JP2015167552A - mutant xylanase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide xylanase with less saccharification residue and lower lignin adhesiveness.SOLUTION: This invention relates to a method for producing mutant xylanase catalytic domains, including either: replacing a region corresponding to the amino acid sequence represented by positions 118-143 of a specific sequence with the amino acid sequence represented by positions 118-143 of the specific sequence or an amino acid sequence having at least 90% of the identity thereof, in the amino acid sequence of the catalytic domains of xylanases belonging to glycoside hydrolase family 11, with the proviso that the identity of the amino acid sequence of the catalytic domains and the amino acid sequence represented by the specific sequence is less than 90%; or replacing a region corresponding to the amino acid sequence represented by positions 144-172 of the specific sequence with the amino acid sequence represented by positions 144-172 of the specific sequence or an amino acid sequence having at least 90% of the identity thereof. The mutant xylanase mutated by the method is also provided.

Description

  The present invention relates to mutant xylanases.

  Biomass is an organic resource derived from renewable organisms excluding fossil resources. Among them, cellulosic biomass is attracting attention. Development of technology to produce sugar by decomposing cellulose and producing useful resources such as alternatives to petroleum resources and biofuels by chemical conversion and fermentation technology using microorganisms from the obtained sugar It has been broken.

  Cellulosic biomass is composed mainly of cellulose, hemicellulose, and lignin. It is known that such biomass is decomposed in a complex format by synergistic action of cellulase that decomposes cellulose, hemicellulase that decomposes hemicellulose, and the like. For effective utilization of cellulosic biomass, it is necessary to develop a saccharifying enzyme capable of decomposing cellulose and hemicellulose with high efficiency.

  For saccharification of cellulosic biomass, a saccharification enzyme cocktail based on cellulase has been conventionally used. In recent years, it has been reported that the addition of hemicellulases such as xylanase and arabinofuranosidase to saccharification enzyme cocktails containing cellulase as the main component improves the saccharification rate, and the importance of hemicellulase in cellulosic biomass saccharification Sexuality was suggested (Patent Document 1).

  In saccharification of cellulosic biomass, saccharification enzyme is adsorbed on cellulose or hemicellulose as a substrate, and sugar is produced by decomposing them. Such adsorption of the enzyme to cellulose or hemicellulose substrate involved in sugar production is called productive adsorption. In contrast, adsorption of the enzyme to components that are not degraded by saccharifying enzymes, such as lignin, ash, and other components, is referred to as non-specific adsorption. The saccharification efficiency of an enzyme is considered to depend on factors such as the specific activity of the enzyme, heat resistance, and non-specific adsorption, and these factors are noted in the development of saccharifying enzymes.

  The residual component after saccharifying cellulosic biomass with a saccharifying enzyme is mainly composed of lignin. However, it is known that saccharification efficiency is reduced by adsorbing saccharifying enzymes to lignin in cellulosic biomass. In cellulosic biomass saccharification, nonspecific adsorption of saccharifying enzymes to lignin is an undesirable property. So far, it has been reported that cellobiohydrolase (CBH) II has reduced nonspecific adsorption to non-cellulose materials due to modifications such as substitution of negatively charged amino acids and removal of positively charged amino acids. (Patent Document 2).

Special table 2011-515089 gazette Special table 2011-523854 gazette

  A xylanase belonging to the glycoside hydrolase family 11 (hereinafter sometimes referred to as GH11 xylanase) is a hemicellulase that can be used for saccharification of cellulosic biomass. However, many GH11 xylanases have high adsorptivity to saccharification residues and lignin, and when used for biomass saccharification, there is a disadvantage that the saccharification rate is reduced by nonspecific adsorption. Adsorption sites for glycation residues and lignin in GH11 xylanase are still unknown, and no attempt has been made to reduce adsorptivity by enzyme modification.

  The present inventors have found that among GH11 xylanases, the xylanase derived from bacteria belonging to the genus Xylanimonas has low adsorptivity to saccharification residue or lignin. In addition, the present inventors have identified a site involved in adsorption of glycation residue or lignin in GH11 xylanase. Furthermore, the present inventors have reduced the adsorptivity of saccharification residue or lignin by mutating (recombinant) xylanase in which the site involved in adsorption of glycation residue or lignin of GH11 xylanase is replaced with the corresponding site of xylanase derived from genus Xylanimonas. I found out.

Accordingly, in one aspect, the present invention is a method for producing a mutant xylanase catalytic domain comprising:
In the amino acid sequence of the catalytic domain of xylanase belonging to glycoside hydrolase family 11 (however, the identity between the amino acid sequence of the catalytic domain and the amino acid sequence represented by SEQ ID NO: 2 is less than 90%)
A region corresponding to the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 is substituted with the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto, or ,
Substituting the region corresponding to the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 with the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto Provide a way.

In another aspect, the present invention provides a method for producing a mutant xylanase comprising the following:
In xylanases belonging to glycoside hydrolase family 11,
The amino acid sequence of the catalytic domain of the xylanase is shown at positions 118 to 143 of SEQ ID NO: 2 in the amino acid sequence of the catalytic domain (the identity between the amino acid sequence of the catalytic domain and SEQ ID NO: 2 is less than 90%). Replacing the region corresponding to the amino acid sequence with the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto, or
A region corresponding to the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 in the amino acid sequence of the catalytic domain is the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 or an amino acid having at least 90% identity thereto A method is provided that comprises replacing with a sequence.

In another aspect, the present invention provides a method for reducing glycation residue adsorptivity of a xylanase catalytic domain, comprising:
In the amino acid sequence of the catalytic domain of xylanase belonging to glycoside hydrolase family 11 (however, the identity between the amino acid sequence of the catalytic domain and the amino acid sequence represented by SEQ ID NO: 2 is less than 90%)
A region corresponding to the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 is substituted with the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto, or ,
Substituting the region corresponding to the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 with the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto Provide a way.

In another aspect, the present invention provides a method for reducing xylanase saccharification residue adsorption, comprising:
In xylanases belonging to glycoside hydrolase family 11,
The amino acid sequence of the catalytic domain of the xylanase is shown at positions 118 to 143 of SEQ ID NO: 2 in the amino acid sequence of the catalytic domain (the identity between the amino acid sequence of the catalytic domain and SEQ ID NO: 2 is less than 90%). Replacing the region corresponding to the amino acid sequence with the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto, or
A region corresponding to the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 in the amino acid sequence of the catalytic domain is the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 or an amino acid having at least 90% identity thereto A method is provided that comprises replacing with a sequence.

In a further aspect, the invention provides a mutant xylanase catalytic domain, in order from the N-terminus,
(A) an amino acid sequence represented by positions 1 to 46 of SEQ ID NO: 4 or an amino acid sequence having at least 90% identity thereto,
(B) an amino acid sequence represented by positions 47 to 113 of SEQ ID NO: 4 or an amino acid sequence having at least 60% identity thereto,
(C) the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 or the amino acid sequence having at least 90% identity thereto, and the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 or at least 60% identical thereto Or an amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 or an amino acid sequence having at least 60% identity thereto, and an amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 or at least An amino acid sequence having 90% identity,
And (d) the amino acid sequence shown at positions 168 to 187 of SEQ ID NO: 4, or an amino acid sequence having at least 60% identity thereto,
A mutant xylanase catalytic domain is provided.

  In a further aspect, the present invention provides a mutant xylanase comprising the above mutant xylanase catalytic domain and a carbohydrate binding module.

  In a further aspect, the present invention provides a polynucleotide encoding the mutant xylanase catalytic domain or the mutant xylanase.

  In a further aspect, the present invention provides a vector comprising the above polynucleotide.

  In a further aspect, the present invention provides a method for producing a transformant for introducing the above vector into a host.

  In a further aspect, the present invention provides a biomass saccharifying agent comprising the mutant xylanase catalytic domain or the mutant xylanase.

  In a further aspect, the present invention provides a method for producing sugar from biomass using the biomass saccharifying agent.

  The present invention provides a mutant xylanase catalytic domain with reduced adsorption to saccharification residues and lignin. By using a mutant xylanase containing the catalytic domain, biomass containing lignin can be saccharified more efficiently and sugar can be produced from biomass more efficiently and cheaply than conventional xylanases. Can do.

Saccharification residue adsorption rate of xylanase catalytic domain. Saccharification residue adsorption rate of xylanase catalytic domain. The amount of sugar produced from xylan in the presence of lignin by the xylanase-containing enzyme composition.

(1. Definition)
As used herein, nucleotide and amino acid sequence identity is calculated by the Lipman-Pearson method (Science, 1985, 227: 1435-1441). Specifically, it is calculated by performing an analysis with Unit size to compare (ktup) as 2, using a homology analysis (Search homology) program of genetic information processing software Genetyx-Win.

  In this specification, the “corresponding position” or “corresponding region” on the amino acid sequence is present in each amino acid sequence by comparing the target amino acid sequence with a reference sequence (for example, the amino acid sequence shown by SEQ ID NO: 2). Can be determined by aligning the sequences so as to give the greatest homology to the conserved amino acid residues. The alignment can be performed using known algorithms, the procedures of which are known to those skilled in the art. For example, the alignment can be performed manually based on the above-described Lipman-Pearson method or the like, but the Clustal W multiple alignment program (Thompson, JD et al, 1994, Nucleic Acids Res. 22: 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 [www. ddbj.nig.ac.jp/Welcome-j.html]).

  In this specification, the “glycoside hydrolase family 11” (or sometimes GH11) is a family classified by CARBOHYDRATE-ACTIVE ENZYME CAZY-Home ([www.casey.org/]). Means the 11th group. In the present specification, a xylanase (or GH11 xylanase) belonging to glycoside hydrolase family 11 is a protein having about 200 amino acids, has a β-jelly roll structure, and has endo-β-1,4-xylanase activity or endo-β-. It is a family of hemicellulases having 1,3-xylanase activity (the forefront of biomass degrading enzyme research-centering on cellulase and hemicellulase-Akihiko Kondo, Yoshihiko Amano, Hiroshi Tamaru Co., Ltd.)

  In the present specification, “xylanase” refers to an enzyme having an activity of hydrolyzing a xylose β-1,4-glycoside bond in xylan. More specifically, it refers to an enzyme consisting of a “Catalytic Domain (CD)” or a CD bound via a linker and a “Carbohydrate binding module (CBM)”.

  In the present specification, “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 by reacting with the protein using xylan as a substrate and measuring the amount of xylan degradation product produced. For example, the xylanase activity of a protein can be measured by quantifying the reducing end of a sugar produced from xylan by the 3,5-dinitrosalicylic acid (DNS) method. More specifically, soluble xylan, sodium acetate buffer (pH 5.0), and target protein are mixed, reacted at 50 ° C., and then DNS solution (solution containing sodium hydroxide, dinitrosalicylic acid, sodium potassium tartrate). Is added and allowed to react at 99.9 ° C., and then the absorbance of the reaction solution at a wavelength of 540 nm is measured. By standardizing the measured value with a calibration curve created using xylose, the reducing end of the produced sugar can be quantified. Specific procedures for measuring xylanase activity by the DNS method are described in detail in Examples below.

  In the present specification, the term “xylanase catalytic domain” or simply “catalytic domain” (both may be referred to as CD) refers to a domain possessing xylanase activity, which is included in xylanase. Examples of naturally occurring CDs include a domain derived from Xylanimonas cellulolytica (DSM 15894) consisting of the amino acid sequence shown in SEQ ID NO: 2, a thermobifida fusca consisting of the amino acid sequence shown in SEQ ID NO: 4 ( Thermobifida fusca) domains derived from YX, and regions possessing the xylanase activity of other GH11 xylanases corresponding to regions of those domains.

  In the present specification, the “carbohydrate binding module (CBM)” refers to a domain that is covalently bound to CD via a linker and binds to a xylanase catalytic substrate (eg, xylan). Examples of naturally occurring CBMs include a module derived from Xylanimonas cellulolytica (DSM 15894) consisting of the amino acid sequence represented by SEQ ID NO: 5, a module derived from Thermobifida fusca XY consisting of the amino acid sequence represented by SEQ ID NO: 6, And other GH11 xylanase substrate binding regions corresponding to those module regions.

  In the present specification, “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 (stem) 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. Among these, from the viewpoint of availability and raw material cost, wood, processed or pulverized wood, plant stems, leaves, fruit bunches and the like are preferable, bagasse, EFB, oil palm (stem) are more preferable, and bagasse is further. preferable. You may use the said biomass individually or in mixture of 2 or more types. The biomass may be dried.

In the present specification, the “saccharification residue” refers to a solid content remaining after saccharification of cellulose and hemicellulose in biomass with a saccharification enzyme. The xylanase “adsorbability of saccharification residue” is high, for example, by reacting the saccharification residue with xylanase, adsorbing xylanase to the saccharification residue, and then measuring the xylanase activity remaining in the supernatant of the reaction solution, It can calculate by calculating | requiring the relative activity with respect to xylanase activity when not making it react with this saccharification residue. For example, the saccharification residue adsorption rate can be expressed by the following formula.

Saccharification residue adsorption rate (%) = 100-relative specific activity (%)
Relative specific activity (%) = (xylanase activity of enzyme sample after reaction with saccharification residue / xylanase activity of enzyme sample unreacted with saccharification residue) × 100

A xylanase having a lower relative specific activity has a higher saccharification residue adsorbability. As the saccharification residue, for example, lignin or a solid residue after saccharification of an alkali mixed crushed bagasse at 50 ° C. for 24 hours using a known cellulase or cellulase preparation (for example, Cellic (registered trademark) CTec2 manufactured by Novozymes) is used. be able to. For the reaction between the saccharification residue and xylanase, for example, treatment at 50 ° C. for 1 hour can be employed. Specific procedures for measuring xylanase saccharification residue adsorptivity are described in detail in Examples below.

(2. Method for producing mutant xylanase)
In one embodiment, the mutant xylanase of the present invention is produced by substituting a region involved in saccharification residue adsorption in the catalytic domain of GH11 xylanase with a corresponding region of the xylanase catalytic domain from Xylanimonas cellulolytica. It is a replacement xylanase. As a result of intensive studies by the present inventors, the region involved in adsorption of glycation residue in GH11 xylanase is a specific region in the catalytic domain, for example, the Thermobifida fusca-derived xylanase catalytic domain consisting of the amino acid sequence represented by SEQ ID NO: 4. Was revealed to be the region from position 114 to position 167 of the amino acid sequence. Further, by substituting the region with a region corresponding to the xylanase catalytic domain derived from Xylanimonas cellulosilytica having low saccharification residue adsorptivity, that is, the region at positions 118 to 172 of the amino acid sequence represented by SEQ ID NO: 2, It was revealed that saccharification residue adsorptivity is reduced. Furthermore, it has been clarified that the same saccharification residue adsorptivity reducing effect can be obtained even when the region is partially substituted. Therefore, the region involved in glycation residue adsorptivity in GH11 xylanase corresponds to the region of positions 114 to 167 of the amino acid sequence shown in SEQ ID NO: 4 (or the region of positions 118 to 172 of the amino acid sequence shown in SEQ ID NO: 2). A part or all of this region is a region corresponding to the xylanase catalytic domain derived from Xylanimonas cellulosilytica (that is, a corresponding part of the region from position 118 to position 172 of the amino acid sequence represented by SEQ ID NO: 2 or By substituting all of them, a mutant xylanase with improved saccharification residue adsorption can be obtained.

Accordingly, in one aspect, the present invention provides a GH11 xylanase comprising a fragment consisting of part or all of the amino acid sequence shown at positions 118 to 172 of SEQ ID NO: 2, or a fragment having at least 90% sequence identity with the fragment. A method for producing a mutant xylanase catalytic domain comprising substituting a region corresponding to part or all of the amino acid sequence represented by positions 118 to 172 of SEQ ID NO: 2 in the amino acid sequence of the catalytic domain of
In another embodiment, the present invention relates to a fragment consisting of a part or all of the amino acid sequence shown at positions 118 to 172 of SEQ ID NO: 2, or a fragment having at least 90% sequence identity with the GH11 xylanase. And a method for producing a mutant xylanase, comprising substituting a region corresponding to part or all of the amino acid sequence shown at positions 118 to 172 of SEQ ID NO: 2 in the amino acid sequence of the catalytic domain of the xylanase.

The mutated xylanase or its catalytic domain has a reduced saccharification residue adsorptivity compared to the xylanase before its mutation or its catalytic domain.
Accordingly, in yet another aspect, the present invention relates to a fragment consisting of a part or all of the amino acid sequence shown at positions 118 to 172 of SEQ ID NO: 2, or a fragment having at least 90% sequence identity with the fragment, Reduction of glycan residue adsorptivity of xylanase catalytic domain comprising substitution of a region corresponding to part or all of the amino acid sequence shown at positions 118 to 172 of SEQ ID NO: 2 in the amino acid sequence of catalytic domain of GH11 xylanase Provide a method.
In yet another aspect, the present invention relates to a fragment comprising a part or all of the amino acid sequence shown at positions 118 to 172 of SEQ ID NO: 2, or a fragment having at least 90% sequence identity with the GH11 xylanase. Reducing the adsorption property of xylanase to a glycation residue, comprising substituting a region corresponding to part or all of the amino acid sequence shown at positions 118 to 172 of SEQ ID NO: 2 in the amino acid sequence of the catalytic domain of the xylanase Provide a method.

  In one embodiment of the method of the present invention, the fragment consisting of part or all of the amino acid sequence shown at positions 118 to 172 of SEQ ID NO: 2 is from the amino acid sequence shown at positions 118 to 172 of SEQ ID NO: 2. A partial sequence of the amino acid sequence shown at positions 118 to 172 of SEQ ID NO: 2 and 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, Examples include fragments of 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54 amino acid residues. Preferably, a fragment consisting of the amino acid sequence shown at positions 118 to 172 of SEQ ID NO: 2, a fragment consisting of the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2, and the amino acid sequence shown at positions 135 to 140 of SEQ ID NO: 2 A fragment consisting of the amino acid sequence shown at positions 133 to 163 of SEQ ID NO: 2, a fragment consisting of the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2, and the positions 150 to 165 of SEQ ID NO: 2. A fragment consisting of an amino acid sequence.

  In a preferred embodiment of the method of the present invention, as an example of a fragment having at least 90% sequence identity with a fragment consisting of part or all of the amino acid sequence shown at positions 118 to 172 of SEQ ID NO: 2, 90% or more, preferably 92% or more, more preferably 95% or more, still more preferably 96% or more, and a fragment containing part or all of the amino acid sequence shown at positions 118 to 172 of SEQ ID NO: 2 Preferably, a fragment having a sequence identity of 98% or more; a fragment containing part or all of the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 and 90% or more, preferably 92% or more, more preferably 96% 90% or more of a fragment having the above sequence identity; a fragment containing a part or all of the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2, A fragment having a sequence identity of 3% or more, more preferably 96% or more; a sequence of 90% or more, preferably 93% or more, more preferably 96% or more with the amino acid sequence shown at positions 133 to 163 of SEQ ID NO: 2. Fragments having identity; fragments having a sequence identity of 90% or more, preferably 93% or more with the amino acid sequence shown at positions 150 to 165 of SEQ ID NO: 2, and the like.

  Preferably, the fragment having at least 90% sequence identity with the fragment consisting of a part or all of the amino acid sequence shown at positions 118 to 172 of SEQ ID NO: 2 is the xylanase activity and the amino acid sequence shown by SEQ ID NO: 2 A fragment obtained from a region corresponding to the region corresponding to positions 118 to 172 of the amino acid sequence represented by SEQ ID NO: 2 in a polypeptide having an adsorption rate of glycation residue equivalent to the xylanase catalytic domain consisting of Here, the equivalent saccharification residue adsorption rate means that the adsorption rate of 15 mg / mL saccharification residue measured according to Reference Examples 4 to 6 described later is 20% or less, preferably 10% or less, or in SEQ ID NO: 2. It means that it is less than 150%, preferably less than 120%, with respect to the adsorption rate of the glycation residue of the xylanase catalytic domain comprising the amino acid sequence shown.

  In the method of the present invention, the GH11 xylanase before mutation (hereinafter also referred to as parent GH11 xylanase), which becomes the parent xylanase of the mutant xylanase of the present invention, is a xylanase derived from Xylanimonas genus, a xylanase derived from Thermobifida, or Trichoderma. Examples include xylanase derived from Bacillus, xylanase derived from Aspergillus, xylanase derived from Clostridium, xylanase derived from Cellulomonas, and the like. Preferably, in the above method, the amino acid sequence of the catalytic domain of the parental GH11 xylanase has less than 90% identity with the amino acid sequence represented by SEQ ID NO: 2, more preferably 85% or less, still more preferably 80% or less It is. The xylanase catalytic domain consisting of the amino acid sequence represented by SEQ ID NO: 2 is a catalytic domain of xylanase derived from Xylanimonas cellulolytica (DSM 15894).

  In the method of the present invention, preferably, a region corresponding to positions 1 to 50 of SEQ ID NO: 2 in the amino acid sequence of the catalytic domain of the parent GH11 xylanase is further represented by the amino acid sequence represented by positions 1 to 46 of SEQ ID NO: 4, Or a fragment comprising an amino acid sequence having at least 90%, for example, 90% or more, preferably 92% or more, more preferably 94% or more, still more preferably 96% or more, still more preferably 97.5% or more. Replace with. Preferably, the fragment consisting of the amino acid sequence having at least 90% identity with the amino acid sequence represented by positions 1 to 46 of SEQ ID NO: 4 is the amino acid sequence represented by SEQ ID NO: 4 in the polypeptide of the xylanase catalytic domain. It may be a fragment obtained from the region corresponding to the region 1 to 46 position.

  In another embodiment of the method of the present invention described above, the C-terminus of the catalytic domain of the parental GH11 xylanase has an amino acid sequence represented by positions 188 to 198 of SEQ ID NO: 4 or at least 60%, for example, 60% or more. Furthermore, a fragment comprising an amino acid sequence having preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, still more preferably 95% or more is arranged. For example, the fragment replaces the C-terminal region of the region substituted with a fragment consisting of a part or all of the amino acid sequence shown at positions 118 to 172 of SEQ ID NO: 2 and the fragment corresponding thereto, or The fragment is added to the C-terminal side of the substituted region.

  Therefore, in a preferred embodiment of the method of the present invention, the catalytic domain of the parental GH11 xylanase before mutation is at least 90%, for example, 90% or more, preferably 92% or more with the amino acid sequence represented by SEQ ID NO: 4 More preferably 94% or more, still more preferably 96% or more, still more preferably 98%, even more preferably 99% or more. Examples of the catalytic domain of the xylanase consisting of the amino acid sequence represented by SEQ ID NO: 4 include the catalytic domain of Thermobifida fusca XY xylanase.

  Preferably, the amino acid sequence of the catalytic domain of the parental GH11 xylanase before mutation in the method of the present invention is a position corresponding to position 85 and / or a position corresponding to position 174 of the amino acid sequence represented by SEQ ID NO: 4 (in other words, , A position corresponding to position 89 and / or a position corresponding to position 179 of the amino acid sequence represented by SEQ ID NO: 2 may each have a glutamic acid residue. Glutamate residues present at these positions are believed to be involved in xylanase activity (Acta Crystallographica Section D Biological Crystallography, 2006, 62: 784-792).

(3. Mutant xylanase)
By mutating the catalytic domain of the parent GH11 xylanase as described above, a mutant xylanase catalytic domain with reduced saccharification residue adsorptivity can be produced.

  In the mutant xylanase catalytic domain of the present invention, the region corresponding to part or all of the amino acid sequence shown at positions 118 to 172 of SEQ ID NO: 2 in the amino acid sequence of the catalytic domain of GH11 xylanase is 118 to It consists of an amino acid sequence consisting of part or all of the amino acid sequence shown at position 172 or an amino acid sequence having at least 90% identity with it. Moreover, the mutant xylanase catalytic domain of the present invention has xylanase activity, and its adsorptivity to saccharification residues is reduced as compared to the parental GH11 xylanase before mutation.

  Furthermore, in the amino acid sequence of the mutant xylanase catalytic domain of the present invention, the region corresponding to positions 1 to 50 of SEQ ID NO: 2 is the amino acid sequence represented by positions 1 to 46 of SEQ ID NO: 4 or at least 90% identity thereto It preferably consists of an amino acid sequence having

Accordingly, in a preferred embodiment, the mutant xylanase catalytic domain of the present invention consists of the following amino acid sequences in order from the N-terminus:
(A) an amino acid sequence represented by positions 1 to 46 of SEQ ID NO: 4 or an amino acid sequence having at least 90% identity thereto;
(B) an amino acid sequence represented by positions 47 to 113 of SEQ ID NO: 4 or an amino acid sequence having at least 60% identity thereto;
(C) The following:
(C1) The amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 or the amino acid sequence having at least 90% identity thereto and the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 or at least 60% identical thereto Or (c2) an amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 or an amino acid sequence having at least 60% identity thereto and an amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 Or an amino acid sequence having at least 90% identity thereto, or (c3) an amino acid sequence represented by positions 118 to 172 of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto;
And (d) the amino acid sequence shown at positions 168 to 187 of SEQ ID NO: 4, or an amino acid sequence having at least 60% identity thereto.

  In a preferred embodiment, (a) is an amino acid sequence represented by positions 1 to 46 of SEQ ID NO: 4, or 90% or more, preferably 92% or more, more preferably 94% or more, and still more preferably 96% or more. The amino acid sequence is preferably composed of an amino acid sequence having a sequence identity of 97.5% or more. In a more preferred embodiment, the above (a) is an amino acid sequence represented by positions 1 to 46 of SEQ ID NO: 4.

  In a preferred embodiment, the above (b) is an amino acid sequence represented by positions 47 to 113 of SEQ ID NO: 4, or 60% or more, preferably 70% or more, more preferably 80% or more, and further preferably 90% or more. More preferably 95% or more, and still more preferably 98% or more of the amino acid sequence having a sequence identity, and in a more preferred embodiment, (b) above is the amino acid shown at positions 47 to 113 of SEQ ID NO: 4 Is an array. Preferably, in (b) above, a glutamic acid residue may be present at a position corresponding to position 85 of the amino acid sequence represented by SEQ ID NO: 4.

  In a preferred embodiment, the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 in (c1) above or the amino acid sequence having at least 90% identity thereto is the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2. And the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 is more preferably 92% or more, more preferably 96% or more. Further, the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 in the above (c1) or the amino acid sequence having at least 60% identity thereto is the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2, % Or more, preferably 70% or more, more preferably 80% or more, and still more preferably 90% or more amino acid sequences.

  In a preferred embodiment, the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 in (c2) above or the amino acid sequence having at least 90% identity thereto is the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2. The amino acid sequence is preferably 93% or more, more preferably 96% or more, and still more preferably an amino acid sequence represented by positions 144 to 172 of SEQ ID NO: 2. In addition, the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 in the above (c2) or the amino acid sequence having at least 60% identity thereto is the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2, % Or more, preferably 70% or more, more preferably 80% or more, and still more preferably 90% or more amino acid sequences.

  In a preferred embodiment, the amino acid sequence shown at positions 118 to 172 of SEQ ID NO: 2 in (c3) above or the amino acid sequence having at least 90% identity thereto is the amino acid shown at positions 118 to 172 of SEQ ID NO: 2. Preferably 92% or more, more preferably 95% or more, still more preferably 96% or more, and still more preferably 98% or more, and more preferably at positions 118 to 172 of SEQ ID NO: 2. It is the amino acid sequence shown.

  In a preferred embodiment, the above (d) is an amino acid sequence represented by positions 168 to 187 of SEQ ID NO: 4, or 60% or more, preferably 70% or more, more preferably 80% or more, and still more preferably 90% or more. Even more preferably, it consists of an amino acid sequence having a sequence identity of 95% or more. In a more preferred embodiment, the above (d) is an amino acid sequence represented by positions 168 to 187 of SEQ ID NO: 4. Preferably, in (d) above, a glutamic acid residue may be present at a position corresponding to position 174 of the amino acid sequence represented by SEQ ID NO: 4.

  In a preferred embodiment, (a) is an amino acid sequence represented by positions 1 to 46 of SEQ ID NO: 4, or an amino acid sequence having at least 90% identity with the amino acid sequence, and (b) is 47 to 47 of SEQ ID NO: 4. The amino acid sequence shown at position 113 or an amino acid sequence having at least 90% identity thereto, and (d) above is the amino acid sequence shown at positions 168 to 187 of SEQ ID NO: 4 or at least 90% identity It is an amino acid sequence having sex. In a more preferred embodiment, (a) is an amino acid sequence represented by positions 1 to 46 of SEQ ID NO: 4, and (b) is an amino acid sequence represented by positions 47 to 113 of SEQ ID NO: 4, and (D) is an amino acid sequence represented by positions 168 to 187 of SEQ ID NO: 4.

  In one embodiment, the catalytic domain of the mutated xylanase of the present invention consisting of (a) to (d) above is further (e) an amino acid sequence represented by positions 188 to 198 of SEQ ID NO: 4 or at least It may further comprise an amino acid sequence having an identity of 60%, such as 60% or more, preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, and even more preferably 95% or more.

  In another preferred embodiment, the catalytic domain of the mutant xylanase of the present invention has an amino acid sequence represented by SEQ ID NO: 7, 8, 11, 12, 13 or 14, or at least 90%, such as 90% or more, preferably Is at least 92%, more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, still more preferably at least 97.5%, even more preferably at least 98%, even more preferably at least 99% It consists of an amino acid sequence having the above identity.

  The present invention also provides a mutant xylanase comprising the mutant xylanase catalytic domain of the present invention. The mutated xylanase of the present invention comprises the above-described mutated xylanase catalytic domain of the present invention and a carbohydrate binding module, has xylanase activity, and has no mutated xylanase catalytic domain of the present invention. Xylanase with reduced adsorptivity.

  The mutant xylanase of the present invention or the catalytic domain thereof is a solid residue after saccharification of an alkali mixed crushed bagasse using a cellulase preparation Cellic (registered trademark) CTec2 manufactured by Novozymes according to Reference Examples 4 to 6 described below at 50 ° C. for 24 hours. When the saccharification residue adsorption rate is calculated by reacting the saccharification residue 15 mg / mL with the mutant xylanase of the present invention or its catalytic domain at 50 ° C. for 1 hour, the saccharification residue adsorption rate is preferably It is 80% or less, more preferably 60% or less, further preferably 50% or less, even more preferably 40% or less, still more preferably 30% or less, and still more preferably 20% or less. Alternatively, the glycation residue adsorption rate of the mutated xylanase catalytic domain of the present invention is less than 150%, preferably less than 120%, relative to the glycation residue adsorption rate of the xylanase catalytic domain consisting of the amino acid sequence shown in SEQ ID NO: 2.

(4. Vectors and transformants)
The mutant xylanase of the present invention can be produced, for example, by a transformant introduced with a gene encoding the mutant xylanase of the present invention or a catalytic domain thereof (hereinafter also referred to as CD of the present invention). More specifically, the mutant xylanase or CD of the present invention is obtained by introducing a vector containing a polynucleotide encoding the mutant xylanase or CD into a host to obtain a transformant, and then culturing the transformant. Can be produced. That is, if the transformant is cultured in an appropriate medium, the gene encoding the mutant xylanase or CD of the present invention contained in the transformant is expressed, and the mutant xylanase or CD of the present invention is produced. The mutant xylanase or CD of the present invention can be obtained by isolating or purifying the produced mutant xylanase or CD from the culture.

  Accordingly, the present invention further provides a vector comprising a polynucleotide encoding the mutant xylanase or CD of the present invention. The present invention further provides a method for producing a transformant, which comprises introducing a polynucleotide encoding the mutant xylanase or CD of the present invention or a vector containing them into a host. Furthermore, the present invention provides a transformant containing the polynucleotide or vector. Furthermore, the present invention provides a method for producing a mutant xylanase or a catalytic domain thereof, comprising culturing the transformant.

  The polynucleotide encoding the CD of the present invention can be synthesized genetically or chemically based on the amino acid sequence of the CD. For example, a polynucleotide encoding the CD of the present invention can be prepared by the following procedure: A polynucleotide encoding a catalytic domain of the parent GH11 xylanase, for example, a catalytic domain consisting of the amino acid sequence shown in SEQ ID NO: 4. Prepare. On the other hand, a polynucleotide encoding a xylanase catalytic domain (hereinafter sometimes referred to as Xcel-CD-Xyn) consisting of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence 90% or more identical thereto is prepared. Then, by cleaving and ligating both polynucleotides according to a conventional method, the corresponding region in the polynucleotide encoding the parental GH11 xylanase catalytic domain is replaced with the predetermined region in the polynucleotide encoding Xcel-CD-Xyn. For example, a polynucleotide encoding the CD of the present invention can be prepared. A known reagent or kit (for example, In-Fusion (registered trademark) HD Cloning Kit; Takara Bio) can be used for cleavage and ligation of the polynucleotide.

  The polynucleotide (SEQ ID NO: 1) encoding the amino acid sequence represented by SEQ ID NO: 2 can be isolated from Xylanimonas cellulolytica (DSM 15894) or the like. The polynucleotide (SEQ ID NO: 3) encoding the amino acid sequence represented by SEQ ID NO: 4 can be isolated from Thermobifida fusca XY and the like. Isolation of a polynucleotide from the bacterium can be performed using any method used in the art. For example, after extracting the whole genome DNA of the bacterium, the target nucleic acid is selectively amplified by PCR using a primer designed based on the sequence of SEQ ID NO: 1 or 3, and the amplified nucleic acid is purified, A polynucleotide encoding the amino acid sequence represented by the number 2 or 4 can be obtained.

  A polynucleotide encoding an amino acid sequence 90% or more identical to the amino acid sequence shown in SEQ ID NO: 2 is not suitable for the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 isolated by the above-mentioned procedure. It can be produced by introducing a mutation by a known mutagenesis method such as genetic mutagenesis. For example, after mutating into a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 by the above-mentioned known method and expressing the obtained polynucleotide to obtain a protein, the xylanase activity and glycation residue adsorption rate of the protein And a polynucleotide encoding a protein having the desired xylanase activity and glycation residue adsorption property can be selected. Preferably, the adsorption rate of 15 mg / mL saccharification residue measured according to Reference Examples 4 to 6 described later is 20% or less, more preferably 10% or less, or the saccharification residue adsorption rate is an amino acid represented by SEQ ID NO: 2. A polynucleotide encoding a protein having a xylanase activity of less than 150%, preferably less than 120% with respect to the xylanase catalytic domain consisting of the sequence may be selected.

  The polynucleotide encoding the mutant xylanase of the present invention can be synthesized genetically or chemically based on its amino acid sequence. Alternatively, the polynucleotide encoding the mutant xylanase of the present invention can be prepared by linking the above-mentioned polynucleotide encoding the CD of the present invention to a polynucleotide encoding a carbohydrate binding module. A polynucleotide encoding a carbohydrate binding module can be isolated from a known microorganism having xylanase, for example, Xylanimonas cellulolytica (DSM 15894), Thermobifida fusca XY, and the like. Alternatively, a gene of GH11 xylanase containing a catalytic domain and a carbohydrate binding module is cloned, and a part of the catalytic domain coding region on the gene is replaced with a predetermined region of a polynucleotide encoding Xcel-CD-Xyn. Thus, a polynucleotide encoding the mutant xylanase of the present invention can be prepared.

  The type of vector into which the polynucleotide encoding the mutant xylanase or CD of the present invention is to be introduced is not particularly limited, and vectors usually used for protein production such as plasmids, cosmids, phages, viruses, YACs, BACs and the like can be mentioned. It is done. A plasmid vector is preferable. For example, commercially available plasmid vectors for protein expression such as shuttle vectors pHY300PLK, pUC19, pUC119, and pBR322 (all manufactured by Takara Bio Inc.) can be preferably used.

  The vector may contain a DNA fragment containing a DNA replication initiation region or a DNA region containing a replication origin. Alternatively, in the vector, in order to secrete a promoter region, terminator region, or expressed protein for initiating transcription of the gene upstream of the gene encoding the mutant xylanase or CD of the present invention. A control sequence such as a secretory signal region may be operably linked. Alternatively, a marker gene (for example, a resistance gene of a drug such as ampicillin, neomycin, kanamycin, chloramphenicol) for selecting a host into which the vector has been appropriately introduced may be further incorporated. Alternatively, when an auxotrophic strain is used as a host into which the vector of the present invention is introduced, a vector containing a gene encoding the required nutrition may be used.

  Ligation of a sequence encoding xylanase or its catalytic domain and the above-described regulatory sequence or marker gene sequence can be performed by a method such as SOE (splicing by overlap extension) -PCR (Gene, 1989, 77: 61-68). it can. Procedures for introducing gene sequences into plasmid vectors are well known in the art. The types of control sequences such as the promoter region, terminator, and secretion signal region are not particularly limited, and a commonly used promoter or secretion signal sequence can be appropriately selected and used depending on the host to be introduced.

  Examples of the control sequences include S237egl promoter and signal sequence (Biosci, Biotechnol, Biochem, 2000, 64 (11): 2281-2289), and Bacillus sp. KSM-S237 strain (FERM BP-7875) or Bacillus sp. Examples thereof include a transcription initiation control region, a translation initiation region, and a secretory signal peptide region of a cellulase gene derived from KSM-64 strain (FERM BP-2886). Alternatively, a promoter that expresses a saccharifying enzyme such as cellobiohydrolase, endoglucanase, β-glucosidase, xylanase, and β-xylosidase may be used. Alternatively, promoters of metabolic pathway enzymes such as pyruvate decarboxylase, alcohol dehydrogenase, and pyruvate kinase may be used.

  Examples of the transformant host into which the vector is introduced include microorganisms such as bacteria and filamentous fungi. Examples of bacteria include bacteria belonging to the genus Staphylococcus, Enterococcus, Listeria, Bacillus, etc. Among these, Bacillus bacteria such as Bacillus subtilis Alternatively, a mutant strain thereof (for example, a protease 9-deficient strain KA8AX described in JP-A-2006-174707) is preferable. Examples of filamentous fungi include Trichoderma, Aspergillus, Rizhopus, etc. Among these, Trichoderma is preferable from the viewpoint of enzyme productivity.

  As a method for introducing a vector into a host, a method usually used in the art such as a protoplast method and an electroporation method can be used. By selecting an appropriately introduced strain using marker gene expression, auxotrophy, etc. as an index, the target transformant introduced with the vector can be obtained.

  When the thus obtained transformant introduced with the vector containing the polynucleotide encoding the mutant xylanase or CD of the present invention is cultured in an appropriate medium, the gene on the vector is expressed, and the present invention Of mutant xylanase or CD. A medium used for culturing the transformant can be appropriately selected by those skilled in the art according to the type of the transformant. The produced mutant xylanase or CD of the present invention can be isolated or purified from the culture by a conventional method. At this time, when the mutant xylanase or CD gene of the present invention and a secretory signal sequence are operably linked on the vector, the produced xylanase or CD is secreted outside the cell body, so that it is easier. Can be recovered. The recovered xylanase or CD may be further purified by a known means.

(5. Biomass saccharification method or sugar production method)
The mutant xylanase or CD of the present invention has a lower saccharification residue adsorption rate and improved biomass saccharification activity than conventional xylanases and xylanase preparations. Therefore, the mutant xylanase or CD of the present invention can be preferably used for saccharification of biomass or for production of sugar from biomass.
Therefore, the present invention further provides a biomass saccharifying agent containing the mutant xylanase of the present invention or a catalytic domain thereof as an active ingredient.
Furthermore, the present invention provides a biomass saccharification method using the mutant xylanase of the present invention or a catalytic domain thereof, or the biomass saccharifying agent of the present invention.
Furthermore, the present invention provides a method for producing sugar from biomass using the mutant xylanase of the present invention or a catalytic domain thereof, or the biomass saccharifying agent of the present invention.

  The biomass saccharifying agent containing the mutant xylanase or CD of the present invention as an active ingredient is preferably an enzyme composition for biomass saccharification (hereinafter sometimes referred to as the enzyme composition of the present invention). The enzyme composition of the present invention contains the mutant xylanase or CD of the present invention, and 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. Examples of the cellulase used in the enzyme composition of the present invention include commercially available cellulase preparations and cellulases derived from animals, plants and microorganisms. In the enzyme composition of 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 one or more selected from the group consisting of cellobiohydrolase and endoglucanase.

  Specific examples of cellulase preparations that can be used in combination with the mutant xylanase or CD of the present invention include Celclast (registered trademark) 1.5 L (manufactured by Novozymes), TP-60 (manufactured by Meiji Seika Co., Ltd.), Cellic (registered trademark). Examples include, but are not limited to, CTec2 (manufactured by Novozymes), AcceleraseTMDUET (manufactured by Genencor), and Ultraflo (registered trademark) L (manufactured by Novozymes).

  In the total protein amount of the enzyme composition of the present invention, the content of the mutant xylanase or CD of the present invention is preferably 0.5 to 70% by mass in terms of the amount of CD from the viewpoint of improving saccharification efficiency. 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 composition of the present invention is preferably 10 to 99% by mass, more preferably 30 to 95% by mass, and still more preferably 50 to 95% from the viewpoint of improving saccharification efficiency. % By mass. The content of hemicellulase other than the mutant xylanase of the present invention or its catalytic domain in the total protein amount of the biomass saccharifying agent of the present invention is preferably 0.01 to 30% by mass from the viewpoint of improving saccharification efficiency. More preferably, it is 0.1-20 mass%, More preferably, it is 0.5-20 mass%.

  In the enzyme composition of the present invention, the protein amount ratio of the mutant xylanase or CD of the present invention to the above cellulase is preferably 0.01 to 100 as the mass ratio of [CD / cellulase] from the viewpoint of improving saccharification efficiency. More preferably, it is 0.05-5, More preferably, it is 0.05-1, More preferably, it is 0.05-0.5.

  The method for producing sugar from biomass according to the present invention includes a step of saccharifying biomass with the mutant xylanase of the present invention, the CD of the present invention, or the enzyme composition of the present invention. In the sugar production method of the present invention, by using the mutant xylanase or CD of the present invention having low saccharification residue adsorptivity, the non-specific adsorption of xylanase is reduced, thereby improving the xylan saccharification rate. Furthermore, in the sugar production method of the present invention, cellulose saccharification efficiency is also improved. The reason for this cellulose saccharification efficiency improvement is not clear, but by using the mutant xylanase or CD of the present invention, the hemicellulose in the biomass is efficiently decomposed, and the saccharification enzyme is likely to come into contact with cellulose. This is presumably because saccharification efficiency is improved.

  The biomass to which the biomass saccharifying agent of the present invention is applied or used in the sugar production method of the present invention is as described above in the section (1. Definition). From the viewpoints of availability, raw material costs, and saccharification efficiency, the biomass is preferably wood, processed or crushed wood, plant stems, leaves, or fruit bunches. Bagasse, EFB, oil palm (stem) ) Is more preferable, and bagasse is more preferable. The said biomass may be used individually or in mixture of 2 or more types. The biomass may be dried.

  The biomass saccharification method and the sugar production method of the present invention are characterized by improving biomass pulverization efficiency and improving saccharification efficiency or sugar production efficiency (that is, shortening the sugar production time). It is preferable to further include a step of pretreating the biomass before the step of saccharification with the enzyme composition.

  Examples of the pretreatment include one or more selected from the group consisting of alkali treatment, pulverization treatment, and hydrothermal treatment. As the pretreatment, alkali treatment is preferable from the viewpoint of improving saccharification efficiency, and from the viewpoint of further improving saccharification efficiency, alkali treatment and pulverization treatment are preferably performed, and alkali treatment and pulverization treatment are more preferably performed simultaneously. .

  The alkali treatment refers to reacting biomass with a basic compound described later. Examples of the alkali treatment method include a method of immersing biomass in an alkali solution containing a basic compound to be described later (hereinafter sometimes referred to as “immersion treatment”), or a mixture of biomass and a basic compound. And the like (hereinafter, sometimes referred to as “alkali mixed pulverization process”).

  The pulverization process means that the biomass is mechanically pulverized into small particles. By making the biomass into smaller particles, the saccharification efficiency is further improved. Moreover, when the crystal structure of the cellulose contained in biomass is destroyed by the pulverization process, the saccharification efficiency is still improved. The pulverization process can be performed using a known pulverizer. There is no restriction | limiting in particular in the grinder used, What is necessary is just an apparatus which can make biomass small particles. The grinding treatment may be combined with the alkali treatment with the basic compound described above. The pulverization process may be performed before or after the alkali treatment, or may be performed in parallel with the pulverization process, for example, the alkali mixed pulverization process described above. In the alkali mixed pulverization treatment, for example, biomass immersed in an alkali solution may be subjected to pulverization treatment (wet pulverization), or solid alkali and biomass may be pulverized together (dry pulverization). Grinding is preferred.

  The hydrothermal treatment refers to heat treatment of biomass in the presence of moisture. The hydrothermal treatment can be performed using a known reaction apparatus, and the reaction apparatus used is not particularly limited. Preferably, in the hydrothermal treatment, the coarsely pulverized biomass is made into a water slurry dispersed in water, and this is heat-treated. From the viewpoint of improving the fluidity of the slurry, the content of biomass in the water slurry is preferably 1 to 500 g / L, more preferably 5 to 400 g / L, and still more preferably 8 to 300 g / L.

  The biomass saccharification method and the sugar production method of the present invention include a step of saccharifying biomass, preferably the pretreated biomass, with the mutant xylanase, CD or enzyme composition of the present invention (in the present specification, “saccharification treatment”). May be included).

  The conditions for the saccharification treatment are not particularly limited as long as the mutant xylanase and CD of the present invention and other enzymes used together are not inactivated. Appropriate conditions can be appropriately determined by those skilled in the art depending on the type of biomass, the procedure of the pretreatment step, and the type of enzyme used.

  In the saccharification treatment, it is preferable to add the mutant xylanase, CD, or enzyme composition of the present invention to a suspension containing biomass. The content of the biomass in the suspension is preferably 0.5 to 20% by mass, more preferably 3 to 15% by mass, more preferably 3 to 15% by mass, from the viewpoint of improving saccharification efficiency or sugar production efficiency (that is, shortening the sugar production time). Preferably it is 5-10 mass%.

  The amount of the mutant xylanase or CD of the present invention to be used for the suspension is appropriately determined depending on the pretreatment conditions and the type and properties of the enzyme used together. Preferably, it is 0.04 to 600% by mass, more preferably 0.1 to 100% by mass, and still more preferably 0.1 to 50% by mass.

  The reaction pH for the saccharification treatment is preferably pH 4 to 9, more preferably pH 5 to 8, and further preferably pH 5 from the viewpoints of improving saccharification efficiency or sugar production efficiency (that is, shortening the sugar production time) and reducing production costs. ~ 7.

  The reaction temperature of the saccharification treatment is preferably 20 to 90 ° C., more preferably 25 to 85, from the viewpoints of improvement of saccharification efficiency, improvement of saccharification efficiency or sugar production efficiency (that is, shortening of sugar production time), and reduction of production cost. ° C, more preferably 30 to 80 ° C, even more preferably 40 to 75 ° C, still more preferably 45 to 65 ° C, still more preferably 50 to 65 ° C, still more preferably 50 to 60 ° C. 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 or saccharide production efficiency (that is, shortening saccharide production time) and reducing production costs. Therefore, it is preferably 1 to 5 days, more preferably 1 to 4 days, and further preferably 1 to 3 days.

  As another exemplary embodiment of the present invention described above, the following is further disclosed herein.

<1> A method for producing a mutant xylanase catalytic domain, comprising:
In the amino acid sequence of the catalytic domain of xylanase belonging to glycoside hydrolase family 11 (however, the identity between the amino acid sequence of the catalytic domain and the amino acid sequence represented by SEQ ID NO: 2 is less than 90%)
A region corresponding to the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 is substituted with the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto, or ,
Substituting the region corresponding to the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 with the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto ,Method.

<2> A method for producing a mutant xylanase, comprising the following:
In xylanases belonging to glycoside hydrolase family 11,
The amino acid sequence of the catalytic domain of the xylanase is shown at positions 118 to 143 of SEQ ID NO: 2 in the amino acid sequence of the catalytic domain (the identity between the amino acid sequence of the catalytic domain and SEQ ID NO: 2 is less than 90%). Replacing the region corresponding to the amino acid sequence with the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto, or
A region corresponding to the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 in the amino acid sequence of the catalytic domain is the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 or an amino acid having at least 90% identity thereto A method comprising replacing with a sequence.

<3> A method for reducing glycation residue adsorptivity of a xylanase catalytic domain, comprising:
In the amino acid sequence of the catalytic domain of xylanase belonging to glycoside hydrolase family 11 (however, the identity between the amino acid sequence of the catalytic domain and the amino acid sequence represented by SEQ ID NO: 2 is less than 90%)
A region corresponding to the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 is substituted with the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto, or ,
Substituting the region corresponding to the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 with the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto ,Method.

<4> A method for reducing saccharification residue adsorptivity of xylanase, comprising:
In xylanases belonging to glycoside hydrolase family 11,
The amino acid sequence of the catalytic domain of the xylanase is shown at positions 118 to 143 of SEQ ID NO: 2 in the amino acid sequence of the catalytic domain (the identity between the amino acid sequence of the catalytic domain and SEQ ID NO: 2 is less than 90%). Replacing the region corresponding to the amino acid sequence with the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto, or
A region corresponding to the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 in the amino acid sequence of the catalytic domain is the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 or an amino acid having at least 90% identity thereto A method comprising replacing with a sequence.

  <5> Adsorption rate for saccharification residue generated after cellulase saccharification treatment of alkali mixed crushed bagasse is preferably 80% or less, more preferably 60% or less, further preferably 50% or less, even more preferably 40% or less, The method according to <3> or <4>, preferably 30% or less, more preferably 20% or less.

<6> Preferably, the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto is:
It has at least 90%, preferably 92% or more, more preferably 96% or more identity with the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2, or the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2. Amino acid sequence,
And preferably, the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto is:
It has at least 90%, preferably 93% or more, more preferably 96% or more identity with the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 or the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2. Amino acid sequence,
The method according to any one of <1> to <5>.

<7> Preferably, in the amino acid sequence of the catalytic domain of xylanase belonging to the glycoside hydrolase family 11, a region corresponding to the amino acid sequence represented by positions 118 to 172 of SEQ ID NO: 2 is substituted with the following amino acid sequence: The method according to any one of <1> to <6>:
At least 90%, preferably 92% or more, more preferably 95% or more, and still more preferably 96 with the amino acid sequence shown at positions 118 to 172 of SEQ ID NO: 2, or the amino acid sequence shown at positions 118 to 172 of SEQ ID NO: 2. Amino acid sequence having% or more, preferably 98% or more identity.

<8> The method according to any one of <1> to <7>, wherein the catalytic domain of the xylanase belonging to the glycoside hydrolase family 11 is preferably:
(1a) It is a catalytic domain of a xylanase derived from Xylanimonas, a xylanase derived from Thermobifida, a xylanase derived from Trichoderma, a xylanase derived from Bacillus, an xylanase derived from Aspergillus, a xylanase derived from Clostridium, or a xylanase derived from Cellulomonas;
(1b) the identity with the amino acid sequence represented by SEQ ID NO: 2 is 85% or less, preferably 80% or less; or (1c) the amino acid sequence represented by SEQ ID NO: 4, or at least 90%, preferably It consists of amino acid sequences having an identity of 92% or more, more preferably 94% or more, still more preferably 96% or more, even more preferably 98%, still more preferably 99% or more.

<9> Preferably, the catalytic domain of xylanase belonging to the glycoside hydrolase family 11 has a glutamic acid residue at a position corresponding to position 85 and / or a position corresponding to position 174 of SEQ ID NO: 4, <1> to < 8. The method according to any one of 8>.

<10> Preferably, in the amino acid sequence of the catalytic domain of xylanase belonging to the glycoside hydrolase family 11, a region corresponding to the amino acid sequence represented by positions 1 to 50 of SEQ ID NO: 2 is substituted with the following amino acid sequence: The method according to any one of <1> to <9>:
At least 90%, preferably 92% or more, more preferably 94% or more, and still more preferably 96 with the amino acid sequence shown at positions 1 to 46 of SEQ ID NO: 4, or the amino acid sequence shown at positions 1 to 46 of SEQ ID NO: 4 % Amino acid sequence, more preferably 97.5% identity or more.

<11> Preferably, the region corresponding to the amino acid sequence represented by positions 1 to 50 of SEQ ID NO: 2 in the amino acid sequence of the catalytic domain after substitution is composed of the following amino acid sequences, <1> to <10> The method according to any one of:
At least 90%, preferably 92% or more, more preferably 94% or more, and still more preferably 96 with the amino acid sequence shown at positions 1 to 46 of SEQ ID NO: 4, or the amino acid sequence shown at positions 1 to 46 of SEQ ID NO: 4 % Amino acid sequence, more preferably 97.5% identity or more.

<12> Preferably, any one of <1> to <11>, wherein a fragment comprising the following amino acid sequence is added to the C-terminal of the region corresponding to the amino acid sequence represented by positions 118 to 172 of SEQ ID NO: 2. Method described in:
An amino acid sequence represented by positions 188 to 198 of SEQ ID NO: 4, or at least 60%, preferably 70% or more, more preferably 80% or more, more preferably 90% or more, still more preferably 95% or more. Amino acid sequence having.

<13> The method according to any one of <1> to <12>, wherein a xylanase catalytic domain comprising the following amino acid sequence is preferably produced:
90% or more, preferably 92% or more with the amino acid sequence represented by SEQ ID NO: 7, 8, 11, 12, 13 or 14, or the amino acid sequence represented by SEQ ID NO: 7, 8, 11, 12, 13 or 14 Preferably 94% or higher, more preferably 95% or higher, even more preferably 96% or higher, still more preferably 97.5% or higher, even more preferably 98% or higher, still more preferably 99% or higher. Amino acid sequence.

<14> a mutant xylanase catalytic domain, in order from the N-terminus,
(A) an amino acid sequence represented by positions 1 to 46 of SEQ ID NO: 4 or an amino acid sequence having at least 90% identity thereto,
(B) an amino acid sequence represented by positions 47 to 113 of SEQ ID NO: 4 or an amino acid sequence having at least 60% identity thereto,
(C) the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 or the amino acid sequence having at least 90% identity thereto, and the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 or at least 60% identical thereto Or an amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 or an amino acid sequence having at least 60% identity thereto, and an amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 or at least An amino acid sequence having 90% identity,
And (d) the amino acid sequence shown at positions 168 to 187 of SEQ ID NO: 4, or an amino acid sequence having at least 60% identity thereto,
A mutant xylanase catalytic domain consisting of

<15> Preferably, the mutant xylanase catalytic domain according to <14>, wherein (a) to (d) are as follows:
(A): the amino acid sequence represented by positions 1 to 46 of SEQ ID NO: 4 or at least 90%, preferably 92% or more, more preferably 94% or more, still more preferably 96% or more, still more preferably 97.5% Amino acid sequences having the above identity,
(B): the amino acid sequence represented by positions 47 to 113 of SEQ ID NO: 4 or at least 60%, preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, even more preferably 95% or more An amino acid sequence having an identity of 98% or more, still more preferably an amino acid sequence represented by the amino acid sequence represented by positions 47 to 113 of SEQ ID NO: 4;
(C) is any of the following (c1) to (c3):
(C1) the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 or an amino acid sequence having at least 90%, preferably 92% or more, more preferably 96% or more identity thereto, and 144 to 172 of SEQ ID NO: 2 Or an amino acid sequence having at least 60%, preferably 70% or more, more preferably 80% or more, and still more preferably 90% or more, or (c2) 118 to 143 of SEQ ID NO: 2 An amino acid sequence represented by the position or an amino acid sequence having at least 60%, preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more thereof, and positions 144 to 172 of SEQ ID NO: 2 Or at least 90%, preferably 93% or more, more preferably 96% The amino acid sequence having the above identity, or (c3) the amino acid sequence shown at positions 118 to 172 of SEQ ID NO: 2 or at least 90%, preferably 92% or more, more preferably 95% or more, and still more preferably 96 Amino acid sequences having% or more, even more preferably 98% or more identity;
(D): Amino acid sequence shown at positions 168 to 187 of SEQ ID NO: 4 or at least 60%, preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, even more preferably 95% or more Amino acid sequences having the identity of

<16> Preferably, at the C-terminal, (e) the amino acid sequence represented by positions 188 to 198 of SEQ ID NO: 4, or at least 60%, preferably 70% or more, more preferably 80% or more, and still more preferably 90 The mutant xylanase catalytic domain according to <14> or <15>, further comprising an amino acid sequence having an identity of at least%, even more preferably at least 95%.

<17> The mutant xylanase catalytic domain according to <15> or <16>, wherein (c) is preferably (c3).

<18> A mutant xylanase catalytic domain consisting of the amino acid sequence represented by SEQ ID NO: 7, 8, 11, 12, 13 or 14, or an amino acid sequence having 90% or more identity to them.

<19> The mutant xylanase catalytic domain according to <18>, preferably consisting of the following amino acid sequence:
Amino acid sequence represented by SEQ ID NO: 7, 8, 11, 12, 13 or 14; or 90% or more, preferably 92% or more, and the amino acid sequence represented by SEQ ID NO: 7, 8, 11, 12, 13 or 14; Preferably 94% or higher, more preferably 95% or higher, even more preferably 96% or higher, still more preferably 97.5% or higher, even more preferably 98% or higher, still more preferably 99% or higher. Amino acid sequence.

<20> A mutant xylanase comprising the mutant xylanase catalytic domain according to any one of <14> to <19> above and a carbohydrate binding module.

<21> A polynucleotide encoding the mutant xylanase catalytic domain according to any one of <14> to <19> or the mutant xylanase according to <20>.

<22> A vector comprising the polynucleotide according to <21> above.

<23> A method for producing a transformant, wherein the vector according to <22> is introduced into a host.

<24> A transformant comprising the polynucleotide according to <21> above or the vector according to <22> above.

<25> A biomass saccharifying agent comprising the mutant xylanase catalytic domain according to any one of <14> to <19> or the mutant xylanase according to <20>.

<26> The biomass saccharifying agent according to <25>, preferably further comprising cellulase.

<27> A biomass saccharification method using the mutant xylanase catalytic domain according to any one of <14> to <19> or the mutant xylanase according to <20>.

<28> The method according to <27>, preferably further using cellulase.

<29> Use of the mutant xylanase catalytic domain according to any one of the above <14> to <19> or the biomass saccharification of the mutant xylanase according to <20>.

<30> Use according to <29>, preferably in combination with cellulase.

<31> Biomass using the mutant xylanase catalytic domain according to any one of <14> to <19>, the mutant xylanase according to <20>, or the biomass saccharifying agent according to <25> or <26> Of sugar production from

  EXAMPLES Hereinafter, an Example is shown and this invention is demonstrated more concretely.

<Reference Example 1 PCR reaction>
In the polymerase chain reaction (PCR) for DNA fragment amplification in the following examples, an Applied Biosystems 2720 thermal cycler (Applied Biosystems) was used, and PrimeSTAR Max Premix (Takara Bio) or PrimeSTAR GXL DNA Polymerase (Takara Bio). DNA amplification was performed using the attached reagents. The PCR reaction solution composition was 1 μL of appropriately diluted template DNA, 20 pmol each of sense primer and antisense primer, and 10 μL PrimeStar Max Premix, so that the total reaction solution volume was 20 μL. PCR reaction conditions were 98 ° C. for 10 seconds, 55-60 ° C. for 10 seconds and 72 ° C. for 10 to 90 seconds (adjusted according to the target amplification product. Standard is 10 seconds per kb). This was done by repeating 30 times.

<Reference Example 2 Protein Concentration Measurement (Lowry Method)>
The Lowry method was used for protein quantification. The enzyme solution was diluted with ion-exchanged water to an appropriate concentration, and then quantified using a DC protein assay (BioRad). Reagent S was mixed at a ratio of 20 μL to 1 mL of Reagent A, and 25 μL of this mixed solution was added to 5 μL of protein. Further, 200 μL of Reagent B (color developing solution) was added, and left at room temperature for 15 minutes. Absorbance at 750 nm was measured with a microplate reader (Versa max, Molecular Devices), and the protein concentration was quantified from a BSA calibration curve.

<Reference Example 3 Measurement of Xylanase Activity>
The xylanase activity was measured by quantifying the reducing end of the produced sugar by the 3,5-dinitrosalicylic acid (DNS) method. Final concentration soluble xylan 1% (w / v) (2% (w / v) xylan from beechwood (Sigma Aldrich) is boiled for 1 minute, allowed to stand at room temperature, and then the supernatant obtained by centrifugation is soluble Xylan), 50 mM sodium acetate buffer (pH 5.0), and 10 μL of enzyme were mixed and reacted at 50 ° C. for 10 minutes. After the reaction, 100 μL of DNS solution (sodium hydroxide (Wako Pure Chemical Industries) 1.6% (w / v), dinitrosalicylic acid (Wako Pure Chemical Industries) 0.5% (w / v), sodium potassium tartrate ( 30.0% (w / v)) was added and reacted at 99.9 ° C. for 10 minutes. By measuring the absorbance at a wavelength of 540 nm with a microplate reader (Molecular Device Japan), the reducing end of the produced sugar was quantified. Xylose was used for the calibration curve.

<Reference Example 4 Preparation of Biomass>
(1) Bagasse preparation Bagasse (sugar cane pomace, holocellulose content 71.3 mass%, crystallinity 29%, moisture content 7.0 mass%) is placed in a vacuum dryer VO-320 (Advantech Toyo). The dried bagasse was dried under reduced pressure for 2 hours under conditions of nitrogen flow to obtain a dry bagasse having a holocellulose content of 71.3% by mass, a crystallinity of 29% and a water content of 2.0% by mass.
(2) Mixing and grinding treatment 100 g of the obtained dry bagasse and 8.8 g of granular sodium hydroxide “Tosoh Pearl” (manufactured by Tosoh Corporation) with a particle size of 0.7 mm (0.5 mol with respect to 1 mol of AGU constituting holocellulose) Equivalent amount), batch type vibration mill MB-1 (Central Chemical Machine: Container total volume 3.5L, φ30mm, length 218mm as medium, 13 SUS304 rods with circular cross section, rod filling rate 57% ) And pulverized for 2 hours to obtain a bagasse pulverized product (average particle size of about 16.6 μm).

<Reference Example 5 Saccharification Residue>
Alkaline mixed ground bagasse 5% (w / v) prepared in Reference Example 4, cellulase preparation Cellic (registered trademark) CTec2 (Novozymes) 5 mg / g-biomass, 30 μg / mL tetracycline hydrochloride (wako), 0.6 μg / 10 mL of the solution containing mL erythromycin (wako) was reacted in a 25 mL free-standing centrifuge tube at 50 ° C. and 150 rpm (Tytech: BR-40F) for 24 hours to saccharify bagasse. After completion of the reaction, the supernatant was removed by centrifugation (11000 rpm × 10 minutes) and washed twice with 50 mM sodium acetate buffer (pH 5.0) to recover the solid content. The obtained saccharification residue (hereinafter referred to as CTec2 saccharification residue) was used for the following xylanase adsorptive analysis.

<Reference Example 6 Adsorbability Analysis for Saccharification Residue>
The CTec2 saccharification residue was used as a substrate (saccharification residue), and the adsorption behavior of xylanase was analyzed. Substrate of a predetermined concentration (a saccharification residue suspension was dispensed and centrifuged to obtain a water-containing solid content only. The mass of the saccharification residue was calculated from a calibration curve), 50 mM sodium acetate buffer, xylanase 50 to 200 μg / 0.5 to 1 mL of the reaction solution containing mL was shaken in a 2 mL Eppendorf tube or a 96-well deep well plate (Thermo SIENTIFIC) at 50 ° C. and 150 rpm for 1 hour. After completion of the reaction, the supernatant was collected by centrifugation (11000 rpm, 5 minutes, 4 ° C.), and the xylanase activity was measured by the DNS method described above. As shown by the following formula, the activity at the substrate concentration of 0 mg / mL was defined as 100, and the xylanase relative specific activity of the supernatant of the reaction solution reacted with the substrate was calculated. By subtracting the obtained activity value from 100, the saccharification residue adsorption rate of xylanase was calculated.
Saccharification residue adsorption rate (%) = 100-relative specific activity (%)
Relative specific activity (%) = (xylanase activity of reaction supernatant with addition of saccharification residue / xylanase activity of reaction supernatant without addition of saccharification residue) × 100

<Reference Example 7 Analysis of produced sugar>
In the analysis of the amount of reducing sugar in the supernatant after the xylan saccharification reaction, the amounts of xylose, xylobiose and xylotriose were quantified by HPLC. For HPLC, a Dionex DX500 chromatography system, a CarboPacPA1 column (4 × 250 mm), an ED40 pulsed amperometric detector was used, and the eluent was A: 100 mM sodium hydroxide aqueous solution, B: 1 M sodium acetate / A 100 mM sodium hydroxide aqueous solution, C: ultrapure water was used.

<Example 1 Preparation of Mutant Xylanase>
(1) Preparation of xylanase gene-containing vector Xylanimonas cellulosilytica DSM15894 strain (Xcel) and Thermobifida fusca YX strain (Tfu) were mixed with Trypticase Soy Agar medium (3.0% Trypticase Soy, 2.0% Agar), respectively. And inoculated at 28 ° C. for 1 day. Genomic DNA was obtained from the cells obtained by the culture using an UltraClean ^™ Microbial DNA Isolation Kit (Mo Bio Laboratories, Inc.).

  Xcel xylanase (Xcel-Xyn) gene region using forward primer Xcel-Xyn F (SEQ ID NO: 18) and reverse primer Xcel-Xyn R (SEQ ID NO: 19) shown in Table 1 using the obtained genomic DNA as a template An approximately 1.0 kbp fragment (A) of (SEQ ID NO: 15) was amplified. Similarly, a vector containing an approximately 1.0 kbp fragment (B) of the Tfu xylanase (Tfu-Xyn) gene region (SEQ ID NO: 16) was prepared. As the primer, forward primer Tfu-Xyn F (SEQ ID NO: 20) and reverse primer Tfu-Xyn R (SEQ ID NO: 21) were used.

  The S237 alkaline cellulase gene derived from the Bacillus genus KSM-S237 strain (FERM BP-7875) (see Japanese Patent Laid-Open No. 2000-210081) (SEQ ID NO: 17) at the BamHI restriction enzyme breakpoint of the shuttle vector pHY300PLK (Takara Bio) Using the recombinant plasmid pHY-S237 into which the DNA fragment to be encoded was inserted as a template, using the forward primer pHY-S237 F (SEQ ID NO: 22) and reverse primer pHY-S237 R (SEQ ID NO: 23) shown in Table 1, S237 An approximately 5.6 kbp fragment (C) in the region excluding the alkaline cellulase structural gene was amplified. The amplified gene fragment was purified with High Pure PCR Product Purification kit (Roche).

  Purified DNA fragment (A) or (B) 1 μL and DNA fragment (C) 1 μL, 5 × In-Fusion (registered trademark) HD Enzyme Premi included in In-Fusion (registered trademark) HD Cloning Kit (Takara Bio) × 2 μL and DNase-free water 6 μL were mixed and reacted at 50 ° C. for 15 minutes, and the xylanase gene fragment was cloned into a vector. Then, using 2 μL of the reaction solution, competent cell E.I. 20 μL of E. coli DH5α (Takara Bio) was transformed. An LB agar medium containing 50 ppm ampicillin sodium (Wako Pure Chemical Industries) and 2.0% agar (Wako Pure Chemical Industries) was used as the regeneration medium for the transformant. 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 then the plasmid was recovered and purified from the obtained bacterial cell using High Pure Plasmid Isolation kit (Roche).

(2) Preparation of xylanase catalytic domain gene In order to prepare an expression vector for a gene encoding xylanase catalytic domain (CD), the above-mentioned Xcel-Xyn gene or the recombinant plasmid inserted with Tfu-Xyn gene was used as a template. Forward primer Xcel-CD-Xyn F (SEQ ID NO: 24) and reverse primer Xcel-CD-Xyn R (SEQ ID NO: 25) shown in Table 1 or forward primer Tfu-CD-Xyn F (SEQ ID NO: 26) and reverse primer About 5.6 to 6.0 kbp fragment containing a gene encoding Xcel-Xyn or Tfu-Xyn CD was amplified by PCR using Tfu-CD-Xyn R (SEQ ID NO: 27). Primers were prepared according to the manual of PrimeSTAR (registered trademark) Mutagenesis Basal Kit (Takara Bio). The gene fragment was purified with High Pure PCR Product Purification kit (Roche). Using 2 μL of the purified PCR fragment, competence and cell E. coli were used. 20 μL of E. coli DH5α (Takara Bio) was transformed.
Hereinafter, the Xcel-derived xylanase CD may be referred to as Xcel-CD-Xyn. The Tfu-derived xylanase catalyst CD is sometimes referred to as Tfu-CD-Xyn.

(3) Preparation of mutant xylanase gene A chimeric xylanase of Xcel-CD-Xyn and Tfu-CD-Xyn was prepared. PCR was performed using the above-described plasmid introduced with the gene encoding Xcel-CD-Xyn and the plasmid introduced with the gene encoding Tfu-CD-Xyn as a template, using the primers described in Tables 2-3, DNA encoding a partial fragment of the catalytic domain was amplified. In the same manner as in (2) above, DNA encoding each partial fragment is ligated onto a plasmid vector in a predetermined order, and the resulting plasmid is used to transform E. coli, select a transformant, and Purification was performed.

(4) Production of mutant xylanase The purified plasmid was introduced into a host bacterium according to the protoplast transformation method (Mol. Gen. Genet., 1979, 168: 111-115). As hosts, nine extracellular proteases and lytic enzymes from Bacillus subtilis strain 168 were deleted, and ΔDpr9lyt ↑ prsA strain, which improved protein folding efficiency, nine extracellular proteases and extracellular cellulase from 168 strains. The 874 · Dpr7 · eglS ↑ prsA strain which had been deleted and improved the protein folding efficiency was used. Transformant regeneration medium includes tetracycline-containing DM3 regeneration agar medium (Bactocasamino acid (Difco) 0.5% (w / v), yeast extract (Difco) 0.5% (w / v), disodium succinate Hexahydrate (Wako Pure Chemical Industries) 4.05% (w / v), dipotassium monohydrogen phosphate (Wako Pure Chemical Industries) 0.35% (w / v), dipotassium dihydrogen phosphate (wa) Kojun Pharmaceutical) 0.15% (w / v), Glucose (Wako Pure Chemical Industries) 0.1% (w / v), Magnesium Chloride (Wako Pure Chemical Industries) 20 mM, Bovine Serum Albumin (Wako Pure Chemical Industries) 0.01% (w / v), trypan blue 0.005% (w / v) (ACROS ORGANICS), tetracycline hydrochloride (Wako Pure Chemical Industries) 0.005% (w / v), carboxymethylcellulose 1.0 % (W / v), cold 1.0% (w / v)) was used.

  The transformant grown on the DM3 regeneration agar medium was inoculated into 5 mL of LB (15 μg / mL tetracycline / large test tube) and cultured with shaking at 30 ° C. and 120 rpm for 16 hours. 600 μL of the culture solution was added to 30 mL of 2 × L Mal medium (bactotryptone 2% (w / v), yeast extract 1.0% (w / v), sodium chloride 1.0% (w / v), manganese sulfate pentahydrate Inoculated into Japanese product (Wako Pure Chemical Industries) 75ppm, maltose monohydrate (Wako Pure Chemical Industries) 7.5% (w / v), tetracycline hydrochloride 15ppm) at 30 ° C, 120rpm for 2-3 days Shake culture (PRECi: PRXYg-98-R type) was performed. The supernatant of the obtained culture broth was collected by centrifugation (8000 rpm, 15 minutes). The obtained supernatant was desalted and buffer-substituted with Econo-Pac (registered trademark) 10DG Columns (BIO RAD). A 50 mM Tris-HCl (pH 7.5) buffer was used as a replacement buffer.

  The structure of the obtained chimeric xylanase CD is shown in Table 4.

<Example 2 Adsorption property of saccharification residue of mutant xylanase>
Under the conditions of Reference Example 6, the adsorption rate for the CTec2 saccharification residue was analyzed for each mutant xylanase CD prepared in Example 1. The CTec2 saccharification residue concentration was 15 or 58 mg / mL. The results are shown in FIG. The mutant xylanase CD (TTXXX, TXXXX) of the present invention having the Xcel-CD-Xyn sequence in the (c1) to (c2) regions had a reduced glycation residue adsorption rate compared to Tfu-CD-Xyn. . From these results, it was confirmed that the region corresponding to the region of positions 114 to 167 of the amino acid sequence of Tfu-CD-Xyn represented by SEQ ID NO: 4 is involved in the adsorption of xylanase saccharification residue, and this region is expressed as Xcel-CD-Xyn. It was shown that glycan residue adsorption by xylanase can be reduced by substituting the derived sequence.

<Example 3 Adsorption of saccharification residue of mutant xylanase>
With respect to the mutant xylanase CD having regions (c1) to (c2) having different sequences, the saccharification residue adsorption rate was analyzed under the same conditions as in Example 2. The results are shown in FIG. The xylanase CD (XXTXT, XXXTX, XXXTT, XXXXT) having the Xcel-CD-Xyn sequence in any of the (c1) and (c2) regions is the sequence of Tfu-CD-Xyn in the (c1) to (c2) regions. The adsorption rate of the saccharification residue was reduced as compared with those having XXTT (XXTTTT, XXTTX).

<Example 4 Saccharification of xylan in the presence of lignin>
(1) Preparation of soluble xylan 2% (w / v) xylan from beechwood (Sigma Aldrich) is boiled for 1 minute, allowed to stand at room temperature, and the supernatant obtained by centrifugation (8000 rpm, 10 minutes) is acceptable Soluble xylan was used.

(2) Production of sugar 50 mM sodium acetate buffer, soluble xylan 1% (w / v), lignin 0-2 mg / mL, cellulase 1 mg / g-biomass, xylanase 1 mg / g-biomass, 30 μg / mL tetracycline hydrochloride ( 1 ml of a solution containing wako) and 0.6 μg / mL erythromycin (wako) was reacted in a 2 mL Eppendorf tube at 50 ° C. and 150 rpm (Tytech: BR-40F) to saccharify xylan. As the xylanase, the mutant xylanase CD (TXXXX, TXXXX, XXTTTT), Xcel-CD-Xyn (SEQ ID NO: 2) or Tfu-CD-Xyn (SEQ ID NO: 4) prepared in Example 1 was used. As the cellulase, a commercially available cellulase preparation (Celcrust (registered trademark); Novozymes) was used.

  After completion of the reaction, the supernatant was recovered by centrifuging the reaction solution at 11000 rpm for 5 minutes at 4 ° C. The amount of reducing sugar in the supernatant was quantified by the method described in Reference Example 7. The results are shown in FIG. The amount of sugar produced from xylan in the presence of lignin increased up to 3.5 times compared to the enzyme composition containing Tfu-CD-Xyn when the enzyme composition containing the mutant xylanase CD of the present invention was used. . From this, it was shown that the mutant xylanase of the present invention is suitable for saccharification of biomass containing saccharification residues such as lignin.

Claims (18)

A method for producing a mutant xylanase catalytic domain comprising:
In the amino acid sequence of the catalytic domain of xylanase belonging to glycoside hydrolase family 11 (however, the identity between the amino acid sequence of the catalytic domain and the amino acid sequence represented by SEQ ID NO: 2 is less than 90%)
A region corresponding to the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 is substituted with the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto, or ,
Substituting the region corresponding to the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 with the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto ,Method. A method for producing a mutant xylanase comprising:
In xylanases belonging to glycoside hydrolase family 11,
The amino acid sequence of the catalytic domain of the xylanase is shown at positions 118 to 143 of SEQ ID NO: 2 in the amino acid sequence of the catalytic domain (the identity between the amino acid sequence of the catalytic domain and SEQ ID NO: 2 is less than 90%). Replacing the region corresponding to the amino acid sequence with the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto, or
A region corresponding to the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 in the amino acid sequence of the catalytic domain is the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 or an amino acid having at least 90% identity thereto A method comprising replacing with a sequence. A method for reducing the adsorption of saccharification residues of a xylanase catalytic domain, comprising:
In the amino acid sequence of the catalytic domain of xylanase belonging to glycoside hydrolase family 11 (however, the identity between the amino acid sequence of the catalytic domain and the amino acid sequence represented by SEQ ID NO: 2 is less than 90%)
A region corresponding to the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 is substituted with the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto, or ,
Substituting the region corresponding to the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 with the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto ,Method. A method for reducing the saccharification residue adsorptivity of xylanase, comprising:
In xylanases belonging to glycoside hydrolase family 11,
The amino acid sequence of the catalytic domain of the xylanase is shown at positions 118 to 143 of SEQ ID NO: 2 in the amino acid sequence of the catalytic domain (the identity between the amino acid sequence of the catalytic domain and SEQ ID NO: 2 is less than 90%). Replacing the region corresponding to the amino acid sequence with the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto, or
A region corresponding to the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 in the amino acid sequence of the catalytic domain is the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 or an amino acid having at least 90% identity thereto A method comprising replacing with a sequence.   The region corresponding to the amino acid sequence represented by positions 1 to 50 of SEQ ID NO: 2 in the amino acid sequence of the catalytic domain after substitution is the amino acid sequence represented by positions 1 to 46 of SEQ ID NO: 4 or 90% or more identity thereto The method according to claim 1, comprising an amino acid sequence having   The amino acid sequence of the catalytic domain of the xylanase belonging to the glycoside hydrolase family 11 is the amino acid sequence represented by SEQ ID NO: 4 or an amino acid sequence having 90% or more identity thereto. Method.   The xylanase catalytic domain consisting of the amino acid sequence represented by SEQ ID NO: 7, 8, 11, 12, 13 or 14 or an amino acid sequence having 90% or more identity with them is produced. The method according to claim 1. A mutated xylanase catalytic domain, in order from the N-terminus;
(A) an amino acid sequence represented by positions 1 to 46 of SEQ ID NO: 4 or an amino acid sequence having at least 90% identity thereto,
(B) an amino acid sequence represented by positions 47 to 113 of SEQ ID NO: 4 or an amino acid sequence having at least 60% identity thereto,
(C) the amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 or the amino acid sequence having at least 90% identity thereto, and the amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 or at least 60% identical thereto Or an amino acid sequence shown at positions 118 to 143 of SEQ ID NO: 2 or an amino acid sequence having at least 60% identity thereto, and an amino acid sequence shown at positions 144 to 172 of SEQ ID NO: 2 or at least An amino acid sequence having 90% identity,
And (d) the amino acid sequence shown at positions 168 to 187 of SEQ ID NO: 4, or an amino acid sequence having at least 60% identity thereto,
A mutant xylanase catalytic domain consisting of   The mutant xylanase catalytic domain according to claim 8, further comprising (e) an amino acid sequence represented by positions 188 to 198 of SEQ ID NO: 4 or an amino acid sequence having at least 60% identity thereto at the C-terminus.   The mutant xylanase catalytic domain according to claim 8 or 9, wherein (c) is an amino acid sequence represented by positions 118 to 172 of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto.   A mutant xylanase catalytic domain comprising the amino acid sequence represented by SEQ ID NO: 7, 8, 11, 12, 13 or 14, or an amino acid sequence having 90% or more identity with them.   A mutant xylanase comprising the mutant xylanase catalytic domain according to any one of claims 8 to 11 and a carbohydrate binding module.   A polynucleotide encoding the mutant xylanase catalytic domain according to any one of claims 8 to 11 or the mutant xylanase according to claim 12.   A vector comprising the polynucleotide according to claim 13.   A method for producing a transformant, wherein the vector according to claim 14 is introduced into a host.   A biomass saccharifying agent comprising the mutant xylanase catalytic domain according to any one of claims 8 to 11 or the mutant xylanase according to claim 12.   The biomass saccharifying agent of claim 16, further comprising cellulase.   The manufacturing method of the saccharide | sugar from biomass using the biomass saccharifying agent of Claim 16 or 17.