JP2017035001A - Heat resistant xylanase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a xylanase that can more efficiently glycosylate biomass and has excellent thermal stability.SOLUTION: The present invention provides (1) a xylanase selected from the following proteins (a) to (c): (a) a protein having a kinase activity and consisting of a specific amino acid sequence in which one or more of alanine at positions 77, 214, and 328 substituted with proline; (b) a protein having a kinase activity and consisting of an amino acid sequence having 90% or more identity except for the amino acids at positions 77, 214 and 328 in the amino acid sequence of the protein of the (a); and (c) a protein having a kinase activity and consisting of an amino acid sequence having one or several amino acids being deleted, substituted, or added at positions other than positions 77, 214, and 328 in the amino acid sequence of the protein of the (a).SELECTED DRAWING: None

Description

  The present invention relates to a xylanase having heat resistance.

  A technique for producing sugar from cellulose in a biomass material containing cellulose (hereinafter sometimes referred to as “biomass”) and converting it into ethanol, lactic acid or the like by a fermentation method has been known. Furthermore, in recent years, development of technology that efficiently uses biomass as a resource has attracted attention from the viewpoint of tackling environmental problems.

  Biomass is composed of cellulose fibers and hemicellulose and lignin mainly containing xylan surrounding them. In order to increase the saccharification efficiency of cellulose and hemicellulose in biomass, it is necessary to develop an enzyme that hydrolyzes cellulose and hemicellulose. It is also necessary to remove lignin from the biomass.

  Conventionally, saccharification of biomass by cellulase and enzymatic degradation of biomass by hemicellulase and ligninase have been performed. For example, an enzyme composition in which hemicellulase such as xylanase derived from Trichoderma reesei or arabinofuranosidase is added to cellulase has been developed (Patent Document 1). Moreover, the manufacturing method of the saccharide | sugar by making the protein which has the xylanase activity derived from the microorganisms group which exists in bagasse compost, and the said protein and cellulase react with biomass resources is known (patent document 2). Furthermore, xylanase preparations (such as Cellic (registered trademark) HTec) manufactured and sold by Novozymes are also known.

However, conventional biomass saccharification methods using hemicellulase or xylanase have not yet been satisfactory in terms of saccharification efficiency, monosaccharide productivity, or enzyme cost.
Furthermore, the conventional xylanase has a relatively narrow optimum pH range and is inferior in terms of heat stability, and thus has limited applications.

Special table 2011-515089 gazette JP 2012-029678 A

  The present invention relates to providing a xylanase that can saccharify biomass more efficiently and has excellent thermal stability.

  The present inventors have found a novel xylanase derived from bacteria belonging to the genus Cellulomonas that can exhibit a high xylanase activity in a wide pH range, and have already filed a patent application (Japanese Patent Application No. 2012-119034). As a result of further investigation, it was found that the thermal stability can be improved by substituting the amino acid residue at a predetermined position of the xylanase with another amino acid residue.

That is, the present invention relates to the following (1) to (6).
(1) A xylanase selected from proteins represented by the following (a) to (c).
(A) A protein having an xylanase activity, comprising an amino acid sequence in which any one or more of alanine at positions 77, 214 and 328 is substituted with proline in the amino acid sequence represented by SEQ ID NO: 2.
(B) A protein comprising an amino acid sequence having 90% or more identity other than the amino acids at positions 77, 214 and 328 with the amino acid sequence of the protein of (a) and having xylanase activity.
(C) in the amino acid sequence of the protein of (a), consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added at positions other than the 77th, 214th and 328th amino acids; And a protein having xylanase activity.
(2) A gene encoding the xylanase according to any one of (1).
(3) A recombinant vector containing the gene of (2).
(4) A transformant obtained by introducing the recombinant vector of (3) into a host.
(5) An enzyme preparation for biomass saccharification containing the protein of (1).
(6) A method for producing sugar, comprising a step of saccharifying biomass with the enzyme preparation of (5).

  The xylanase of the present invention has a wide range of applications because it acts over a relatively wide range of pH conditions and has excellent xylanase activity at 50-65 ° C. Therefore, if this is used, biomass can be efficiently saccharified even under severe conditions.

  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 Lippman-Person method (Lipman-Pearson method; Science, 227, 1435, (1985)) and homology of genetic information processing software Genetyx-Win (Ver. 5.1.1; software development). Using an analysis (Search homology) program, it can be calculated by performing an analysis with Unit size to compare (ktup) of 2.

  As used herein, “xylanase activity” refers to an activity of hydrolyzing a xylose β-1,4-glycoside bond in xylan. The xylanase activity of a protein can be determined, for example, by reacting with the protein using xylan as a substrate and measuring the amount of xylan degradation product produced. Specific procedures for measuring xylanase activity are described in detail in the examples below.

  In the present specification, the “amino acid residue at the corresponding position” is identified by comparing the target amino acid sequence with a reference sequence using a known algorithm, and maximizing the conserved amino acid residue present in the amino acid sequence of each xylanase. This can be done by aligning the sequences so as to confer homology. By aligning the amino acid sequence of xylanase in this way, it is possible to determine the position of the homologous amino acid residue in the sequence of each xylanase regardless of insertion or deletion in the amino acid sequence. 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. Clustal W is, for example, the European Bioinformatics Institute (EBI, [www.ebi.ac.uk/index.html]) and the Japan DNA Data Bank (DDBJ, [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 ".

The xylanase of the present invention includes proteins represented by the following (a) to (c).
(A) A protein having an xylanase activity, comprising an amino acid sequence in which any one or more of alanine at positions 77, 214 and 328 is substituted with proline in the amino acid sequence represented by SEQ ID NO: 2.
(B) A protein comprising an amino acid sequence having 90% or more identity other than the amino acids at positions 77, 214 and 328 with the amino acid sequence of the protein of (a) and having xylanase activity.
(C) in the amino acid sequence of the protein of (a), consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added at positions other than the 77th, 214th and 328th amino acids; And a protein having xylanase activity.

Examples of the “amino acid sequence represented by SEQ ID NO: 2” shown in (a) include a xylanase derived from a bacterium belonging to the genus Cellulomonas, specifically, an amino acid sequence derived from Cellulomonas fimi ATCC484 xylanase (Japanese Patent Application No. 2012-1119034).
In the amino acid sequence represented by SEQ ID NO: 2, the amino acids at position 77 (77th from the N-terminal), position 214 (214th from the N-terminal) and position 328 (position 328 from the N-terminal) are alanine. The xylanase represented by (a) has a xylanase consisting of the amino acid sequence represented by SEQ ID NO: 2 as a reference xylanase, and any one or more of alanine residues at positions 77, 214 and 328 of the xylanase are proline residues Is a mutant (also referred to as mutant xylanase). The amino acid residue substitution may be naturally occurring or artificially introduced.
Such a mutant xylanase has improved heat resistance compared to the reference xylanase.

  In the present invention, in addition to the xylanase shown in (a), in addition to the substitution of the amino acid residue at the position shown in (a) as in (b) and (c), the xylanase activity is not impaired. Insofar as xylanases with mutations (eg deletions, substitutions or additions) at any other position are included. The mutation at the arbitrary position may be naturally occurring or artificially introduced.

In (b), “the amino acid sequence having 90% or more identity with the amino acid sequence of the protein of (a) other than the amino acids at positions 77, 214, and 328” includes the amino acid of the protein of (a) Among the sequences, amino acid sequences having 90% or more, preferably 95% or more, more preferably 98% or more identity with the remaining amino acid sequences excluding the amino acids at positions 77, 214 and 328 are mentioned. .
In the xylanase represented by (b), a protein having xylanase activity consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2 is used as a reference xylanase, position 77 of SEQ ID NO: 2, Proteins comprising an amino acid sequence in which any one or more alanines at positions corresponding to positions 214 and 328 are substituted with proline and having xylanase activity are included.

In (c), “in the amino acid sequence of the protein of (a), an amino acid sequence in which one or several amino acids are deleted, substituted or added at positions other than the amino acids at positions 77, 214 and 328” In the amino acid sequence of the protein of (a), in the remaining amino acid sequence excluding the amino acids at positions 77, 214 and 328, 1 to 10 amino acids, preferably 1 to 5, more preferably 1 An amino acid sequence in which ˜3, more preferably 1-2 are deleted, substituted, or added is mentioned. Here, “amino acid deletion” means deletion or disappearance of an amino acid residue in the sequence, and “amino acid substitution” means that an amino acid residue in the sequence is replaced with another amino acid residue. By “amino acid addition” is meant that an amino acid residue has been added. “Addition” includes insertion of another amino acid residue between amino acids in the sequence.
In the xylanase represented by (c), a protein having a xylanase activity consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added to the amino acid sequence represented by SEQ ID NO: 2 is used as a reference xylanase. And a protein having an xylanase activity comprising an amino acid sequence in which one or more alanine residues at positions corresponding to positions 77, 214, and 328 of SEQ ID NO: 2 are substituted with proline residues. The

  Arbitrary mutations made in the proteins having the xylanase activity of (b) and (c) above are, for example, ultraviolet irradiation or site-directed mutagenesis (Site-Directed Mutagenesis) to the gene encoding the target xylanase. It can be produced by introducing a mutation by a known mutagenesis method such as, expressing a gene having the mutation, and selecting a protein having a desired xylanase activity. The procedure for producing such a mutant is well known to those skilled in the art.

The gene encoding the xylanase of the present invention is a gene encoding the protein represented by the above (a) to (c), and specific examples thereof include the following genes (d) to (f). The invention is not limited to this. In the present invention, the “gene encoding xylanase” means any nucleic acid fragment (including DNA, mRNA, artificial nucleic acid, etc.) encoding the amino acid sequence of xylanase. The “gene” according to the present invention may include other base sequences such as an untranslated region (UTR) in addition to the open reading frame.
(D) In the base sequence represented by SEQ ID NO: 1, at least one base selected from alanine selected from positions 229 to 231, 640 to 642, and 982 to 984 of SEQ ID NO: 1 encodes proline. A gene consisting of a base sequence substituted with a base and encoding a protein having xylanase activity.
(E) In the base sequence of the gene of (d), the identity is 90% or more, preferably 95 except for the bases corresponding to positions 229 to 231, 640 to 642 and 982 to 984 of SEQ ID NO: 1. %, More preferably 98% or more of a base sequence, and a gene encoding a protein having xylanase activity.
(F) In the base sequence of the gene of (d), one or several, preferably at positions other than the bases corresponding to positions 229 to 231, 640 to 642, and 982 to 984 of SEQ ID NO: 1, A gene encoding a protein having a base sequence in which 1 to 15 bases, more preferably 1 to 6 bases are deleted, substituted or added, and having xylanase activity. “Addition” includes insertion of another base between bases in the sequence.

  The xylanase of the present invention can be obtained, for example, by the following method. That is, the gene encoding the cloned reference xylanase is subjected to the mutation of the present invention using various mutagenesis techniques known in the art. For example, with respect to the gene encoding xylanase represented by SEQ ID NO: 1, the base sequence encoding the amino acid residue to be replaced is mutated to the base sequence encoding the amino acid residue after substitution. Then, by transforming an appropriate host using the obtained mutant gene, culturing the recombinant host, and collecting from the culture, the mutation in which the amino acid residue to be replaced is replaced with the desired amino acid residue Xylanase can be obtained.

  The gene encoding the reference xylanase can be isolated from bacteria of the genus Cellulomonas, such as Cellulomonas fimi (ATCC 484), using any method used in the art. For example, the gene encoding the xylanase consisting of the amino acid sequence shown in SEQ ID NO: 2 is obtained by extracting the total genomic DNA of the Cellulomonas genus bacteria and then performing PCR using a primer designed based on the base sequence of SEQ ID NO: 1 Can be obtained by selectively amplifying and purifying the amplified gene. Alternatively, the gene can be synthesized genetically or chemically based on the amino acid sequence of the xylanase of the present invention.

  As a means for mutating a gene encoding a reference xylanase, basically, PCR amplification using a gene encoding a reference xylanase (for example, a DNA comprising the base sequence represented by SEQ ID NO: 1) as a template DNA or various DNA polymerases Based on the replication reaction, various site-directed mutagenesis methods well known to those skilled in the art 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 can be most commonly performed using a mutation primer containing a nucleotide mutation to be introduced. Such a mutation primer is annealed to a region containing a nucleotide sequence encoding the amino acid residue to be substituted in the reference xylanase gene, and replaced in place of the nucleotide sequence (codon) encoding the amino acid residue to be substituted. What is necessary is just to design so that the base sequence which has the nucleotide sequence (codon) which codes a subsequent amino acid residue may be included. 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.

  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.). By using a primer set containing a mutation primer and carrying out site-specific mutagenesis as described above using a reference xylanase gene as a template DNA, a gene encoding a xylanase into which the target mutation has been introduced can be obtained.

  As a more specific method for preparing a gene encoding the xylanase of the present invention, first, amino acids 77, 214 and 328 in the amino acid sequence shown in SEQ ID NO: 2 are substituted with proline using the recovered plasmid as a template. A DNA fragment is amplified by PCR using an oligonucleotide containing a base sequence encoding the amino acid sequence as a primer. Here, as a preferable example of the PCR reaction conditions, a heat denaturation reaction for converting a double-stranded DNA into a single strand is performed at 98 ° C. for 10 seconds, and an annealing reaction for hybridizing the primer pair to the single-stranded DNA at 50 ° C. An extension reaction for 15 seconds is carried out at 68 ° C. for 9 minutes, and one cycle of these is carried out for 40 cycles.

The DNA amplified by the PCR reaction is treated with a DpnI enzyme that specifically cleaves methylated DNA, and E. coli is transformed with the enzyme, and selected on a plate medium containing antibiotics. By extracting a plasmid from the transformed Escherichia coli, a gene encoding a xylanase into which the target amino acid mutation has been introduced can be obtained.

  Production of xylanase using the obtained gene encoding the xylanase of the present invention can be performed by, for example, transforming a host bacterium by ligating the gene with a DNA vector capable of stably amplifying, or maintaining the mutated gene stably. And inoculating the host strain into a medium containing an assimilable carbon source, nitrogen source and other essential nutrients, and culturing according to a conventional method.

  The type of vector is not particularly limited, and examples thereof include vectors usually used for protein production, such as plasmids, cosmids, phages, viruses, YACs, and BACs. A plasmid vector is preferable. For example, a commercially available plasmid vector for protein expression such as shuttle vector pHY300PLK (Takara Bio), pUC19 (Takara Bio), pUC119 (Takara Bio), pBR322 (Takara Bio) and the like can be suitably used.

  The vector may include a DNA fragment containing a DNA replication initiation region and a DNA region containing a replication origin. Further, in the above vector, upstream of the gene encoding the xylanase of the present invention, a promoter region for initiating transcription of the gene, a secretory signal region for secreting the expressed protein outside the cell, etc. The arrays may be operably linked. Alternatively, a drug resistance gene (ampicillin, neomycin, kanamycin, chloramphenicol, etc.) for selecting a microorganism into which the plasmid of the present invention has been appropriately introduced may be further incorporated. Examples of the control sequences include S237egl promoter and signal sequence (Biosci. Biotechnol. Biochem., 64 (11): 2281-9, 2000), and Bacillus sp. KSM-237 strain (FERM BP-7875) and Bacillus Examples thereof include a transcription initiation regulatory region, a translation initiation region and a secretory signal peptide region of a cellulase gene derived from sp. KSM-64 strain (FERM BP-2886). Alternatively, as the control region, the base sequence of base numbers 1 to 662 of the base sequence represented by SEQ ID NO: 3, the base sequence of base numbers 1 to 696 of the cellulase gene comprising the base sequence represented by SEQ ID NO: 4, or these Having a sequence identity of 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, and even more preferably 98% or more, and a transcription initiation control function, Examples thereof include a base sequence having a translation initiation control function and a function as a secretory signal peptide. Ligation of the xylanase gene with the above regulatory sequences and drug resistance gene can be performed by a method such as SOE (splicing by overlap extension) -PCR (Gene, 77, 61, 1989). The procedure for introducing a gene into a plasmid vector is well known in the art. In addition, the expression that the gene and the control sequence are “operably linked” means that the gene is arranged so that it can be expressed under the control of the control region.

  Examples of microorganisms for introducing the constructed vector include Staphylococcus genus, Enterococcus genus, Listeria genus, bacteria belonging to the genus Bacillus, etc., among these, Bacillus genus bacteria such as Bacillus subtilis or mutants thereof (for example, And a protease 9-deficient strain KA8AX described in JP-A-2006-174707) are preferred. As the introduction method, a method usually used in the art such as a protoplast method and electroporation can be used. A strain into which the introduction has been appropriately carried out can obtain the desired transformant by selecting drug resistance or the like as an index.

  When the transformant thus obtained is cultured in an appropriate medium, the gene encoded on the vector contained in the transformant is expressed, and the xylanase of the present invention is produced. The medium used for the culture can be appropriately selected by those skilled in the art according to the type of transformant. The protein of the present invention can be obtained by isolating or purifying the produced xylanase from the culture by a conventional method. At this time, when the xylanase gene and the secretory signal sequence are operably linked on the vector, the produced xylanase is secreted outside the cell body, so that recovery becomes easier. The recovered xylanase may be further purified by a known means.

Since the xylanase of the present invention acts in a relatively wide range of pH conditions and has an excellent xylanase activity at 50 to 65 ° C., the composition containing the xylanase of the present invention is used for biomass saccharification. It can be an enzyme preparation.
Here, “biomass” refers to cellulosic and / or lignocellulosic biomass containing hemicellulose components produced by plants and algae. Specific examples of biomass include various types of wood obtained from conifers such as larch and cedar, and broad-leaved trees such as oil palm (stems) and cypress; processed or pulverized products of wood such as wood chips; wood pulp produced from wood Pulp such as cotton linter pulp obtained from fibers around cotton seeds; Paper such as newspaper, cardboard, magazines, fine paper; bagasse (sugar cane residue), palm empty fruit bunch (EFB), rice straw Stalks of plants such as corn stalks or leaves, leaves, fruit bunches, etc .; plant shells such as rice husks, palm husks, coconut husks, algae and the like. Of these, from the viewpoints of availability and raw material costs, wood, processed or pulverized wood, plant stems, leaves, fruit bunches, etc. are preferred, bagasse, EFB, oil palm (stem) are more preferred, and bagasse is further. preferable. You may use the said biomass individually or in mixture of 2 or more types. Moreover, said biomass may be dried.

In addition to the xylanase of the present invention, the enzyme preparation for biomass saccharification preferably further contains cellulase from the viewpoint of improving saccharification efficiency. Here, cellulase refers to an enzyme that hydrolyzes the glycosidic bond of β-1,4-glucan of cellulose, and is a generic term for enzymes called endoglucanase, exoglucanase or cellobiohydrolase, β-glucosidase, and the like. It is. As the cellulase used in the present invention, commercially available cellulase preparations and cellulases derived from animals, plants and microorganisms may be mentioned.
In the present invention, these cellulases may be used alone or in combination of two or more. From the viewpoint of improving saccharification efficiency, the cellulase preferably contains endoglucanase.

  Specific examples of the cellulase include cellulase derived from Trichoderma reesei; cellulase derived from Trichoderma viride; Bacillus sp. KSM-N145 (FERM P-19727) and Bacillus sp. ) KSM-N252 (FERM P-17474), Bacillus sp. KSM-N115 (FERM P-19726), Bacillus sp. KSM-N440 (FERM P-19728), Bacillus sp (Bacillus sp.) .) Cellulases derived from various Bacillus strains such as KSM-N659 (FERM P-19730); thermostable cellulases derived from Pyrococcus horikoshii; cellulases derived from Humicola insolens; Etc., and the like. Among these, cellulase derived from Trichoderma reesei, Trichoderma viride, or Humicola insolens is preferable from the viewpoint of improving saccharification efficiency. Specific examples of cellulase preparations containing the cellulase include Cellcrust (registered trademark) 1.5 L (manufactured by Novozymes), TP-60 (manufactured by Meiji Seika Co., Ltd.), Cellic (registered trademark) CTec2 (manufactured by Novozymes), Examples include Accelerase ™ DUET (manufactured by Genencor) and Ultraflo (registered trademark) L (manufactured by Novozymes). Of these, Cellic (registered trademark) CTec2 and AcceleraseTM DUET (manufactured by Genencor) are preferable, and Cellic (registered trademark) CTec2 is more preferable from the viewpoint of improving saccharification efficiency, reducing manufacturing costs, and obtaining availability.

  Specific examples of β-glucosidase, which is a kind of cellulase, include β-glucosidase derived from Aspergillus niger (for example, Novozymes Novozyme 188 and Megazyme β-glucosidase), and Trichoderma lysase (Trichoderma lysase). reesei) or Penicillium emersonii-derived β-glucosidase. Among these, from the viewpoint of improving saccharification efficiency, Novozyme 188 and β-glucosidase derived from Trichoderma reesei are preferable, and β-glucosidase derived from Trichoderma reesei is more preferable.

  Specific examples of endoglucanase, which is a kind of cellulase, include Trichoderma reesei, Acremonium celluloriticus, Humicola insolens, Clostridium thermocellum, Bacillus. ), Enzymes derived from Thermobifida, Cellulomonas, and the like. Among these, from the viewpoint of improving saccharification efficiency, endoglucanases derived from Trichoderma lysae, Humicola insolens, Bacillus and Cellulomonas are preferable, and endoglucanases derived from Trichoderma lyse are more preferable.

  Moreover, the enzyme preparation of the present invention may contain a hemicellulase other than the xylanase of the present invention. Here, hemicellulase refers to an enzyme that hydrolyzes hemicellulose, and is a general term for enzymes called xylanase, xylosidase, galactanase, and the like. Specific examples of hemicellulases other than the xylanase of the present invention include a hemicellulase derived from Trichoderma reesei; a xylanase derived from Bacillus sp. KSM-N546 (FERM P-19729); an Aspergillus niger ), Trichoderma viride, Humicola insolens, or xylanase from Bacillus alcalophilus; Thermomyces, Aureobasidium, Streptomyces, Cross (Clostridium), Thermomotoga, Thermoascus, Caldocellum, or Thermomonospora xylanase, Bacillus pumilus Bacillus pumilus) derived from β- xylosidase include Serenomonasu Ruminantiumu (Selenomonas ruminantium) from β- xylosidase. Among these, from the viewpoint of improving saccharification efficiency, the enzyme preparation of the present invention preferably contains a xylanase derived from Bacillus sp. Alternatively, it is more preferable to contain a xylalase derived from Trichoderma viride or β-xylosidase derived from Selenomonas ruminantium.

The content of the xylanase of the present invention in the total protein amount of the enzyme preparation of the present invention is preferably 0.5% by mass or more, more preferably 1% by mass or more, further preferably 2% by mass from the viewpoint of improving saccharification efficiency. % And preferably 70% by mass or less, more preferably 50% by mass or less, still more preferably 40% by mass or less, and still more preferably 30% by mass or less. Moreover, Preferably it is 0.5-70 mass%, More preferably, it is 1-50 mass%, More preferably, it is 2-40 mass%, More preferably, it is 2-30 mass%.
The content of the cellulase in the total protein amount of the enzyme preparation of the present invention is preferably 10% by mass or more, more preferably 30% by mass or more, and further preferably 50% by mass or more, from the viewpoint of improving saccharification efficiency. And preferably 99% by mass or less, more preferably 95% by mass or less. Moreover, Preferably it is 10-99 mass%, More preferably, it is 30-95 mass%, More preferably, it is 50-95 mass%.
Further, the content of the endoglucanase in the total protein amount of the enzyme preparation of the present invention is preferably 1% by mass or more, more preferably 5% by mass or more, and further preferably 10% by mass from the viewpoint of improving saccharification efficiency. And preferably 70% by mass or less, more preferably 50% by mass or less, and still more preferably 40% by mass or less. Moreover, Preferably it is 1-70 mass%, More preferably, it is 5-50 mass%, More preferably, it is 10-40 mass%.
Further, the content of hemicellulase other than the xylanase of the present invention in the total protein amount of the enzyme preparation of the present invention is preferably 0.01% by mass or more, more preferably 0.8% from the viewpoint of improving saccharification efficiency. 1 mass% or more, More preferably, it is 0.5 or more mass%, Preferably it is 30 mass% or less, More preferably, it is 20 mass% or less. Moreover, Preferably it is 0.01-30 mass%, More preferably, it is 0.1-20 mass%, More preferably, it is 0.5-20 mass%.

  In the enzyme preparation of the present invention, the protein amount ratio of the xylanase of the present invention to the cellulase (xylanase / cellulase of the present invention) is preferably 0.01 or more, more preferably 0.05 or more, from the viewpoint of improving saccharification efficiency. And preferably 100 or less, more preferably 5 or less, still more preferably 1 or less, and still more preferably 0.5 or less. Moreover, Preferably it is 0.01-100, More preferably, it is 0.05-5, More preferably, it is 0.05-1 and More preferably, it is 0.05-0.5.

  In the enzyme preparation of the present invention, the protein amount ratio of the xylanase of the present invention to the above endoglucanase (xylanase / endoglucanase of the present invention) is preferably 0.05 or more, more preferably 0.1, from the viewpoint of improving saccharification efficiency. And preferably 10 or less, more preferably 5 or less, still more preferably 2 or less, and still more preferably 1 or less. Moreover, Preferably it is 0.05-10, More preferably, it is 0.1-5, More preferably, it is 0.1-2, More preferably, it is 0.2-1.

The sugar production method of the present invention includes a step of saccharifying biomass with the xylanase or enzyme preparation of the present invention.
The conditions for the saccharification treatment are not particularly limited as long as the xylanase of the present invention and other enzymes added at the same time are not inactivated, and those skilled in the art appropriately determine the type of biomass, the procedure of the pretreatment step, and the type of enzyme used. However, it is preferable to add the xylanase or enzyme preparation to a suspension containing biomass. The content of biomass in the suspension is preferably 0.5% by mass or more, more preferably 3% by mass or more, and further preferably 5% by mass, from the viewpoint of improving saccharification efficiency and productivity (reducing production time). And preferably 20% by mass or less, more preferably 15% by mass or less, and still more preferably 10% by mass or less. Moreover, Preferably it is 0.5-20 mass%, More preferably, it is 3-15 mass%, More preferably, it is 5-10 mass%.
The amount of xylanase or enzyme preparation used for the suspension is appropriately determined depending on the pretreatment conditions, the type and properties of the enzyme to be blended, and is preferably 0.04% by mass or more, more preferably based on the biomass mass. Is 0.1% by mass or more, and preferably 600% by mass or less, more preferably 100% by mass or less, and still more preferably 50% by mass or less. Moreover, Preferably it is 0.04-600 mass%, More preferably, it is 0.1-100 mass%, More preferably, it is 0.1-50 mass%.
The reaction pH during the saccharification treatment is preferably pH 4 or more, more preferably pH 5 or more, preferably pH 9 or less, from the viewpoints of improving saccharification efficiency, improving productivity (shortening production time), and reducing production costs. More preferably, it is pH 8 or less, More preferably, it is pH 7 or less. Moreover, it is preferably pH 4-9, more preferably pH 5-8, and still more preferably pH 5-7.
The reaction temperature during the saccharification treatment is preferably 20 ° C or higher, more preferably 25 ° C or higher, more preferably 30 or higher, from the viewpoints of improving saccharification efficiency, improving productivity (shortening production time), and reducing production costs. Even more preferably 40 ° C or higher, still more preferably 45 ° C or higher, still more preferably 50 ° C or higher, and 90 ° C or lower is preferable, more preferably 85 ° C or lower, still more preferably 80 ° C or lower, still more preferably 75 ° C. or lower, more preferably 65 ° C. or lower, still more preferably 60 ° C. or lower. Moreover, 20-90 degreeC is preferable, More preferably, it is 25-85 degreeC, More preferably, it is 30-80 degreeC, More preferably, it is 40-75 degreeC, More preferably, it is 45-65 degreeC, More preferably, it is 45-60 degreeC Further more preferably, it is 50-60 degreeC. The reaction time of the saccharification treatment can be appropriately set according to the type or amount of biomass, the amount of enzyme, etc., but from the viewpoint of improving saccharification efficiency, improving productivity (shortening production time), and reducing production cost, Preferably it is 1-5 days, More preferably, it is 1-4 days, More preferably, it is 1-3 days.

  In the sugar production method of the present invention, from the viewpoints of improving grinding efficiency, improving saccharification efficiency, and improving production efficiency (shortening production time), before the step of saccharifying biomass with the xylanase or enzyme preparation of the present invention, It is preferable to further include a step of pretreating the biomass. Examples of the pretreatment include one or more selected from the group consisting of alkali treatment, pulverization treatment, and hydrothermal treatment. Since the xylanase of the present invention has high enzyme activity even in the alkaline region, the pretreatment is preferably alkali treatment from the viewpoint of improving saccharification efficiency, and alkali treatment and pulverization treatment are performed from the viewpoint of further improving saccharification efficiency. It is preferable to perform the alkali treatment and the pulverization treatment at the same time.

With respect to the above-described embodiment, the following aspects are disclosed in the present invention.
<1> A xylanase selected from proteins represented by (a) to (c) below.
(A) A protein having an xylanase activity, comprising an amino acid sequence in which any one or more of alanine at positions 77, 214 and 328 is substituted with proline in the amino acid sequence represented by SEQ ID NO: 2.
(B) A protein comprising an amino acid sequence having 90% or more identity other than the amino acids at positions 77, 214 and 328 with the amino acid sequence of the protein of (a) and having xylanase activity.
(C) in the amino acid sequence of the protein of (a), consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added at positions other than the 77th, 214th and 328th amino acids; And a protein having xylanase activity.
<2> A gene encoding the xylanase according to <1> above.
<3> A recombinant vector containing the gene of <2> above.
<4> A transformant obtained by introducing the recombinant vector of <3> above into a host.
<5> The transformant according to <4>, wherein the host is a microorganism.
<6> An enzyme preparation for saccharification of biomass containing the protein of <1> above.

<7> The enzyme preparation for biomass saccharification according to <6>, further including cellulase.
<8> The enzyme preparation for biomass saccharification according to <7>, wherein the cellulase contains endoglucanase.
<9> The content of the endoglucanase in the total protein amount of the enzyme preparation is preferably 1% by mass or more, more preferably 5% by mass or more, further preferably 10% by mass or more, and preferably 70%. % By mass or less, more preferably 50% by mass or less, further preferably 40% by mass or less, preferably 1 to 70% by mass, more preferably 5 to 50% by mass, and further preferably 10 to 40% by mass. <8> Enzyme preparation for biomass saccharification.
<10> The protein amount ratio of the protein of <1> to the cellulase (the protein / the cellulase) is preferably 0.01 or more, more preferably 0.05 or more, and preferably 100 or less, more preferably Is 5 or less, more preferably 1 or less, still more preferably 0.5 or less, and preferably 0.01 to 100, more preferably 0.05 to 5, further preferably 0.05 to 1. More preferably, the enzyme preparation for biomass saccharification according to the above <7> to <9>, which is 0.05 to 0.5.
<11> The protein amount ratio of the protein of <1> to the endoglucanase (the protein / the endoglucanase) is preferably 0.05 or more, more preferably 0.1 or more, and preferably 10 or less. More preferably, it is 5 or less, More preferably, it is 2 or less, More preferably, it is 1 or less, Preferably it is 0.05-10, More preferably, it is 0.1-5, More preferably, it is 0.1-2, More The enzyme preparation for biomass saccharification according to <8> to <9> above, more preferably 0.2 to 1.
<12> The biomass is selected from the group consisting of processed or pulverized wood, pulp, paper, bagasse, rice straw, corn stalk or leaf, palm empty fruit bunch (EFB), plant shells, and algae At least one selected from the group consisting of wood, processed or pulverized wood, plant stems, leaves, and fruit bunches, more preferably bagasse, EFB, oil palm (stem) The enzyme preparation for biomass saccharification according to the above <6> to <11>, which is at least one selected from the group, more preferably bagasse.

<13> A method for producing sugar, comprising a step of saccharifying biomass with the enzyme preparation of <6> to <12> above.
<14> The method according to <13>, wherein, in the saccharification treatment, the protein of <1> or the enzyme preparation of <6> to <12> is added to a suspension containing the biomass.
<15> The method according to <14>, wherein the biomass content in the suspension is 0.5 to 20% by mass, preferably 3 to 15% by mass, and preferably 5 to 10% by mass.
<16> The amount of the protein <1> or the enzyme preparation <6> to <12> used in the suspension is preferably 0.04% by mass or more, more preferably, based on the mass of the biomass. Is 0.1% by mass or more, and preferably 600% by mass or less, more preferably 100% by mass or less, still more preferably 50% by mass or less, and preferably 0.04 to 600% by mass, more preferably Is 0.1-100 mass%, More preferably, it is 0.1-50 mass%, The method as described in said <14> or <15>.

<17> The reaction pH during the saccharification treatment is preferably pH 4 or more, more preferably pH 5 or more, preferably pH 9 or less, more preferably pH 8 or less, further preferably pH 7 or less, and preferably pH 4 to 9, The method of said <13>-<16> which is more preferably pH 5-8, More preferably, it is pH 5-7.
<18> The reaction temperature during the saccharification treatment is preferably 20 ° C. or higher, more preferably 25 ° C. or higher, still more preferably 30 or higher, still more preferably 40 ° C. or higher, still more preferably 45 ° C. or higher, still more preferably. 50 ° C or higher and preferably 90 ° C or lower, more preferably 85 ° C or lower, still more preferably 80 ° C or lower, even more preferably 75 ° C or lower, still more preferably 65 ° C or lower, still more preferably 60 ° C or lower. 20 to 90 ° C is preferable, more preferably 25 to 85 ° C, still more preferably 30 to 80 ° C, still more preferably 40 to 75 ° C, still more preferably 45 to 65 ° C, and still more preferably 45 to 65 ° C. The method of <13> to <17> above, wherein the temperature is 60 ° C, still more preferably 50-60 ° C.

EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, the technical scope of this invention is not limited by this Example.
For the polymerase chain reaction (PCR) for DNA fragment amplification in the following examples, an Applied Biosystems 2720 thermal cycler (Applied Biosystems) is used, and PrimeSTAR Max Premix GXL (Takara Bio) and attached reagents are used. DNA amplification was performed. The PCR reaction solution composition was 1 μL of appropriately diluted template DNA, 10 pmol of each of the sense primer and antisense primer, and 1 μL of PrimeSTAR Max Premix GXL, so that the total amount of the reaction solution was 50 μL. PCR reaction conditions were carried out by repeating 40 steps of three steps of temperature changes of 98 ° C. for 10 seconds, 50 ° C. for 15 seconds and 68 ° C. for 9 minutes.

Example 1 Cloning of Cellulomonas-derived cellulase gene
1-1 Genomic DNA extraction
Cellulomonas fimi ATCC 484 strain was inoculated into Growth medium (1.0% Polypeptone, 0.2% Yeast extract, 0.1% MgSO4 · 7H2O, pH 7.0) and cultured at 30 ° C. for 1 day. Genomic DNA was obtained from the cells obtained by the culture using UltraClean ™ Microbial DNA Isolation Kit (manufactured by Mo Bio Laboratories, Inc.).

1-2 Vector production
Using the genomic DNA obtained in 1-1 as a template, using the forward primer 1 (SEQ ID NO: 5) and reverse primer 1 (SEQ ID NO: 6) shown in Table 1, the xylanase gene region on the genome (SEQ ID NO: 2) An approximately 1.4 kbp fragment (A) was amplified. Further, at the BamHI restriction enzyme cleavage point of shuttle vector pHY300PLK (Takara Bio), S237 alkaline cellulase gene derived from Bacillus bacterium KSM-S237 strain (FERM BP-7875) (see JP 2000-210081) (SEQ ID NO: 3) ) Using the recombinant plasmid pHY-S237 into which the DNA fragment encoding) was inserted as a template, using the forward primer 2 (SEQ ID NO: 7) and reverse primer 2 (SEQ ID NO: 8) shown in Table 1, the S237 alkaline cellulase structural gene The approximately 5.6 kbp fragment (B) was amplified. The amplified gene fragment was purified with High Pure PCR Product Purification kit (Roche).

  Mix 1 μL of purified DNA fragment (A) and 1 μL of DNA fragment (B), 2 μL of 5 × In-Fusion HD Enzyme Premix included in In-Fusion HD Cloning Kit (Takara Bio), 6 μL of DNase-free water, and add 50 μC For 15 minutes. Thereafter, 2 μL of the reaction solution was used to transform 20 μL of competent cell E. coli HB101 (Takara Bio). An LB agar medium containing 50 ppm ampicillin sodium (Wako Pure Chemical Industries) and 2.0% agar (Wako Pure Chemical Industries) was used as a regeneration medium for transformants. From the transformants obtained as ampicillin resistant strains, a strain carrying the plasmid into which the target gene was inserted was selected by colony PCR. The selected transformant was cultured using the same LB agar medium (37 ° C., 1 day), and the plasmid was recovered and purified from the obtained bacterial cell using High Pure Plasmid Isolation kit (Roche).

1-3 Transformation into Host Bacteria The plasmid was introduced into the host bacteria according to the protoplast transformation method (Mol. Gen. Genet., 168, 111 (1979)). At this time, Bacillus subtilis 168 protease 9-deficient strain (KA8AX) (JP 2006-174707) was used as the host bacterium. Transformant regeneration medium includes tetracycline-containing DM3 regeneration agar medium (xylan from beechwood (Sigma-Aldrich) 1.0% (w / v), bactocasamino acid (Difco) 0.5% (w / v), yeast extract (Difco) 0.5% (w / v), L-tryptophan (Wako Pure Chemical Industries) 0.01% (w / v), disodium succinate hexahydrate (Wako Pure Chemical Industries) 8.1% (w / v), monophosphate Dipotassium hydrogen (Wako Pure Chemical Industries) 0.35% (w / v), Monopotassium dihydrogen phosphate (Wako Pure Chemical Industries) 0.15% (w / v), Glucose (Wako Pure Chemical Industries) 0.5% (w / v) ), Magnesium chloride (Wako Pure Chemical Industries) 20mM, bovine serum albumin (Wako Pure Chemical Industries) 0.01% (w / v), trypan blue 0.01% (w / v) (ACROS ORGANICS), tetracycline hydrochloride (Wako Pure Chemicals) Industrial) 0.005% (w / v), agar 1.0% (w / v)).

Example 2 Xylanase Production
After seed culture using 5 mL of 15 ppm tetracycline-containing LB medium grown on DM3 regenerated agar medium (30 ° C., 250 rpm, 16 hours), 0.6 mL of the seed culture solution was added to 20 mL of 2 × L Mal medium (Bactotripton 2 % (w / v), yeast extract 1.0% (w / v), sodium chloride 1.0% (w / v), manganese sulfate pentahydrate (Wako Pure Chemical Industries) 75ppm, maltose monohydrate (Wako Pure Chemical) (Industry) 7.5% (w / v), tetracycline hydrochloride 15ppm], and main culture was performed (30 ° C, 230rpm, 3 days) .After culture, by centrifugation (11,000rpm, 15 minutes, 4 ° C) A culture supernatant was obtained, and 3 mL of the culture supernatant was prepared by preparing a recombinant xylanase crude enzyme solution by exchanging the buffer to 20 mM Tris-HCl pH 7.5 using a desalting column Econo-Pac 10DG column (Biorad). .

Example 3 Mutation Introduction 77-Pro induced Cfi Fw (SEQ ID NO: 9) and 77-Pro shown in Table 2 below (actually shown) using as a template a gene encoding a wild-type xylanase consisting of the base sequence shown in SEQ ID NO: 1 Induced Cfi Rv (SEQ ID NO: 10) primer pair, 214-Pro induced Cfi Fw (SEQ ID NO: 11) and 214-Pro induced Cfi Rv (SEQ ID NO: 12) primer pair, 328-Pro induced Cfi Fw (SEQ ID NO: 13) And 328-Pro induced Cfi Rv (SEQ ID NO: 14) using a primer pair, the 229-231th, 640-642th, 982-984th of the base sequence of the wild-type xylanase shown in SEQ ID NO: 1 A DNA fragment containing a gene encoding a mutant xylanase (represented as A77P, A214P, or A328P, respectively) in which each base gcc (alanine; A) was replaced with ccg (proline; P) was amplified. The obtained PCR reaction solution was treated with DpnI to digest the template DNA, and then transformed into Escherichia coli to construct a plasmid expressing the mutant xylanase. The obtained plasmid was confirmed to have a gene encoding a mutant xylanase inserted by a DNA sequencing method.

Example 4 Analysis of Enzymatic Properties of Mutant Xylanase
4-1 Heat resistance The heat resistance was measured. First, the crude enzyme solution was appropriately diluted with 50 mM acetate buffer and incubated at 62-67 ° C. (in increments of 1 ° C.) for 20 minutes using a Veriti thermal cycler (Applied Biosystems). Simultaneously, 20 μL of the synthetic substrate 1 mM p-nitrophenylxylobioside solution, 20 μL of 250 mM acetate buffer and 50 μL of water were mixed to prepare 90 μL of the substrate solution. 10 μL of the crude enzyme solution after incubation was added to the substrate solution, and reacted at 50 ° C. for 10 minutes. After the reaction, 100 μL of an aqueous calcium carbonate solution was added, and the absorbance at 420 nm was measured with an absorption microplate reader VersaMax (Molecular Device Japan). In addition, after adding 100 microliters of calcium carbonate aqueous solution to 90 microliters of substrate solutions, 10 microliters of enzyme solutions were added, and what performed the same operation was made into the blank. Then, a calibration curve created with the p-nitrophenol solution was prepared, and the amount of p-nitrophenol produced was calculated. The value obtained by subtracting the background derived from the substrate solution was defined as the amount of p-nitrophenol produced.
Table 3 shows the results of comparison between the mutant xylanase and the wild type.

  As shown in Table 3, it was confirmed that the xylanase of the present invention showed higher heat resistance than the wild type.

4-2 Optimum reaction pH
The optimum pH was measured. Synthetic substrate 1 mM p-nitrophenylxylobioside solution 20 μL, 250 mM phosphate-citrate buffer (pH 4, 5, 6, 7, 8) 20 μL and water 50 μL were mixed to prepare substrate solution 90 μL. 10 μL of a crude enzyme solution diluted to an appropriate concentration was added to the substrate solution and reacted at 50 ° C. for 10 minutes. After the reaction, 100 μL of an aqueous calcium carbonate solution was added, and the absorbance at 420 nm was measured with an absorption microplate reader VersaMax (Molecular Device Japan). In addition, after adding 100 microliters of calcium carbonate aqueous solution to 90 microliters of substrate solutions, 10 microliters of enzyme solutions were added, and what performed the same operation was made into the blank. Then, a calibration curve created with the p-nitrophenol solution was prepared, and the amount of p-nitrophenol produced was calculated. The value obtained by subtracting the background derived from the substrate solution was defined as the amount of p-nitrophenol produced. The amount of sugar produced at each pH when the sample showing the maximum amount of sugar produced was taken as 100% was determined as the relative activity. Table 4 shows the optimum pH results for the degradation of p-nitrophenylxylobioside by mutant xylanase and wild-type xylanase.

  From Table 4, the xylanase of the present invention showed the maximum activity at pH 6.0 and maintained at least 70% of the maximum activity at pH 5.0 to 7.0.

4-3 Specific activity analysis The specific activity of xylan was measured and compared with the wild type. A substrate solution 90 μL was prepared by mixing 2.0 μl (w / v) xylan from beechwood 50 μL, 250 mM acetate buffer (pH 5.0) 20 μL and water 20 μL. 10 μL of the crude enzyme solution of the mutant diluted to an appropriate concentration was added and reacted at 50 ° C. for 10 minutes. After the reaction, 100 μL of DNS solution was added to stop the reaction, and the reaction was carried out at 100 ° C. for 5 minutes. After cooling, the absorbance at 540 nm was measured with an absorbance microplate reader. A blank was prepared by adding 100 μL of the DNS solution to 90 μL of the substrate solution, adding 10 μL of the enzyme solution, and performing the same operation. The amount of xylooligosaccharides produced by preparing a calibration curve prepared with a xylose solution was calculated, and the value obtained by subtracting the background sugar derived from the substrate solution was taken as the amount of produced sugars. Thereafter, the xylanase activity of the diluted enzyme solution was calculated from the amount of sugar produced, and the relative activity was measured with the wild-type activity as 100%.
Table 5 shows the results of specific activities related to the degradation of xylan by mutant xylanase and wild-type xylanase.

4-4 Optimum reaction temperature The optimum reaction temperature was measured. Synthetic substrate 1 mM p-nitrophenylxylobioside solution 20 μL, 250 mM acetate buffer 20 μL and water 50 μL were mixed to prepare 90 μL of substrate solution. 10 μL of a crude enzyme solution diluted to an appropriate concentration was added to the substrate solution, and reacted at 45 to 70 ° C. (in increments of 5 ° C.) for 10 minutes using a Veriti thermal cycler (Applied Biosystems). After the reaction, 100 μL of an aqueous calcium carbonate solution was added, and the absorbance at 420 nm was measured with an absorption microplate reader VersaMax (Molecular Device Japan). In addition, after adding 100 microliters of calcium carbonate aqueous solution to 90 microliters of substrate solutions, 10 microliters of enzyme solutions were added, and what performed the same operation was made into the blank. Then, a calibration curve created with the p-nitrophenol solution was prepared, and the amount of p-nitrophenol produced was calculated. The value obtained by subtracting the background derived from the substrate solution was defined as the amount of p-nitrophenol produced. The production amount at each temperature when the sample showing the maximum production amount of p-nitrophenylxylobioside was taken as 100% was determined as the specific activity.
Table 6 shows the results of the optimum reaction temperature for the mutant xylanase and the wild type xylanase.

  The xylanase of the present invention had the highest xylan decomposition activity around 50-60 ° C, and showed 60% or more of the maximum activity in the range of 45-65 ° C.

Claims (7)

A xylanase selected from the proteins represented by the following (a) to (c).
(A) a protein having an amino acid sequence in which at least one of alanine at positions 77, 214 and 328 is substituted with proline in the amino acid sequence represented by SEQ ID NO: 2 and having xylanase activity;
(B) A protein comprising an amino acid sequence having 90% or more identity other than the amino acids at positions 77, 214 and 328 with the amino acid sequence of the protein of (a) and having xylanase activity.
(C) in the amino acid sequence of the protein of (a), consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added at positions other than the 77th, 214th and 328th amino acids; And a protein having xylanase activity.   A gene encoding the xylanase according to claim 1.   A recombinant vector containing the gene according to claim 2.   A transformant obtained by introducing the recombinant vector according to claim 3 into a host.   The transformant according to claim 4, wherein the host is a microorganism.   An enzyme preparation for biomass saccharification, comprising the protein according to claim 1.   A method for producing sugar, comprising a step of saccharifying biomass with the enzyme preparation according to claim 6.