of glycoside hydrolases, is suitable for blending in laundry detergents because it acts on amorphous regions of single cotton fibers and effectively removes sebum stains and the like in single fibers.

Bacillus as an enzyme that exhibits an excellent effect as an enzyme for detergents such as laundry detergent
sp. Alkaline cellulase derived from KSM-N257 strain has been developed and patent applications have been filed (Patent Documents 1 and 2). The alkaline cellulase derived from the KSM-N257 strain is an enzyme belonging to glycoside hydrolase family 8 (GH family 8). The family of glucoside hydrolases is a group of enzymes characterized by cleaving β-1,4 bonds such as β-1,4 glucan, xylan, chitosan, and lichenan. The enzyme derived from the KSM-N257 strain is also a glycoside hydrolase having a lichenan degrading activity in addition to a cellulose degrading activity such as carboxymethyl cellulose and crystalline cellulose. Regarding the enzyme derived from the KSM-N257 strain (hereinafter referred to as N257 glycoside hydrolase), cellulase derived from Bacillus circulans (Non-patent Document 1) and Bacillus sp. Analysis of amino acids at the catalytic site and conserved amino acids has been performed by comparison of amino acid sequences with cellulase derived from KSM-330 strain (Non-patent Document 2) and X-ray analysis of enzyme protein crystals (Non-patent Document 3) .

  Furthermore, for N257 glycoside hydrolase, the substitution of a specific amino acid residue on the amino acid sequence with another amino acid residue improves the stability and specific activity of the enzyme in a liquid detergent, or heat resistance. It has been found that the properties are improved (Patent Document 3, Patent Document 4).

  In recent years, in liquid detergents, so-called concentrated liquid detergents that can be used in an amount that is about half that of conventional liquid detergents are commercially available. Such concentrated liquid detergents contain a high-concentration surfactant. Yes. Therefore, the conditions are extremely severe for the enzyme, and further improvement of the surfactant resistance is required in addition to the improvement of the enzyme activity and heat resistance.

JP 2001-309781 A JP 2002-85078 A Japanese Patent Application Laid-Open No. 2014-10777 JP 2014-10777 A

Bueno et al., Nucleic Acids Research, 18, 4248, 1990. Ozaki et al. Gen. Microbiol. , 137, 41-48, 1991 Hakamada et al., Biochimica et Biophysica Acta, 1570, 174-180, 2002.

  The present invention relates to providing a mutant glycoside hydrolase having excellent heat resistance and stability in a liquid detergent, particularly excellent stability to a surfactant.

  The present inventor further examined N257 glycoside hydrolase. As a result, multiple mutants in which a plurality of amino acid residues on the amino acid sequence of the glycoside hydrolase are substituted with other amino acid residues have excellent heat resistance. It has been found that the stability in liquid detergents is improved, and in particular, the surfactant resistance is greatly improved.

That is, the present invention relates to the following 1) to 7).
1) In the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence, the amino acid residues at positions 138, 301, and 310 or corresponding positions are as follows: Mutant glycoside hydrolase that is a residue.
(A) Position 138 or a position corresponding thereto: histidine (b) Position 301 or a position corresponding thereto: histidine (c) Position 310 or a position corresponding thereto: glutamic acid 2) Gene encoding the above-mentioned mutant glycoside hydrolase .
3) A recombinant vector containing the above gene.
4) A transformant containing the above recombinant vector.
5) A method for producing a mutant glycoside hydrolase using the transformant.
6) A liquid detergent composition containing the mutant glycoside hydrolase.
7) In the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence, the amino acid residues at positions 138, 301, and 310 or corresponding positions are represented by the following amino acids: A method for improving the surfactant resistance of glycoside hydrolase, comprising substituting a residue.
(A) Position 138 or a position corresponding thereto: histidine (b) Position 301 or a position corresponding thereto: histidine (c) Position 310 or a position corresponding thereto: glutamic acid

  The mutant glycoside hydrolase of the present invention has excellent heat resistance, improved stability in a liquid detergent, and particularly excellent surfactant resistance. Therefore, the liquid detergent in which the mutant glycoside hydrolase of the present invention is blended can stably maintain a high enzyme activity and exhibit a high detergency.

  As used herein, the term “amino acid residue” refers to 20 amino acid residues constituting a protein, Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val are meant.

  As used herein, “identity (between amino acid sequences)” refers to the number of full-length amino acid residues in the number of positions where identical amino acid residues are present in both sequences when the two amino acid sequences are aligned. The ratio (%) to the number. Specifically, it is calculated by the Lippmann-Pearson method (Lipman-Pearson method; Science, 227, 1435, (1985)), and homology of genetic information processing software Genetyx-Win (Ver. 11.0.2; software development). Using an analysis (Search homology) program, it can be calculated by performing an analysis with Unit size to compare (ktup) of 2.

  In this specification, unless otherwise defined, “one or several” used for deletion, substitution, addition or insertion of an amino acid residue or base in an amino acid sequence or nucleotide sequence is, for example, 1 to 37, Preferably it may be 1-20, more preferably 1-10. In the present specification, “addition” of an amino acid residue or base includes addition of one to several amino acids or bases to one end and both ends of the sequence.

  In the present specification, “glycoside hydrolase activity” refers to an activity capable of cleaving β-1,4 bonds such as β-1,4 glucan, xylan, chitosan, lichenan, cellulase activity, lichenase activity, and xylanase. Includes activity. As used herein, “glycoside hydrolase” refers to a protein or polypeptide having glycoside hydrolase activity, such as a protein or polypeptide having one or more of cellulase activity, lichenase activity, and xylanase activity. It can be.

  As used herein, a “parent” protein or polypeptide of a given mutant protein or polypeptide is a protein or polypeptide that becomes the mutant protein or polypeptide by making a predetermined mutation in its amino acid residue Say.

The mutant glycoside hydrolase of the present invention comprises three amino acid residues of a glycoside hydrolase consisting of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 80% or more sequence identity with the amino acid residue. A triple substituent substituted with a group, a quadruple substitute obtained by substituting one amino acid residue with another amino acid residue for the triple substitute, and one or more additional places for the quadruple substitute In which multiple amino acid residues are substituted with other amino acid residues.
As a triple substitution product, the amino acid residue at position 138 is changed to histidine, the amino acid residue at position 301 is changed to histidine, and the amino acid residue at position 310 is changed from a glycoside hydrolase consisting of the amino acid sequence shown in SEQ ID NO: 2. It can be obtained by substituting with glutamic acid. Alternatively, the mutant glycoside hydrolase of the present invention is a glycoside hydrolase consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 2, and the amino acid residue at the position corresponding to position 138 is changed to histidine. The amino acid residue at the position corresponding to position 301 is substituted with histidine, and the amino acid residue at the position corresponding to position 310 is replaced with glutamic acid. Such a triple substitution product has improved heat resistance and stability in a liquid detergent, particularly resistance to a surfactant (for example, sodium linear alkylbenzene sulfonate (LAS)).

  The quadruple substitution product is obtained by further substituting the amino acid residue at position 248 or a corresponding position with tyrosine to the above triple substitution product. Such a quadruple substituted product significantly improves stability in a liquid detergent, particularly surfactant resistance.

The multi-substitution is the same as the above-mentioned quadruple substitution, in addition to the 22nd, 80th, 85th, 99th, 120th, 180th, 186th, 239th, 240th, 243th, 259th, 289th, 289th, 349 Examples include those obtained by substituting at least one amino acid residue selected from the group consisting of amino acid residues at positions 376, 376, or positions corresponding thereto with the following amino acid residues (A) to (N). Such a multiple substitution product further improves the surfactant resistance.
(A) Position 22 or a position corresponding thereto: alanine (B) Position 80 or a position corresponding thereto: isoleucine (C) Position 85 or a position corresponding thereto: phenylalanine (D) Position 99 or a position corresponding thereto : Aspartic acid (E) position 120 or a position corresponding thereto: aspartic acid (F) position 180 or a position corresponding thereto: aspartic acid (G) position 186 or a position corresponding thereto: alanine (H) position 239 or Corresponding positions: isoleucine (I) position 240 or corresponding positions: leucine (J) position 243 or corresponding positions: isoleucine (K) position 259 or corresponding positions: asparagine (L) 289 Position or a position corresponding thereto: glutamic acid (M) position 349 or a position corresponding to these: meth Nin (N) 376-position or a position corresponding to, alanine

As the multiple substituents, it is preferable to combine 1 to 4 types of the above-mentioned substitutions (A) to (N) to form a 5-fold, 6-fold, 7-fold or 8-fold substitute. . Examples of such combinations include those shown in the following i) to v).
i) The amino acid residue at position 180 or a position corresponding thereto is the following amino acid residue.
(F) Position 180 or a position corresponding thereto: aspartic acid ii) The amino acid residues at positions 99 and 186 or positions corresponding thereto are the following amino acid residues, respectively.
(D) position 99 or a position corresponding thereto: aspartic acid (G) position 186 or a position corresponding thereto: alanine iii) amino acid residues at positions 22, 180 and 349, or positions corresponding thereto; Each of the following amino acid residues:
(A) Position 22 or a position corresponding thereto: alanine (F) Position 180 or a position corresponding thereto: Aspartic acid (M) Position 349 or a position corresponding thereto: methionine iv) Positions 239, 240 and 259 Or the amino acid residues corresponding to these are the following amino acid residues, respectively.
(H) position 239 or a position corresponding thereto: isoleucine (I) position 240 or a position corresponding thereto: leucine (K) position 259 or a position corresponding thereto: asparagine v) positions 80, 120 and 376, Or the amino acid residue of the position corresponding to these is the following amino acid residue, respectively.
(B) Position 80 or a position corresponding thereto: Isoleucine (E) Position 120 or a position corresponding thereto: Aspartic acid (N) Position 376 or a position corresponding thereto: alanine

  The glycoside hydrolase comprising the amino acid sequence represented by SEQ ID NO: 2 and the glycoside hydrolase comprising the amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 are the mutant glycoside hydrolase of the present invention. The parent glycoside hydrolase. In the following specification, these may be referred to as “the parent glycoside hydrolase of the present invention”.

  Among the parent glycoside hydrolases of the present invention, as the glycoside hydrolase having the amino acid sequence represented by SEQ ID NO: 2, for example, glycoside hydrolase derived from N257 strain [Bacillus sp. KSM-N257 strain (FERM P-17473)] is used. (Patent Documents 1 and 2).

  Among the parent glycoside hydrolases of the present invention, the glycoside hydrolase comprising an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 2 is different from the amino acid sequence shown in SEQ ID NO: 2. , 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, still more preferably 97% or more, still more preferably 98% or more, and the amino acid sequence represented by SEQ ID NO: 2 Preferably, a protein or polypeptide having a sequence identity of 99% or more and having glycoside hydrolase activity, and one to several amino acids are deleted or substituted in the amino acid sequence represented by SEQ ID NO: 2. Or a protein that has been added and has glycoside hydrolase activity, or Ripepuchido and the like.

  In glycoside hydrolase derived from the N257 strain [Bacillus sp. KSM-N257 strain (FERM P-17473)] having the amino acid sequence represented by SEQ ID NO: 2, the amino acids involved in the catalytic activity are glutamic acid at position 63 and asparagine at position 124. It is known that it is an acid (nonpatent literature 3). It can also be seen that glutamic acid at position 253 and asparagine at position 263 are also involved in the catalytic activity as compared to the three-dimensional structure of the cytosinease of Bacillus sp. K17. Therefore, in the parent glycoside hydrolase of the present invention consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 2, preferably corresponds to position 63 of the amino acid sequence shown in SEQ ID NO: 2. The amino acid residue at the position is glutamic acid, the amino acid residue at the position corresponding to position 124 is aspartic acid, the amino acid residue at the position corresponding to position 253 is glutamic acid, and the amino acid at the position corresponding to position 263 The residue is asparagine.

  Examples of glycoside hydrolase that can be the parent glycoside hydrolase of the present invention and has an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 2 include GH family 8 described in Non-Patent Document 3. Other enzymes belonging to: for example, cellulolytic xylanase derived from Bacillus circulans (GenBank accession no. AAP22946, 97% amino acid sequence identity); lichenase derived from Paenibacillus sp. Y412MC10 (GenBank accession no. YP_003296630, amino acid sequence identity) ); Glycosyl hydrolase family 8 derived from Paenibacillus sp. HGF5 (GenBank accession no. ZP_08) 839797, 96% amino acid sequence identity); lichenase from Paenibacillus vortex V453 (GenBank accession no. ZP_078997964, 93% amino acid sequence identity); 83%).

  In this specification, the “amino acid residue at the corresponding position” is identified by comparing the target amino acid sequence with a reference sequence (for example, the amino acid sequence represented by SEQ ID NO: 2) using a known algorithm, This can be done by aligning the sequences so as to give maximum homology to conserved amino acid residues present in the amino acid sequence of the ase. By aligning the amino acid sequence of glycoside hydrolase in this way, it is possible to determine the position of the homologous amino acid residue in each glycoside hydrolase regardless of insertion or deletion in the amino acid sequence. is there. The alignment can be performed manually based on, for example, the above-mentioned Lippmann-Person method, but the Clustal W multiple alignment program (Thompson, JD et al, (1994) Nucleic Acids Res. 22, p. 4673-4680) with default settings. Alternatively, it is possible to use Clustal W2 or Clustal omega, which is a revised version of Clustal W. Clustal W, Clustal W2, and Clustal omega are, for example, European Bioinformatics Institute (EBI, [www.ebi.ac.uk/index.html]) and Japanese DNA operated by the National Institute of Genetics. It can be used from the website of the data bank (DDBJ, [www.ddbj.nig.ac.jp/Welcome-j.html]).

  A person skilled in the art can further fine-tune the alignment obtained above so as to obtain an optimum alignment as necessary. Such an optimal alignment is preferably determined in consideration of amino acid sequence similarity, frequency of inserted gaps, and the like. Here, the similarity of amino acid sequences means the ratio (%) of the number of positions where the same or similar amino acid residues exist in both sequences when two amino acid sequences are aligned to the total number of amino acid residues. . A similar amino acid residue means an amino acid residue having properties similar to each other in terms of polarity and charge among 20 kinds of amino acids constituting a protein and causing so-called conservative substitution. Such groups of similar amino acid residues are well known to those skilled in the art and include, for example, arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; valine, leucine and isoleucine, respectively. However, it is not limited to these.

  The position of the amino acid residue of the target amino acid sequence aligned with the position corresponding to the arbitrary position of the reference sequence by the above alignment is regarded as the “corresponding position” to the arbitrary position, and the amino acid residue is The amino acid residue at the position ".

  In the amino acid sequence shown in SEQ ID NO: 2, position 138 is lysine, position 301 is asparagine, position 310 is asparagine, position 248 is alanine, position 22 is threonine, position 80 is serine, position 85 is tyrosine, position 99 is asparagine, 120 Asparagine, position 180 is glycine, position 186 is threonine, position 239 is methionine, position 240 is serine, position 243 is asparagine, position 259 is lysine, position 289 is lysine, position 349 is threonine, position 376 is threonine However, by using the above method, for example, the above-described glycoside hydrolase cellulolytic xylanase (GenBank: AAP22946), Paenibacillus sp. Y412MC10-derived lichenase (GenBank: YP_003240630), paenibachi Glycosyl hydrolase family 8 derived from SP HGF5 (GenBank: ZP_08283397), lichenase derived from Paenibacillus vortex V453 (GenBank: ZP_078997964), and the following: Each amino acid residue at a position corresponding to the position can be specified.

  The “corresponding position” thus identified is considered to exist at an equivalent position in the three-dimensional structure between proteins that are highly identical in amino acid sequence and are glycoside hydrolases. Therefore, it is presumed that the mutation of the amino acid residue existing at the corresponding position has a similar effect on the specific function of glycoside hydrolase.

Furthermore, the parent glycoside hydrolase of the present invention is more preferably one having any one or more of the following enzymatic properties possessed by the glycoside hydrolase having the amino acid sequence represented by SEQ ID NO: 2.
1) The isoelectric point measured by isoelectric focusing is 9.3;
2) Decomposes carboxymethylcellulose well in liquefied form;
3) Substrate specificity: Decomposes carboxymethylcellulose, lichenan, crystalline cellulose or cellooligosaccharides higher than cellotriose to produce reducing sugars;
4) Optimal reaction pH: works at least at pH 5-10, the optimal pH is 8.5;
5) Optimal reaction temperature: When the reaction is carried out with glycine-sodium hydroxide buffer (pH 8.5), the optimal reaction temperature is 55 ° C;
6) Stable pH range: when treated at 30 ° C. for 60 minutes, it is stable in the range of pH 5-11;
7) Heat resistance: stable to 55 ° C. in 15-minute treatment in Tris-HCl buffer (pH 7.0);
8) Molecular weight: The estimated molecular weight by SDS polyacrylamide gel electrophoresis is 43 kDa.

  In the mutant glycoside hydrolase of the present invention, the above-mentioned substitution of amino acid residues may be naturally occurring or artificially introduced. Furthermore, the mutant glycoside hydrolase of the present invention is not limited to the above-described substitution of amino acid residues, and the mutation at any other position (unless it interferes with the glycoside hydrolase activity and the stability improving effect in liquid detergents) For example, you may have deletion, substitution, addition, insertion). The mutation at the arbitrary position may be naturally occurring or artificially introduced.

  As a means for substituting the amino acid residue of glycoside hydrolase, various mutagenesis techniques known in the art can be used. For example, the nucleotide sequence encoding the amino acid residue to be replaced in the glycoside hydrolase gene encoding the amino acid sequence of the parent glycoside hydrolase of the present invention (hereinafter also referred to as the parent glycoside hydrolase gene) is converted into the amino acid residue after substitution. A mutant glycoside hydrolase in which the amino acid residue to be substituted is substituted with a desired amino acid residue can be obtained by mutating the nucleotide sequence encoding the group and then expressing the mutant glycoside hydrolase from the mutant gene. .

  For example, introduction of a desired mutation into the parent glycoside hydrolase gene is basically based on PCR amplification using the parent glycoside hydrolase gene as a template DNA or replication reactions using various DNA polymerases, and various sites well known to those skilled in the art. Specific mutagenesis can be used. The site-directed mutagenesis method is performed by any method such as inverse PCR method or annealing method (edited by Muramatsu et al., “Revised 4th edition, New Genetic Engineering Handbook”, Yodosha, p. 82-88). Can do. Various commercially available site-specific mutagenesis kits such as QuickChange II Site-Directed Mutagenesis Kit of Stratagene and QuickChange Multi Site-Directed Mutagenesis Kit can also be used as necessary.

  Site-directed mutagenesis into the parent glycoside hydrolase gene can be most commonly performed using a mutation primer containing a nucleotide mutation to be introduced. Such a mutation primer anneals to a region containing a nucleotide sequence encoding the amino acid residue to be replaced in the parent glycoside hydrolase gene, and replaces the nucleotide sequence (codon) encoding the amino acid residue to be replaced. Thus, it may be designed to include a polynucleotide having a nucleotide sequence (codon) encoding the amino acid residue after substitution. Those skilled in the art can appropriately recognize and select a nucleotide sequence (codon) encoding a substitution target and a substituted amino acid residue based on a normal textbook.

Mutagenesis is also carried out by using SPE (splicing by overlap extension) -PCR (DNA) obtained by separately amplifying the upstream and downstream sides of the mutation site using two complementary mutation primers containing the nucleotide mutation to be introduced. Horton
R. M.M. et al. Gene (1989) 77 (1), p. 61-68) can also be used. This mutagenesis procedure using the SOE-PCR method is also described in detail in the examples below.

  The template DNA containing the parent glycoside hydrolase gene is Bacillus sp. KSM-N257 (FERM P-17473), Bacillus circulans, Paenibacillus sp. Y412MC10, Paenibacillus sp. Then, it can be prepared by extracting genomic DNA by a conventional method, or extracting RNA and synthesizing cDNA by reverse transcription. Alternatively, based on the amino acid sequence of the parent glycoside hydrolase, a corresponding nucleotide sequence may be synthesized and used as template DNA.

  Preparation of genomic DNA from the aforementioned Bacillus strain is described, for example, in Pitcher et al. Lett. Appl. Microbiol. 1989, 8: p. 151-156 and the like. The template DNA containing the parent glycoside hydrolase gene may be prepared by inserting a DNA fragment containing the parent glycoside hydrolase gene excised from the prepared cDNA or genomic DNA into an arbitrary vector.

  The primer can be prepared by a known oligonucleotide synthesis method such as phosphoramidite method (Nucleic Acids Research, 17, 7059-7071, 1989). Such primer synthesis can also be prepared using, for example, a commercially available oligonucleotide synthesizer (manufactured by ABI, etc.). A mutant glycoside hydrolase gene into which a target mutation is introduced can be obtained by using a primer set containing a mutation primer and performing the site-specific mutagenesis as described above using the parent glycoside hydrolase gene as a template DNA. . The present invention also relates to the mutant glycoside hydrolase gene thus obtained. In the present invention, the “mutant glycoside hydrolase gene” means any nucleic acid fragment (including DNA, mRNA, artificial nucleic acid, etc.) encoding the amino acid sequence of the mutant glycoside hydrolase gene. The “gene” according to the present invention may include other nucleotide sequences such as an untranslated region (UTR) in addition to the open reading frame.

  A recombinant vector can be prepared by inserting and ligating the obtained mutant glycoside hydrolase gene into an arbitrary vector by a conventional method. The vector used in the present invention is not particularly limited, and may be any vector such as a plasmid, phage, phagemid, cosmid, virus, YAC vector, shuttle vector and the like. Such a vector is not limited, but is preferably a vector that can be amplified in bacteria, in particular, Bacillus bacteria, and is an expression vector that can induce expression of a transgene in Bacillus bacteria. Further preferred. Among them, a shuttle vector, which is a vector that can be replicated in both Bacillus bacteria and other organisms, can be particularly preferably used for recombinant production of a mutant glycoside hydrolase gene. Examples of preferred vectors include, but are not limited to, pHY300PLK (an expression vector capable of transforming both E. coli and Bacillus subtilis; Ishikawa, H. and Shibahara, H., Jpn. J. Genet, (1985) 60. , P. 235-243), pAC3 (Moriyama, H. et al., Nucleic Acids Res. (1988) 16, p. 8732) and the like, pUB110 (Gryczan, T. J. et al., J. MoI. Bacteriol. (1978) 134, p. 318-329), pTA10607 (Bron, S. et al., Plasmid, 18 (1987) p. 8-15), etc. Secretion signal to recombinant protein Grantable secretion vector (Yamane et al., "Bacillus subtilis secretion vector according fusion protein" starch Science, 34. (1987), p.163-170), and the like. In addition, plasmids derived from E. coli (for example, pET22b (+), pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.) can also be used.

  For the purpose of recombinant production of a mutated glycoside hydrolase gene, the vector is preferably an expression vector. Expression vectors include various elements essential for expression in host organisms such as transcription promoters, terminators, and ribosome binding sites, cis elements such as selectable marker genes, polylinkers, and enhancers, poly A addition signals, and ribosome binding sequences (SD sequences). Useful sequences such as

  A transformant can be produced using a recombinant vector containing a mutated glycoside hydrolase gene. In the present invention, a transformant (transformed cell) is prepared by introducing a recombinant vector (specifically, a recombinant expression vector) containing the mutant glycoside hydrolase gene according to the present invention into a host cell, A mutant glycoside hydrolase can be produced by culturing under conditions that induce expression of the recombinant protein.

  Host cells into which the recombinant vector is introduced include bacteria such as E. coli and Bacillus subtilis, microorganisms such as yeast cells, and any cells such as insect cells, animal cells (for example, mammalian cells), plant cells, and the like. Can be used. In the present invention, it is particularly preferable to use Bacillus bacteria such as Bacillus subtilis and mutants thereof.

  For transformation, for example, a well-known transformation technique such as a calcium phosphate method, an electroporation method, a lipofection method, a particle gun method, or a PEG method can be applied. For example, transformation methods applicable to Bacillus bacteria include competent cell transformation methods (Bott. K.F. and Wilson, GA, J. Bacteriol. (1967) 93, 1925), electroporation. (Brigidi. P. et al., FEMS Microbiol. Lett. (1990) 55, 135), protoplast transformation method (Chang, S. and Cohen, SN, Mol. Gen. Genet., (1979). 168, p. 111-115), Tris-PEG method (Takahashi W. et al., J. Bacteriol. (1983) 156, p. 1130-1134).

  Culture of the transformant for production of the recombinant protein can be performed according to a method common to those skilled in the art. For example, a medium for cultivating a transformant based on a microorganism host such as E. coli or yeast cells contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the host microorganism, so that the transformant can be cultured efficiently. Any medium that can be used may be a natural medium or a synthetic medium. Ampicillin, tetracycline, or the like may be added to the medium according to the type of drug selection marker. When culturing a microorganism transformed with an expression vector using an inducible promoter, an inducer may be added to the medium as necessary. For example, when cultivating bacteria or the like transformed with an expression vector using the Lac promoter, a microorganism transformed with isopropyl-1-thio-β-D-galactoside (IPTG) or the like with an expression vector using the trp promoter is cultured. Indole acetic acid (IAA) or the like can be added to the medium. The culture conditions are not particularly limited, but are preferably performed under conditions suitable for the host organism used for transformation. For example, LB medium, 2 × YT medium, 2 × L-maltose medium, CSL fermentation medium, or the like can be used for culturing Bacillus subtilis transformants for producing recombinant proteins.

  The mutant glycoside hydrolase according to the present invention may be expressed from a mutant glycoside hydrolase gene or a transcription product thereof using a cell-free translation system. “Cell-free translation system” is an in vitro transcription translation system or in vitro translation system by adding reagents such as amino acids necessary for protein translation to suspension obtained by mechanically destroying host cells. It is a thing. The expressed mutant glycoside hydrolase can be obtained by using a general method used for protein purification, for example, centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography or the like alone or in combination as appropriate. It can be obtained from a culture solution, a cell disruption solution, or a cell-free translation system. In addition, a solution such as a culture supernatant or a lysate supernatant separated or concentrated by using a centrifugal separation, an ultrafiltration type filter, or the like can be used as a crude enzyme solution as it is. If the expressed mutant glycoside hydrolase is not secreted from inside the cell, the cell may be disrupted before protein separation and purification.

  Preparation of mRNA used in the present invention, preparation of cDNA, PCR, RT-PCR, library preparation, ligation into vector, cell transformation, determination of DNA sequence, nucleic acid chemical synthesis, N-terminal side of protein Experiments such as amino acid sequencing, mutagenesis, protein extraction, etc. can be carried out by the methods described in ordinary experiments. Such experimental documents include, for example, Sambrook et al., Molecular Cloning, A laboratory manual, (2001) 3rd Ed. Sambrook, J .; & Russell, DW. And Cold Spring Harbor Laboratory Press. In particular, for genetic recombination experiments of Bacillus subtilis, see, for example, Hirofumi Yoshikawa "7.2 Bacillus subtilis system" "Second Life Chemistry Laboratory 1. Genetic Research Method II", (1986) Tokyo Chemical Dojinsha (Tokyo), p. . Reference can be made to general experiments on genetic manipulation of Bacillus subtilis such as 150-169.

  Thus, the mutant glycoside hydrolase of the present invention can be obtained. Therefore, the present invention also provides a gene (polynucleotide) encoding the mutant glycoside hydrolase of the present invention, a vector containing the gene, a transformant containing the vector, and a mutant glycoside hydrolase using the transformant. A manufacturing method is provided.

  The mutant glycoside hydrolase of the present invention has glycoside hydrolase activity, preferably one or more enzyme activities selected from the group consisting of alkaline cellulase activity, lichenase activity and xylanase activity, preferably cellulase activity. Preferably, the enzyme activity is higher compared to the parent glycoside hydrolase. In addition, the mutant glycoside hydrolase of the present invention has less decrease in enzyme activity compared to the parent glycoside hydrolase even when stored at a high temperature (60 ° C.) or after long-term storage in a liquid detergent. That is, the mutant glycoside hydrolase of the present invention has improved heat resistance and stability in a liquid detergent compared to the parent glycoside hydrolase. In particular, the resistance to surfactants typified by LAS is greatly improved compared to the parent glycoside hydrolase.

  Accordingly, the present invention also provides a method for improving the surfactant resistance (eg, LAS resistance) of glycoside hydrolase, comprising substituting the amino acid residues of the parent glycoside hydrolase of the present invention as described above.

  The mutant glycoside hydrolase of the present invention is useful as an enzyme for blending various detergents, preferably an enzyme for blending detergents. In particular, the mutant glycoside hydrolase of the present invention has improved stability in liquid detergents, particularly surfactant resistance, and has high enzyme activity and heat resistance. A liquid detergent containing a mutated glycoside hydrolase can exhibit a stronger enzyme detergency. Therefore, the present invention also provides a detergent composition containing the mutant glycoside hydrolase of the present invention.

  The content of the mutant glycoside hydrolase of the present invention in the cleaning composition of the present invention is 0.0001% by mass or more, preferably 0.0001% by mass or more, and 5% by mass or less, preferably 2% by mass of the composition. % Or less. Moreover, it is 0.0001 mass%-5 mass%, Preferably it is 0.0001 mass%-2 mass%. In addition, enzymes having different activities can be blended within a range where they can be uniformly dissolved or dispersed.

  The cleaning composition of the present invention contains a surfactant, water and the like in addition to the glycoside hydrolase of the present invention. As the surfactant, any surfactant such as an anionic surfactant, a nonionic surfactant, an amphoteric surfactant, and a cationic surfactant may be used alone or in combination of two or more. Can be used.

Nonionic surfactants include, for example, the following:
_{R 1 O- (AO) m-} H (R 1 = C8-C22 hydrocarbon, AO = C2-C5 oxyalkylene group, m = 16 to 35) [Patent 2010-275468 JP];
_{R 1 O- (EO) l- (} AO) m- (EO) n-H (R 1 = C8-C18 hydrocarbon, EO = C2 oxyalkylene group, AO = C3-C5 oxyalkylene group, l =. 3 to 30, m = 1 to 5, l + n = 14 to 50) [JP 2010-265445, JP 2011-63784];
_{R 1 O- (EO) m /} (AO) n-H (R 1 = C8-C22 hydrocarbon, EO = C2 oxyalkylene group, AO = C3-C5 oxyalkylene group, m = 10~30, n = 0 -5, EO and AO are random or block bonds) [Japanese Patent Laid-Open No. 2010-189551];
_{R 1 (CO) lO- (EO} ) m / (AO) n-R 2 (R 1 = C8-C22 hydrocarbon, EO = C2 oxyalkylene group, AO = C3-C5 oxyalkylene group, l = 0 to 1 M = 14-50, n = 1-5, R ₂ = hydrogen (1 = 0) or C1-C3 alkyl group, and EO and AO are random or block bonds (Japanese Patent Laid-Open No. 2010-229385);
_{R 1 O- (EO) m- (} AO) n-H (R 1 = C8-C22 hydrocarbon, EO = C2 oxyalkylene group, AO = C3-C5 oxyalkylene group, m = 15~30, n = 1 -5) [Japanese Patent Laid-Open No. 2010-229387];
_{R 1 O- (AO) m /} (Gly) n-H and / or _{R 2 -COO- (AO) p /} (Gly) q-H (R 1 = C8-C22 hydrocarbon group, R ₂ = C7- C21 hydrocarbon group, AO = C2-C3 oxyalkylene group, Gly = glycerol group, m = 0-5, n = 2-10, p = 0-5, q = 2-10, AO and Gly are random or blocked Bonding) [Japanese Patent Laid-Open No. 2010-254881];
_{R 1 -COO- (PO) m /} (EO) n-R 2 (R 1 = C7-C21 hydrocarbon group, COO = carbonyl group, R ₂ = C1-C3 alkyl group, PO = oxypropylene group, EO = Oxyethylene group, m = 0.3-5, n = 8-25, PO and EO are random or block bonds) [Japanese Patent Laid-Open No. 2010-265333];
_{R 1 O- (EO) l- (} PO) m- (EO) n-H (R 1 = C8-C20 hydrocarbon, EO = C2 oxyalkylene group, PO = oxypropylene group, l> = 1, n> = 1, 0 <m <l + n, EO and PO are block bonds) [WO 98/24865];
_{R 1 O- (EO) m- (} PO) n-H ( alkyl or alkenyl group _{R 1 = C10-C16, EO} = ethylene oxide group, PO = propylene oxide group, m = 5~15, n = 1~ 3) [JP-A-8-157867];
_{R 1 (CO) - (EO} ) m-OR 2 (R 1 = C11-C13 linear or branched alkyl or alkenyl group, R ₂ = C1-C3 alkyl group, EO = ethylene oxide group, m = 10 to 20 ) [JP 2008-7706, JP 2009-7451, JP 2009-155594, JP 2009-155606];
_{R 1 (CO) - (AO} ) m-OR 2 (R 1 = C9-C13 linear or branched alkyl group or alkenyl group, AO = C2-C4 oxyalkylene group, R ₂ = C1-C3 alkyl group, m = 5 to 30) [JP 2009-144002 A, JP 2009-173858 A, JP 2010-189612 A]; and fatty acid alkanolamides, fatty acid alkanol glucamides, alkyl polyglucosides, and the like.

  Examples of the anionic surfactant include a carboxylate type anionic surfactant, a sulfonic acid type or sulfate type anionic surfactant, a non-soap anionic surfactant, a linear alkylbenzene sulfonic acid, a benzenesulfonic acid, or Its salt, polyoxyethylene alkyl sulfate ester salt, α-olefin sulfonate, alkylbenzene sulfonate, α-sulfo fatty acid salt, fatty acid soap, phosphate ester surfactant, acyl alaninate, acyl taurate, alkyl Ether carboxylic acid, alcohol sulfate, etc. are mentioned. Preferred examples include quaternary ammonium type surfactants having one long-chain alkyl group having 8 to 22 carbon atoms and tertiary amines having one long-chain alkyl group having 8 to 22 carbon atoms.

  Examples of the cationic surfactant include a quaternary ammonium salt having a long-chain alkyl group, a tertiary amine having one long-chain alkyl group, an alkyltrimethylammonium salt, a dialkyldimethylammonium salt, and an alkylpyridinium salt. . Zwitterionic activators include alkyl acetate betaine, alkanolamidopropyl acetate betaine, alkyl imidazoline, alkyl alanine, etc., alkyl betaine type, alkyl amide betaine type, imidazoline type, alkyl amino sulfone type, alkyl amino carboxylic acid type, alkyl amide carboxylic acid type Examples thereof include amphoteric surfactants of acid type, amide amino acid type or phosphoric acid type. Preferably, sulfobetaine or carbobetaine having an alkyl group having 10 to 18 carbon atoms can be used.

  Preferably, the cleaning composition of the present invention is a liquid detergent composition. The liquid detergent composition may be a concentrated liquid detergent composition. In the present specification, the “concentrated liquid detergent” may mean a liquid detergent containing 40% by mass or more of surfactant and less than 60% by mass of water. Preferably, it may be a liquid detergent containing 40 to 90% by mass of surfactant and 5% by mass or more and less than 60% by mass of water, more preferably 45 to 90% by mass of surfactant and 5% by mass of water. It may be a liquid detergent containing less than 55% by mass, and more preferably a liquid detergent containing 50 to 75% by mass of surfactant and less than 5 to 50% by mass of water. As an example of the “concentrated liquid detergent” as described above, a liquid detergent whose amount may be about half or less than the conventional amount, for example, a liquid detergent for clothing, a standard amount used for 30 L of water in a tank-type washing machine. Can be listed as 7 to 13 g or less.

  The liquid detergent composition of the present invention further comprises components usually used in liquid detergents, such as water-soluble polymers, water-miscible organic solvents, alkaline agents, chelating agents, and other enzymes other than the mutant glycoside hydrolase of the present invention. , Enzyme stabilizers, fluorescent agents, anti-staining agents and dispersants, anti-transfer agents, finishing agents, bleaching agents such as hydrogen peroxide, antioxidants, pH adjusters, buffers, preservatives, perfumes, salts , Alcohols, sugars and the like.

  As the water-soluble polymer, for example, (i) one or more selected from a polyether chain portion including a polymer unit derived from an epoxide having 2 to 5 carbon atoms and (ii) acrylic acid, methacrylic acid and maleic acid A polymer chain part comprising a polymerized unit derived from an unsaturated carboxylic acid monomer, and (i) or (ii) has a graft structure in which either one is a trunk chain and the other is a branch chain And a water-soluble polymer having an alkylene terephthalate unit and / or an alkylene isophthalate unit and an oxyalkylene unit and / or a polyoxyalkylene unit (Japanese Patent Application Laid-Open No. 2009-155606) ) And the like.

  Examples of water-miscible organic solvents include alkanols, alkylene glycols, glycerin, polyalkylene glycols, (poly) alkylene glycol (mono or di) alkyl ethers, alkyl glyceryl ethers, and (poly) alkylene glycol aromatics. Group ethers. C2-C6 alkylene glycols such as ethylene glycol, propylene glycol, butylene glycol, or hexylene glycol, glycerin, polyethylene glycol monophenyl ether, ethylene glycol monobenzyl ether, diethylene glycol monobenzyl ether, and the like are preferable. The content of the water-miscible organic solvent in the liquid detergent composition of the present invention is 1% by mass or more and 40% by mass or less, preferably 35% by mass or less. Moreover, it is 1-40 mass%, Preferably it is 1-35 mass%.

  Examples of the alkali agent include monoethanolamine, diethanolamine, triethanolamine, polyoxyalkyleneamine, and dimethylaminopropylamine as alkanolamines having 1 to 3 C2-C4 alkanols. Monoethanolamine and triethanolamine are preferred. The content of the alkaline agent in the liquid detergent composition of the present invention is 20% by mass or less, preferably 10% by mass or less. Moreover, it is 0-20 mass%, Preferably it is 0-10 mass%.

  Examples of chelating agents include nitrilotriacetic acid, iminodiacetic acid, ethylenediamineacetic acid, diethylenetriaminepentaacetic acid, glycol etherdiaminetetraacetic acid, hydroxyethyliminodiacetic acid, triethylenetetraaminehexaacetic acid, diencoric acid and other aminopolyacetic acid or salts thereof Organic acids such as diglycolic acid, oxydisuccinic acid, carboxymethyloxysuccinic acid, citric acid, lactic acid, tartaric acid, oxalic acid, malic acid, oxydisuccinic acid, gluconic acid, carboxymethylsuccinic acid, carboxymethyltartaric acid, or salts thereof, Aminotri (methylenephosphonic acid), 1-hydroxyethylidene-1,1-diphosphonic acid, ethylenediaminetetra (methylenephosphonic acid), diethylenetriaminepenta (methylenephosphonic acid), these alkali metals or Grade amine salts. The content of the chelating agent in the liquid detergent composition of the present invention is preferably 0.1% by mass or more, and is 5% by mass or less, preferably 4% by mass or less. Moreover, 0.1-5 mass% is preferable, More preferably, it is 0.1-4 mass%.

  Examples of organic acids or salts thereof include polyvalent carboxylic acids such as saturated fatty acids, succinic acid, maleic acid, fumaric acid, or salts thereof; citric acid, malic acid, glycolic acid, p-hydroxybenzoic acid, benzoic acid Or hydroxycarboxylic acids, such as those salts, are mentioned, Citric acid or its salt is especially preferable. Content of the said organic acid or its salt in the liquid detergent composition of this invention is 5 mass% or less, Preferably it is 3 mass% or less. Moreover, it is 0-5 mass%, Preferably it is 0-3 mass%.

  Examples of the recontamination preventive and dispersant include polyacrylic acid, polymaleic acid, carboxymethyl cellulose, polyethylene glycol having a weight average molecular weight of 5000 or more, maleic anhydride-diisobutylene copolymer, maleic anhydride-methyl vinyl ether copolymer, Maleic anhydride-vinyl acetate copolymer, naphthalene sulfonate formalin condensate, and claims 1-21 of JP-A-59-62614 (page 1, column 3, line 5 to page 3, column 4, line 14) Although anti-staining agents such as polymers and dispersants can be mentioned, they may be excluded if they are not suitable for blending.

  Examples of the color transfer inhibitor include polyvinyl pyrrolidone, and the content is preferably 0.01 to 10% by mass.

  As a bleaching agent, it is preferable to mix | blend 1-10 mass% bleaching agents, such as hydrogen peroxide, a percarbonate, and a perborate, for example. When using a bleaching agent, 0.01-10 mass% of bleaching activators (activators), such as tetraacetylethylenediamine (TAED) and Unexamined-Japanese-Patent No. 6-316700 description, can be mix | blended.

  Examples of the fluorescent agent include a biphenyl type fluorescent agent (such as Tinopearl CBS-X) and a stilbene type fluorescent agent (such as a DM type fluorescent dye). It is preferable to blend 0.001 to 2 mass% of the fluorescent agent.

  Examples of the enzyme other than the mutant glycoside hydrolase of the present invention include, for example, protease, other cellulase, other lichenase, other β-glucanase, hemicellulase, lipase, peroxidase, laccase, α-amylase, glucoamylase, and cutinase. , Pectinase, reductase, oxidase, phenol oxidase, ligninase, pullulanase, pectate lyase, xyloglucanase, other xylanases, pectin acetylesterase, polygalacturonase, rhamnogalacturonase, pectin lyase, mannanase, pectin methylesterase, cello Examples of the hydrolase include biohydrolase and transglutaminase, and mixtures of two or more thereof.

  Other components include, for example, boron compounds, calcium ion sources (calcium ion supply compounds), bihydroxy compounds, enzyme stabilizers such as formic acid, antioxidants such as butylhydroxytoluene, distyrenated cresol, sodium sulfite and sodium hydrogensulfite, Solubilizers such as para-toluene sulfonic acid, cumene sulfonic acid, meta-xylene sulfonic acid, benzoate (also effective as a preservative), paraffins such as octane, decane, dodecane and tridecane, and olefins such as decene and dodecene , Water-immiscible organic solvents such as methylene chloride, alkyl halides such as 1,1,1-trichloroethane, terpenes such as D-limonene, dyes, fragrances, antibacterial preservatives, defoamers such as silicone, etc. Is mentioned.

  The liquid detergent composition of the present invention is preferably, but not limited to, for garments or cloth products (sheets, curtains, carpets, wall cloths, etc.). Since the liquid detergent composition according to the present invention contains the mutant glycoside hydrolase of the present invention, the liquid detergent composition can stably contain an enzyme as compared with conventional products, and thus can exhibit high enzyme detergency. .

As exemplary embodiments of the present invention, the following compositions, methods, or uses are further disclosed herein. However, the present invention is not limited to these embodiments.

<1> In the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence, the amino acid residues at positions 138, 301 and 310 or corresponding positions thereof are as follows: Mutant glycoside hydrolase that is an amino acid residue.
(A) Position 138 or a position corresponding thereto: histidine (b) Position 301 or a position corresponding thereto: histidine (c) Position 310 or a position corresponding thereto: glutamic acid <2> Furthermore, position 248 or equivalent <1> mutated glycoside hydrolase, wherein the amino acid residue at the position is tyrosine.
<3> Further, 22nd, 80th, 85th, 99th, 120th, 180th, 186th, 239th, 240th, 243rd, 259th, 289th, 349th, 376th or equivalent <2> The mutated glycoside hydrolase, wherein at least one amino acid residue selected from the group consisting of amino acid residues at positions is the following amino acid residue:
(A) Position 22 or a position corresponding thereto: alanine (B) Position 80 or a position corresponding thereto: isoleucine (C) Position 85 or a position corresponding thereto: phenylalanine (D) Position 99 or a position corresponding thereto : Aspartic acid (E) position 120 or a position corresponding thereto: aspartic acid (F) position 180 or a position corresponding thereto: aspartic acid (G) position 186 or a position corresponding thereto: alanine (H) position 239 or Corresponding positions: isoleucine (I) position 240 or corresponding positions: leucine (J) position 243 or corresponding positions: isoleucine (K) position 259 or corresponding positions: asparagine (L) 289 Position or a position corresponding thereto: glutamic acid (M) position 349 or a position corresponding to these: meth Nin (N) 376-position or a position corresponding to, alanine <4> is selected from the following i) to v), mutation glycoside hydrolase according to claim 3.
i) The amino acid residue at position 180 or a position corresponding thereto is the following amino acid residue.
(F) Position 180 or a position corresponding thereto: aspartic acid ii) The amino acid residues at positions 99 and 186 or positions corresponding thereto are the following amino acid residues, respectively.
(D) position 99 or a position corresponding thereto: aspartic acid (G) position 186 or a position corresponding thereto: alanine iii) amino acid residues at positions 22, 180 and 349, or positions corresponding thereto; Each of the following amino acid residues:
(A) Position 22 or a position corresponding thereto: alanine (F) Position 180 or a position corresponding thereto: Aspartic acid (M) Position 349 or a position corresponding thereto: methionine iv) Positions 239, 240 and 259 Or the amino acid residues corresponding to these are the following amino acid residues, respectively.
(H) position 239 or a position corresponding thereto: isoleucine (I) position 240 or a position corresponding thereto: leucine (K) position 259 or a position corresponding thereto: asparagine v) positions 80, 120 and 376, Or the amino acid residue of the position corresponding to these is the following amino acid residue, respectively.
(B) Position 80 or a position corresponding thereto: isoleucine (E) Position 120 or a position corresponding thereto: Aspartic acid (N) Position 376 or a position corresponding thereto: alanine <5> 90% of the sequence identity The mutant glycoside hydrolase according to any one of <1> to <4>, which is 95% or more, 97% or more, 98% or more, or 99% or more.
<6> The mutant glycoside hydrolase according to any one of <1> to <5>, which has one or more enzyme activities selected from the group consisting of cellulase activity, lichenase activity, and xylanase activity.
<7> A gene encoding a mutant glycoside hydrolase according to any one of <1> to <6>.
<8> A recombinant vector containing the gene of <7>.
<9> A transformant comprising the recombinant vector according to <8>.
<10> A method for producing a mutant glycoside hydrolase using the transformant of <9>.
<11> A detergent composition containing the mutant glycoside hydrolase according to any one of <1> to <6>.
<12> In the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence, the amino acid residues at positions 138, 301 and 310 or the positions corresponding thereto are as follows: A method for improving the surfactant resistance of glycoside hydrolase, comprising substituting an amino acid residue.
(A) Position 138 or a position corresponding thereto: histidine (b) Position 301 or a position corresponding thereto: histidine (c) Position 310 or a position corresponding thereto: glutamic acid <13> Furthermore, position 248 or equivalent <12> The method according to <12>, wherein the amino acid residue at the position is tyrosine.
<14> Further, 22nd, 80th, 85th, 99th, 120th, 180th, 186th, 239th, 240th, 243rd, 259th, 289th, 349th, 376th or equivalent <13> The method according to <13>, wherein at least one amino acid residue selected from the group consisting of amino acid residues at positions is the following amino acid residue.
(A) Position 22 or a position corresponding thereto: alanine (B) Position 80 or a position corresponding thereto: isoleucine (C) Position 85 or a position corresponding thereto: phenylalanine (D) Position 99 or a position corresponding thereto : Aspartic acid (E) position 120 or a position corresponding thereto: aspartic acid (F) position 180 or a position corresponding thereto: aspartic acid (G) position 186 or a position corresponding thereto: alanine (H) position 239 or Corresponding positions: isoleucine (I) position 240 or corresponding positions: leucine (J) position 243 or corresponding positions: isoleucine (K) position 259 or corresponding positions: asparagine (L) 289 Position or a position corresponding thereto: glutamic acid (M) position 349 or a position corresponding to these: meth Nin (N) 376-position or a position corresponding to, alanine <15> is selected from the following i) to v), the method of <14>.
i) The amino acid residue at position 180 or a position corresponding thereto is the following amino acid residue.
(F) Position 180 or a position corresponding thereto: aspartic acid ii) The amino acid residues at positions 99 and 186 or positions corresponding thereto are the following amino acid residues, respectively.
(D) position 99 or a position corresponding thereto: aspartic acid (G) position 186 or a position corresponding thereto: alanine iii) amino acid residues at positions 22, 180 and 349, or positions corresponding thereto; Each of the following amino acid residues:
(A) Position 22 or a position corresponding thereto: alanine (F) Position 180 or a position corresponding thereto: Aspartic acid (M) Position 349 or a position corresponding thereto: methionine iv) Positions 239, 240 and 259 Or the amino acid residues corresponding to these are the following amino acid residues, respectively.
(H) position 239 or a position corresponding thereto: isoleucine (I) position 240 or a position corresponding thereto: leucine (K) position 259 or a position corresponding thereto: asparagine v) positions 80, 120 and 376, Or the amino acid residue of the position corresponding to these is the following amino acid residue, respectively.
(B) Position 80 or a position corresponding thereto: Isoleucine (E) Position 120 or a position corresponding thereto: Aspartic acid (N) Position 376 or a position corresponding thereto: Alanine <16> Surfactant is linear The method according to any one of <12> to <15>, which is sodium alkylbenzenesulfonate.
<17> The method according to any one of <12> to <16>, wherein the sequence identity is 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more.
<18> The method according to any one of <12> to <17>, wherein the glycoside hydrolase has one or more enzyme activities selected from the group consisting of cellulase activity, lichenase activity, and xylanase activity.

  As exemplary embodiments of the present invention, the following compositions, methods, or uses are further disclosed herein. However, the present invention is not limited to these embodiments.

Reference Example 1 Preparation of N257 Glycoside Hydrolase Regarding the method for preparing glycoside hydrolase, N257 glycoside hydrolase derived from Bacillus circulans KSM-N257 strain (FERMP-17473) is shown below as an example.
N257 glycoside hydrolase (SEQ ID NO: 2) encoding gene (SEQ ID NO: 1) [hereinafter also referred to as N257 glycoside hydrolase gene; base sequence is available based on GenBank accession number AB059267; Hakamada et al. , Biochim. Biophys. Acta. 1570, 2002, p. 174-180], the nucleic acid fragment encoding the N257 glycoside hydrolase from the N-terminal amino acid to the terminator sequence was amplified. Furthermore, a gene encoding S237 cellulase (SEQ ID NO: 3) [hereinafter also referred to as S237 cellulase gene; the nucleotide sequence is available based on GenBank accession number AB18420; Hakamada et al. Biosci. Biotechnol. Biochem. 64 (11), 2000, p. 2281-2289; JP-A-2000-210081], the nucleic acid fragment of the promoter and signal sequence was amplified.
Two nucleic acid fragments are ligated by SOE-PCR and introduced into a shuttle vector pHY300PLK (Yakult Head Office; Ishiwa, H. & Shibahara, H., Jpn. J. Genet., 1985, 60, p. 235-243). Thus, an expression plasmid containing the N257 glycoside hydrolase gene was prepared.

Specifically, first, genomic DNA was extracted from a Bacillus sp. KSM-S237 strain by a conventional method, and this was used as a template DNA to prepare primers S237UB1 FW (SEQ ID NO: 4) and S237-PS shown in Table 1. Using a primer set consisting of RV (SEQ ID NO: 5), DNA fragment 1 containing the promoter and signal sequence of the gene encoding S237 cellulase (SEQ ID NO: 3) was amplified.
Next, genomic DNA was extracted from a Bacillus circulans KSM-N257 strain by a conventional method, and this was used as a template DNA to obtain N257mat-Qster FW (SEQ ID NO: 6) and N257-Cryter RV (sequences) shown in Table 1. No. 7) was used to amplify DNA fragment 2 containing the mature protein coding region of N257 glycoside hydrolase gene and the terminator sequence (from base 338 of SEQ ID NO: 1).
Amplification of the DNA fragments 1 and 2 was performed by polymerase chain reaction (PCR) using GeneAmp PCR system (Applied Biosystems) and using Pyrobest DNA polymerase (Takara Bio) and attached reagents. The PCR reaction solution was prepared by mixing 1 μL of appropriately diluted template DNA, 20 pmol each of sense primer and antisense primer, and 2.5 U of Pyrobest DNA polymerase, and adding water to make the total reaction solution volume 50 μL. The PCR reaction is 30 cycles of three stages of temperature changes of 98 ° C. for 10 seconds, 55 ° C. for 30 seconds and 72 ° C. for 1 to 5 minutes (adjusted according to the target amplification product, but 1 minute per 1 kb). After repeating, the reaction was carried out at 72 ° C. for 5 minutes.
Using the DNA fragments 1 and 2 obtained above as a template, DNA fragments containing the S237 cellulase promoter and signal sequence, the N257 mature protein coding region, and the terminator sequence are represented by S237UB1 Fw (SEQ ID NO: 4) and N257 shown in Table 2. -Amplified by SOE-PCR using a primer set consisting of Cryter Rv (SEQ ID NO: 7).

  The shuttle vector pHY300PLK (Yakult Honsha; Ishiwa, H. & Shibahara, H., Jpn. J. Genet., 1985, 60, p. 235-243) was subjected to restriction enzyme treatment with SmaI, and the amplified fragment was inserted into it. A recombinant plasmid pHY-N257 was constructed. The N257 glycoside hydrolase gene fragment inserted in the plasmid was sequenced using a 3100 DNA sequencer (Applied Biosystems), whereby the promoter and signal sequence of the gene encoding S237 cellulase (SEQ ID NO: 3) and the N257 mature protein It was confirmed to have a nucleotide sequence linked to the coding region and terminator sequence. Subsequently, Bacillus subtilis Marburg No. 168 strain (Nature, 390, 1997, p.249)) was transformed by introducing the recombinant plasmid pHY-N257 by protoplast transformation.

In the protoplast transformation method, first, Bacillus subtilis Marburg No. 168 strain: Nature, 390, 1997, p.249 is added to 50 mL of LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl). ) At 37 ° C. for about 2 hours, and when the absorbance at 600 nm reached 0.4, the cells were collected by centrifugation (7000 rpm, 15 minutes) at room temperature. The collected cells were suspended in 5 mL of SMMP [0.5 M sucrose, 20 mM disodium maleate, 20 mM magnesium chloride hexahydrate, 35% (w / v) Antibiotic Medium 3 (Difco)], and then suspended in the SMMP solution. A dissolved 500 μL lysozyme solution (30 mg / mL) was added, and the cells were incubated at 37 ° C. for 1 hour to protoplast the cells. After the incubation, the protoplasts were collected by centrifugation (2800 rpm, 15 minutes) at room temperature and suspended in 5 mL of SMMP to prepare a protoplast solution. To 0.5 mL of protoplast solution, add 10 μL of plasmid solution (including a plasmid vector containing a gene encoding N257 glycoside hydrolase) and 1.5 mL of 40% (w / v) polyethylene glycol (PEG 8000, Sigma), After gently stirring and allowing to stand at room temperature for 2 minutes, 5 mL of SMMP solution was immediately mixed, and protoplasts were collected by centrifugation (2800 rpm, 15 minutes) at room temperature, and resuspended in 1 mL of SMMP solution. The protoplast suspension was shaken (120 rpm) at 37 ° C. for 90 minutes, and then DM3 regenerated agar medium containing tetracycline (15 μg / mL, Sigma) [0.8% (w / v) agar (Wako Pure Chemical Industries), 0 0.5% disodium succinate hexahydrate, 0.5% casamino acid technical (Difco), 0.5% yeast extract, 0.35% monopotassium phosphate, 0.15% dipotassium phosphate, 0.5 % Glucose, 0.4% magnesium chloride hexahydrate, 0.01% bovine serum albumin (Sigma), 0.5% carboxymethyl cellulose, 0.005% trypan blue (Merck) and amino acid mixture (tryptophan, leucine, methionine each 10 μg / mL)] and cultured at 30 ° C. for 72 hours, and the grown colonies were isolated as transformants.
The obtained transformant was cultured overnight at 30 ° C. in 10 mL of LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl), and 0.05 mL of this culture solution was further added to 50 mL of 2 ×. L-maltose medium (2% tryptone, 1% yeast extract, 1% NaCl, 7.5% maltose, 7.5 ppm manganese sulfate 4-5 hydrate, 15 ppm tetracycline) was inoculated and shaken at 30 ° C. for 3 days. Culture was performed. The culture solution containing N257 glycoside hydrolase obtained by the culture was centrifuged to obtain a culture supernatant. It was confirmed by SDS-polyacrylamide gel electrophoresis that the protein contained in the culture supernatant was N257 glycoside hydrolase.

Reference Example 2 Measurement of glycoside hydrolase activity (DNS method)
Cellulase activity was measured using a 96-well plate. 1.0% carboxymethylcellulose (CMC, Nippon Paper Industries) and 50 mM glycine buffer (pH 9.0) were prepared and used as a substrate solution. To 90 μL of this substrate solution, add 10 μL of enzyme solution (culture supernatant) diluted to an appropriate concentration, react at 40 ° C. for 20 minutes, add 100 μL of dinitrosalicylic acid (DNS) solution, and heat-treat at 100 ° C. for 5 minutes. Immediately cooled in ice water. 100 μL of the cooled solution was transferred to a 96-well assay plate (IWAKI), and absorbance at 535 nm was measured using a microplate reader (Molecular Device). DNA engine PTC-200 (Bio-Rad) was used for heat treatment at 40 ° C. and 100 ° C. Multi-dispenser EDR384S (Biotech) was used for sample dilution, addition to the substrate reaction solution, and DNS solution addition. Cellulase activity of glycoside hydrolase is determined by measuring the amount of reducing sugar produced by the enzyme reaction based on the absorbance and setting 1 unit (U) as the activity to release reducing sugar equivalent to 1 μmol of D-glucose per minute under the above conditions. (U) was determined.

Reference Example 3 Measurement of residual activity of glycoside hydrolase after storage in liquid detergent As an enzyme solution, a culture supernatant containing glycoside hydrolase was used. Liquid detergents for evaluation include liquid detergent formulations (nonionic surfactant [carbon number 12 to 16; polyoxyethylene alkyl ether having an average ethylene oxide addition mole number of 12.0] 20% by weight; alkylbenzyldimethylammonium chloride [ Alkyl group having 8 to 18 carbon atoms] 1% by weight; Softanol 70H (made by Nippon Shokubai) 20% by weight; acrylic acid maleic acid copolymer 1.5% by weight; monoethanolamine 1.5% by weight; citric acid 1.15% by weight Butyldiglycol 5% by weight; ethanol 2% by weight; sodium sulfite 0.2% by weight; water 47.65% by weight) (liquid detergent formulation 486 μL, 10% sodium sulfite 5.4 μL, 35 % Calcium chloride 3.1 μL and protease 5.4 μL). 500 μL of the evaluation liquid detergent was dispensed into each well of the 96-well deep well plate, and 43 μL of enzyme solution (culture supernatant) was added to prepare a test sample. Cellulase activity in the test sample was measured immediately after the enzyme solution was added and after storage at 40 ° C. for 7 days. Cellulase activity was measured in the same manner as in Reference Example 2 using a test sample diluted 10-fold with ion exchange water. The ratio of the activity after storage for 3 days to the activity immediately after addition of the enzyme solution was defined as the residual activity of the enzyme.
The protease is an alkaline protease derived from Bacillus SP KSM-KP43 (FERM BP-6532), and an enzyme solution of 80 U / mL was used. The protease activity was determined using the synthetic substrate method described in JP-A-2004-305175.

Reference Example 4 Measurement of residual activity of glycoside hydrolase after incubation An enzyme solution (culture supernatant containing glycoside hydrolase) diluted to an appropriate concentration with 50 mM glycine buffer (pH 9.0) was incubated at 60 ° C. for 10 minutes. . For the incubation operation, a PCR plate and a PCR cycler DNA engine PTC-200 (Bio-Rad) were used. The cellulase activity of the sample before and after incubation was measured by the same method as in Reference Example 2, and the remaining activity after incubation relative to the temperature before incubation (%) [(cellulase activity after incubation) / (cellulase activity before incubation) × 100] Asked.

Reference Example 5 Measurement of residual activity of glycoside hydrolase after incubation in the presence of LAS Dilute to an appropriate concentration with 50 mM glycine buffer (pH 9.0) supplemented with 1% LAS (Linear Alkylbenzene Sulfonete) The enzyme solution (culture supernatant containing glycoside hydrolase) was incubated at 40 ° C. for 60 minutes. For the incubation operation, a PCR plate and a PCR cycler DNA engine PTC-200 (Bio-Rad) were used. The cellulase activity of the sample before and after incubation was measured by the same method as in Reference Example 2, and the remaining activity after incubation relative to the temperature before incubation (%) [(cellulase activity after incubation) / (cellulase activity before incubation) × 100] Asked.

Reference Example 6 Preparation of Random Mutant Random Mutant PCR reaction was performed using the recombinant plasmid pHY-N257 or its mutant plasmid as a template and the primer Xba-F and Bam-R primer sets shown in Table 1 to generate Genemorph II Random. It was performed according to the method of Mutagenesis Kit (Agilent). The PCR reaction solution was prepared by mixing 1 μL of template DNA diluted to 20 μg / mL, 25 pmol each of Xba-F and Bam-R primers, 1 μL of 40 mM dNTP, and 1 μL of DNA polymerase, adding water, and adding the total amount of the reaction solution. Was adjusted to 50 μL. The PCR reaction was carried out under the conditions of 30 cycles of three temperature changes of 98 ° C. for 1 minute, 62 ° C. for 1 minute and 72 ° C. for 1 minute, followed by reaction at 72 ° C. for 10 minutes. The obtained PCR fragment was purified using NucleoFast 96 PCR Plate (MACHREY-NAGEL) and eluted in an appropriate amount of water.
The obtained PCR fragment and the shuttle vector pHY300PLK (Yakult Head Office; Ishiwa, H. & Shibahara, H., Jpn. J. Genet., 1985, 60, p. 235-243) were subjected to restriction enzyme treatment with XbaI and BamHI, respectively. After ligation, a random mutation library of plasmid pHY-N257 was constructed. This library was transformed into E. coli competent cell HB101 (Takara Bio). Transformants were selected by cellulase halo medium containing LAS (1% tryptone peptone, 0.5% yeast extract, 1% sodium chloride, 1.5% agar, 50 ppm tetracycline, 1% carboxymethyl cellulose, 0.01% trypan. Blue, 1% LAS) at 45 ° C. for 2 days. A transformant having a large halo was selected and a plasmid was extracted. The mutation location of the N257 glycoside hydrolase gene inserted into the plasmid was determined using a 3100 DNA sequencer (Applied Biosystems). Subsequently, plasmid pHY-N257 having a mutation extracted from Bacillus subtilis Marburg No. 168 (Nature, 390, 1997, p. 249) was transformed by protoplast transformation.
In the protoplast transformation method, first, Bacillus subtilis Marburg No. 168 strain: Nature, 390, 1997, p.249 is added to 50 mL of LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl). ) At 37 ° C. for about 2 hours, and when the absorbance at 600 nm reached 0.4, the cells were collected by centrifugation (7000 rpm, 15 minutes) at room temperature. The collected cells were suspended in 5 mL of SMMP [0.5 M sucrose, 20 mM disodium maleate, 20 mM magnesium chloride hexahydrate, 35% (w / v) Antibiotic Medium 3 (Difco)], and then suspended in the SMMP solution. A dissolved 500 μL lysozyme solution (30 mg / mL) was added, and the cells were incubated at 37 ° C. for 1 hour to protoplast the cells. After the incubation, the protoplasts were collected by centrifugation (2800 rpm, 15 minutes) at room temperature and suspended in 5 mL of SMMP to prepare a protoplast solution. To 0.5 mL of protoplast solution, add 10 μL of plasmid solution (including a plasmid vector containing a gene encoding N257 glycoside hydrolase) and 1.5 mL of 40% (w / v) polyethylene glycol (PEG 8000, Sigma), After gently stirring and allowing to stand at room temperature for 2 minutes, 5 mL of SMMP solution was immediately mixed, and protoplasts were collected by centrifugation (2800 rpm, 15 minutes) at room temperature, and resuspended in 1 mL of SMMP solution. The protoplast suspension was shaken (120 rpm) at 37 ° C. for 90 minutes, and then DM3 regenerated agar medium containing tetracycline (15 μg / mL, Sigma) [0.8% (w / v) agar (Wako Pure Chemical Industries), 0 0.5% disodium succinate hexahydrate, 0.5% casamino acid technical (Difco), 0.5% yeast extract, 0.35% monopotassium phosphate, 0.15% dipotassium phosphate, 0.5 % Glucose, 0.4% magnesium chloride hexahydrate, 0.01% bovine serum albumin (Sigma), 0.5% carboxymethyl cellulose, 0.005% trypan blue (Merck) and amino acid mixture (tryptophan, leucine, methionine each 10 μg / mL)] and cultured at 30 ° C. for 72 hours, and the grown colonies were isolated as transformants.

Example 1 Production and Evaluation of Glycoside Hydrolase Mutant SG13 In the method described in Reference Example 6, a random mutation in the wild type (parent) N257 glycoside hydrolase mature enzyme region was subjected to pHY-N257 (see Reference Example 1). As a template, Genemorph II Random Mutagenesis Kit (Agilent) was used to obtain mutant SG13 with improved LAS resistance. SG13 is a DNA sequence of N257, A at position 749 is changed to C, G at position 751 is changed to T, A at position 1238 is changed to C, A at position 1265 is changed to G, C at position 1267 is changed to A, amino acid sequence It was confirmed that the mutant was obtained by changing K at position 138 to H, N at position 301 to H, and N at position 310 to E. The plasmid extracted from the SG13 mutant is called pHY-SG13. As in Reference Example 1, plasmid pHY-SG13 extracted from E. coli was introduced into Bacillus subtilis, and the resulting transformant was cultured. From the obtained culture, the cells were removed by centrifugation to obtain a culture supernatant.

  By the method described in Reference Example 3, the residual cellulase activity of the mutant glycoside hydrolase obtained in Example 1 after storage for 7 days was measured. The relative residual activity of SG13 mutant glycoside hydrolase when the residual activity of the parent glycoside hydrolase measured by the same method after 7 days storage was taken as 100% was determined to evaluate the stability of the enzyme.

  Further, the residual activity of the mutant glycoside hydrolase obtained in Example 1 after incubation was measured by the method described in Reference Example 4. The relative residual activity of the SG13 mutant glycoside hydrolase when the residual activity after incubation of the parent glycoside hydrolase measured by the same method was taken as 100% was determined, and the heat resistance of the enzyme was evaluated.

Moreover, the residual activity of the mutant glycoside hydrolase obtained in Example 1 after incubation in the presence of LAS was measured by the method described in Reference Example 5. The relative residual activity of SG13 mutant glycoside hydrolase when the residual activity after incubation of the parent glycoside hydrolase measured by the same method was taken as 100% was determined, and the LAS resistance of the enzyme was evaluated.
The results are shown in Table 2. The SG13 mutant had improved stability, heat resistance and LAS resistance compared to the parent glycoside hydrolase (Table 3).

Example 2 Production and Evaluation of SG13 Glycoside Hydrolase Breakpoint Variant SG13_A248Y (1) Production of SG13 Glycoside Hydrolase Breakpoint Variant SG13_A248Y SG13 glycoside hydrolase is cleaved into two fragments in a liquid detergent. Was confirmed by SDS-PAGE. The amino acid N-terminal analysis of the cleaved two fragments revealed that the amino acid sequence was cleaved between alanine at position 247 and alanine at position 248.
Primer set consisting of primers A248-FW (SEQ ID NO: 10) and A248-RV (SEQ ID NO: 11) shown in Table 1 and PrimeSTAR (SEQ ID NO: 11) shown in Table 1 using plasmid pHY-SG13 extracted from E. coli and diluted sufficiently Inverse PCR was performed using a registered Mutagenesis Basal Kit (Takara Bio). Specifically, 100 pg of pHY-SG13 as a template, 2 μM of each primer (forward, reverse), and 25 μL of PrimeSTAR MAX DNA polymerase attached to Kit were prepared to 50 μL using sterilized water. The reaction was carried out in 30 cycles of denaturation at 98 ° C. for 10 seconds, annealing at 55 ° C. for 5 seconds, and extension at 72 ° C. for 45 seconds. In this PCR system, by performing inverse PCR using a plasmid as a template and a primer with 15 base overlap on the 5 ′ side, a PCR product containing all the sequences of the plasmid with the overlap portion protruding on the 5 ′ side is obtained. Can do. Since this amplification product can take a transformable circular structure, transformation can be performed directly by using this amplification product. In this example, by replacing the 3 ′ base 3 ′ immediately after the overlap sequence of the forward primer with the base sequence of the target amino acid, the reverse primer can be shared when the amino acid residue at the same position is replaced. I made it. By the above method, pHY-SG13_A248Y containing a mutation in which the position corresponding to the alanine residue at position 248 of the SG13 glycoside hydrolase gene was substituted with tyrosine was obtained. After digesting the template plasmid with the restriction enzyme DpnI (TOYOBO), the amplification product was purified using NucleoFast 96 PCR Plate (MACHREY-NAGEL). Next, as in Reference Example 1, the recombinant plasmid pHY-SG13_A248Y was introduced into Bacillus subtilis and the resulting transformant was cultured. From the obtained culture, the cells were removed by centrifugation to obtain a culture supernatant. This culture supernatant contains a mutant glycoside hydrolase (hereinafter also referred to as SG13_A248Y) in which the alanine at position 248 of the amino acid sequence of SG13 glycoside hydrolase (SEQ ID NO: 2) is substituted with tyrosine.

(2) Evaluation of SG13 Glycoside Hydrolase Breakpoint Variant SG13_A248Y By the method described in Reference Example 3, the remaining cellulase activity of SG13_A248Y glycoside hydrolase obtained in Example 2 after storage for 7 days was measured. The relative residual activity of SG13_A248Y mutant glycoside hydrolase when the residual activity after storage for 7 days of SG13 glycoside hydrolase measured by the same method was taken as 100% was determined to evaluate the stability of the enzyme.
Further, the residual activity of the SG13_A248Y mutant glycoside hydrolase obtained in Example 2 after incubation was measured by the method described in Reference Example 4. The relative residual activity of SG13_A248Y mutant glycoside hydrolase when the residual activity after incubation of SG13 glycoside hydrolase measured by the same method was taken as 100% was determined, and the heat resistance of the enzyme was evaluated.
Further, the residual activity of the SG13_A248Y mutant glycoside hydrolase obtained in Example 2 after incubation in the presence of LAS was measured by the method described in Reference Example 5. The relative residual activity of SG13_A248Y mutant glycoside hydrolase when the residual activity after incubation of SG13 glycoside hydrolase measured by the same method was taken as 100% was determined, and the LAS resistance of the enzyme was evaluated.
The results are shown in Table 3. The SG13_A248Y mutant had improved stability, heat resistance and LAS resistance compared to SG13 glycoside hydrolase (Table 4).

Example 3 Production and Evaluation of Random Mutant from SG13_A248Y Glycoside Hydrolase According to the method described in Reference Example 6, a random mutation in the SG13_A248Y glycoside hydrolase mature enzyme region was performed using pHY-SG13_A248Y as a template and Genemorph II Random Mutagenesis Kit (Agilent Mutants with improved LAS resistance were obtained (Table 4). In the same manner as in Reference Example 1, a plasmid extracted from E. coli was introduced into Bacillus subtilis, and the obtained transformant was cultured. From the obtained culture, the cells were removed by centrifugation to obtain a culture supernatant.
By the method described in Reference Example 5, the residual activity of the mutant glycoside hydrolase obtained in Example 3 after incubation in the presence of LAS was measured. The relative residual activity of the mutant glycoside hydrolase was determined when the residual activity after incubation of SG13_A248 glycoside hydrolase measured by the same method was taken as 100%, and the LAS resistance of the enzyme was evaluated.
The results are shown in Table 4. All mutants had improved LAS resistance compared to SG13_A248 glycoside hydrolase (Table 5).

Claims (18)

In the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence, the amino acid residues at positions 138, 301 and 310 or the positions corresponding thereto are the following amino acid residues: A mutated glycoside hydrolase.
(A) Position 138 or a position corresponding thereto: histidine (b) Position 301 or a position corresponding thereto: histidine (c) Position 310 or a position corresponding thereto: glutamic acid   The mutant glycoside hydrolase according to claim 1, wherein the amino acid residue at position 248 or a corresponding position is tyrosine. Furthermore, amino acids at positions 22, 80, 85, 99, 120, 180, 186, 239, 240, 243, 259, 289, 349, 376 or the equivalent positions The mutant glycoside hydrolase according to claim 2, wherein at least one amino acid residue selected from the group consisting of residues is the following amino acid residue.
(A) Position 22 or a position corresponding thereto: alanine (B) Position 80 or a position corresponding thereto: isoleucine (C) Position 85 or a position corresponding thereto: phenylalanine (D) Position 99 or a position corresponding thereto : Aspartic acid (E) position 120 or a position corresponding thereto: aspartic acid (F) position 180 or a position corresponding thereto: aspartic acid (G) position 186 or a position corresponding thereto: alanine (H) position 239 or Corresponding positions: isoleucine (I) position 240 or corresponding positions: leucine (J) position 243 or corresponding positions: isoleucine (K) position 259 or corresponding positions: asparagine (L) 289 Position or a position corresponding thereto: glutamic acid (M) position 349 or a position corresponding to these: meth Nin (N) 376-position or a position corresponding to, alanine The mutant glycoside hydrolase according to claim 3, which is selected from the following i) to v).
i) The amino acid residue at position 180 or a position corresponding thereto is the following amino acid residue.
(F) Position 180 or a position corresponding thereto: aspartic acid ii) The amino acid residues at positions 99 and 186 or positions corresponding thereto are the following amino acid residues, respectively.
(D) position 99 or a position corresponding thereto: aspartic acid (G) position 186 or a position corresponding thereto: alanine iii) amino acid residues at positions 22, 180 and 349, or positions corresponding thereto; Each of the following amino acid residues:
(A) Position 22 or a position corresponding thereto: alanine (F) Position 180 or a position corresponding thereto: Aspartic acid (M) Position 349 or a position corresponding thereto: methionine iv) Positions 239, 240 and 259 Or the amino acid residues corresponding to these are the following amino acid residues, respectively.
(H) position 239 or a position corresponding thereto: isoleucine (I) position 240 or a position corresponding thereto: leucine (K) position 259 or a position corresponding thereto: asparagine v) positions 80, 120 and 376, Or the amino acid residue of the position corresponding to these is the following amino acid residue, respectively.
(B) Position 80 or a position corresponding thereto: Isoleucine (E) Position 120 or a position corresponding thereto: Aspartic acid (N) Position 376 or a position corresponding thereto: alanine   The mutant glycoside hydrolase according to any one of claims 1 to 4, wherein the sequence identity is 90% or more.   The mutant glycoside hydrolase according to any one of claims 1 to 5, which has one or more enzyme activities selected from the group consisting of cellulase activity, lichenase activity and xylanase activity.   A gene encoding the mutant glycoside hydrolase according to any one of claims 1 to 6.   A recombinant vector containing the gene according to claim 7.   A transformant comprising the recombinant vector according to claim 8.   A method for producing a mutant glycoside hydrolase using the transformant according to claim 9.   A detergent composition comprising the mutant glycoside hydrolase according to any one of claims 1 to 6. In the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 80% or more sequence identity with the amino acid sequence, the amino acid residues at positions 138, 301 and 310 or the positions corresponding thereto are the following amino acid residues: A method for improving the surfactant resistance of glycoside hydrolase, which comprises substituting in
(A) Position 138 or a position corresponding thereto: histidine (b) Position 301 or a position corresponding thereto: histidine (c) Position 310 or a position corresponding thereto: glutamic acid   The method according to claim 12, wherein the amino acid residue at position 248 or a position corresponding thereto is tyrosine. Furthermore, amino acids at positions 22, 80, 85, 99, 120, 180, 186, 239, 240, 243, 259, 289, 349, 376 or the equivalent positions The method according to claim 13, wherein at least one amino acid residue selected from the group consisting of residues is the following amino acid residue.
(A) Position 22 or a position corresponding thereto: alanine (B) Position 80 or a position corresponding thereto: isoleucine (C) Position 85 or a position corresponding thereto: phenylalanine (D) Position 99 or a position corresponding thereto : Aspartic acid (E) position 120 or a position corresponding thereto: aspartic acid (F) position 180 or a position corresponding thereto: aspartic acid (G) position 186 or a position corresponding thereto: alanine (H) position 239 or Corresponding positions: isoleucine (I) position 240 or corresponding positions: leucine (J) position 243 or corresponding positions: isoleucine (K) position 259 or corresponding positions: asparagine (L) 289 Position or a position corresponding thereto: glutamic acid (M) position 349 or a position corresponding to these: meth Nin (N) 376-position or a position corresponding to, alanine 15. A method according to claim 14, selected from the following i) to v).
i) The amino acid residue at position 180 or a position corresponding thereto is the following amino acid residue.
(F) Position 180 or a position corresponding thereto: aspartic acid ii) The amino acid residues at positions 99 and 186 or positions corresponding thereto are the following amino acid residues, respectively.
(D) position 99 or a position corresponding thereto: aspartic acid (G) position 186 or a position corresponding thereto: alanine iii) amino acid residues at positions 22, 180 and 349, or positions corresponding thereto; Each of the following amino acid residues:
(A) Position 22 or a position corresponding thereto: alanine (F) Position 180 or a position corresponding thereto: Aspartic acid (M) Position 349 or a position corresponding thereto: methionine iv) Positions 239, 240 and 259 Or the amino acid residues corresponding to these are the following amino acid residues, respectively.
(H) position 239 or a position corresponding thereto: isoleucine (I) position 240 or a position corresponding thereto: leucine (K) position 259 or a position corresponding thereto: asparagine v) positions 80, 120 and 376, Or the amino acid residue of the position corresponding to these is the following amino acid residue, respectively.
(B) Position 80 or a position corresponding thereto: Isoleucine (E) Position 120 or a position corresponding thereto: Aspartic acid (N) Position 376 or a position corresponding thereto: alanine   The method according to any one of claims 12 to 15, wherein the surfactant is sodium linear alkylbenzene sulfonate.   The method according to any one of claims 12 to 16, wherein the sequence identity is 90% or more.   The method according to any one of claims 12 to 17, wherein the glycoside hydrolase has one or more enzyme activities selected from the group consisting of cellulase activity, lichenase activity and xylanase activity.