JP2019134683A - Cellulase - Google Patents

Abstract

To provide cellulase having high binding capacity to a cellulose-based substrate.SOLUTION: Provided is cellulase having a carbohydrate binding module (CBM) of xylanase.SELECTED DRAWING: None

Description

  The present invention relates to a novel cellulase and use thereof.

  Conventionally, in the industrial production of target substances such as L-amino acids by fermentation, components obtained from plant-derived edible resources such as glucose, fructose, sucrose, waste molasses, and starch hydrolysates are used as carbon sources. Have been used. However, due to the recent increase in population and the improvement of living standards in developing countries, practical application studies are underway on the use of plant-derived non-edible biomass raw materials rather than ingredients competing with these foods.

  Such plant-derived non-edible biomass materials are composed of cellulose, hemicellulose, lignin, and the like. Among these, cellulose and hemicellulose are pretreatment steps using heat, acid, etc., cellulase, xylanase, etc. Through the saccharification treatment process of saccharification enzyme, it is converted into 5-monosaccharide and 6-monosaccharide and can be used as a raw material for fermentation (Patent Documents 1 and 2).

  In order to put enzymatic saccharification of plant-derived non-edible biomass raw materials at an industrial level, reduction of enzyme costs is an issue. Reduction of enzyme cost can be achieved, for example, by reducing the amount of saccharifying enzyme used. As a method for reducing the amount of saccharifying enzyme used, there are known methods (Patent Documents 3 and 4) in which saccharifying enzyme is recovered by being bound to a cellulosic substrate such as plant biomass.

Saccharifying enzymes such as cellulase and xylanase can be bound to cellulosic substrates such as plant biomass using a carbohydrate binding module (CBM).
Examples have been reported in which cellulase activity and substrate specificity are improved by modifying the CBM of cellulase (for example, introduction of mutation into CBM or addition of CBM) (Non-Patent Documents 1 to 4).

Japanese National Patent Publication No. 9-507386 Japanese National Patent Publication No. 11-506934 JP 2010-098951 A WO2016 / 043281

Strobel KL et al., Engineering Cel7A Carbohydrate Binding Module and Linker for Reduced Lignin Inhibition. Biotechnol Bioeng. 2015 Nov 30. doi: 10.1002 / bit.25889. Reyes-Ortiz V et al., Addition of a carbohydrate-binding module enhances cellulase penetration into cellulose substrates.Biotechnol Biofuels.2013 Jul 3; 6 (1): 93.doi: 10.1186 / 1754-6834-6-93. Walker JA et al., Multifunctional cellulase catalysis targeted by fusion to different carbohydrate-binding modules. Biotechnol Biofuels. 2015 Dec 21; 8: 220.doi: 10.1186 / s13068-015-0402-0. Tang Z et al., Improving endoglucanase activity by adding the carbohydrate-binding module from Corticium rolfsii.J Microbiol Biotechnol. 2014 Apr; 24 (4): 440-6.

  An object of the present invention is to provide a cellulase having a high binding ability to a cellulosic substrate.

  As a result of diligent research to solve the above problems, the present inventors have replaced β-glucosidase CBM-containing region with xylanase CBM-containing region, thereby binding β-glucosidase to cellulosic substrate. And the present invention has been completed.

That is, the present invention can be exemplified as follows.
[1]
A protein having a carbohydrate binding module of xylanase and having cellulase activity.
[2]
The protein, which is a protein having a carbohydrate binding module of xylanase and a catalytic domain of cellulase.
[3]
The protein, wherein the carbohydrate binding module is a carbohydrate binding module classified into Family 1 of carbohydrate binding modules.
[4]
The protein, wherein the carbohydrate binding module is a carbohydrate binding module of Xyl10A.
[5]
The protein, wherein the carbohydrate binding module comprises the amino acid sequence described in (a), (b), or (c) below:
(A) the amino acid sequence of positions 375 to 403 of SEQ ID NO: 16;
(B) an amino acid sequence having a binding ability to a cellulosic substrate, including substitution, deletion, insertion, or addition of 1 to 5 amino acid residues in the amino acid sequence at positions 375 to 403 of SEQ ID NO: 16;
(C) an amino acid sequence having 90% or more identity to the amino acid sequence at positions 375 to 403 of SEQ ID NO: 16 and having an ability to bind to a cellulosic substrate.
[6]
The protein, wherein the catalytic domain is a catalytic domain of β-glucosidase.
[7]
The protein, wherein the catalytic domain is a catalytic domain of a cellulase classified into family 3 of glycoside hydrolases.
[8]
The protein, wherein the catalytic domain is a catalytic domain of Bgl3A.
[9]
The protein, wherein the catalytic domain comprises an amino acid sequence described in (a), (b), or (c) below:
(A) the amino acid sequence of positions 25 to 601 of SEQ ID NO: 11;
(B) an amino acid sequence having cellulase activity, including substitution, deletion, insertion, or addition of 1 to 10 amino acid residues in the amino acid sequence at positions 25 to 601 of SEQ ID NO: 11;
(C) an amino acid sequence having cellulase activity having 90% or more identity to the amino acid sequence at positions 25 to 601 of SEQ ID NO: 11.
[10]
The protein having a linker region between the carbohydrate binding module and the catalytic domain.
[11]
The protein having a fibronectin type III-like domain.
[12]
The protein which does not have the inherent carbohydrate binding module of the cellulase from which the catalytic domain is derived.
[13]
The protein that is the protein according to the following (a), (b), or (c):
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 34 or 36;
(B) a protein having an amino acid sequence containing substitution, deletion, insertion or addition of 1 to 10 amino acid residues and having cellulase activity in the amino acid sequence shown in SEQ ID NO: 34 or 36;
(C) A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 34 or 36 and having cellulase activity.
[14]
A gene encoding the protein.
[15]
A vector carrying the gene.
[16]
A host having the gene.
[17]
Said host which is a bacterium or a fungus.
[18]
A method for producing a saccharified product, comprising treating a cellulosic substrate with the protein.
[19]
The method, wherein the cellulosic substrate is plant biomass.

  According to the present invention, a cellulase having a high binding ability to a cellulosic substrate is provided. The cellulase is useful, for example, for enzyme recovery and reuse utilizing binding to cellulosic substrates.

The figure which shows the residual rate in the reaction liquid supernatant of each glycolytic enzyme at the time of making the culture supernatant of T. cellulolyticus F09 (DELTA) creA strain | stump | stock react with hydrothermal treatment bagasse. The figure which shows the structure of each Bgl3A and Xyl10A.

  Hereinafter, the present invention will be described in detail.

<1> Chimeric enzyme The present invention provides a chimeric enzyme and a gene encoding the same. The chimeric enzyme provided by the present invention (hereinafter also simply referred to as “chimeric enzyme”) is a cellulase having a carbohydrate binding module (CBM) of xylanase. In other words, the chimeric enzyme is a protein having a xylanase CBM and cellulase activity. A gene encoding a chimeric enzyme is also referred to as a “chimeric enzyme gene”.

<1-1> Cellulase “Cellulase” is a general term for enzymes involved in cellulose degradation. The “cellulase” specifically refers to a protein having an activity of catalyzing a reaction of hydrolyzing a β-1,4 glycoside bond in β-D-glucan. This activity is also referred to as “cellulase activity”. Examples of β-D-glucan include cellulose and cellooligosaccharides such as cellobiose that can be generated as a degradation product thereof.

  The cellulase that can be referred to for specifying the chimeric enzyme is not particularly limited.

Examples of the cellulase include endoglucanase (EC 3.2.1.4), cellobiohydrolase (EC 3.2.1.91 or EC 3.2.1.176), and β-glucosidase (EC 3.2.1.21).

“Endoglucanase” refers to an endo-type cellulase. Specifically, it is a protein having an activity of catalyzing a reaction of randomly hydrolyzing β-1,4 glycoside bonds in β-D-glucan such as cellulose. It may be. This activity is also referred to as “endoglucanase activity”. Examples of endoglucanases include those classified into glycoside hydrolase family 5, 6, 7, 8, 9, 10, 12, 26, 44, 45, 48, 51, 74, or 124. (Http://www.cazy.org/).

The “cellobiohydrolase” refers to an exo-type cellulase, and specifically may be a protein having an activity of catalyzing a reaction in which β-D-glucan such as cellulose is sequentially cleaved from the end to generate a cellobiose unit. . This activity is also referred to as “cellobiohydrolase activity”. Cellobiohydrolase is roughly classified into those acting from the reducing end (EC 3.2.1.91) and those acting from the non-reducing end (EC 3.2.1.176). Examples of cellobiohydrolases acting from the reducing end include those classified into glycoside hydrolase family 5, 6, or 9 (http://www.cazy.org/). Examples of cellobiohydrolases acting from the non-reducing terminal include those classified into glycoside hydrolase family 7, 9, or 48 (http://www.cazy.org/).

  The “β-glucosidase” may be a protein having an activity of catalyzing a reaction in which β-D-glucan such as cellulose and cellooligosaccharide is sequentially cleaved from the non-reducing end to produce β-D-glucose. This activity is also referred to as “β-glucosidase activity”. Examples of β-glucosidase include those classified into glycoside hydrolase family 1, 2, 3, 5, 9, 30, or 116 (http://www.cazy.org/).

An example of cellulase is β-glucosidase. In addition, examples of cellulases include those classified into family 3 of glycoside hydrolases (GH3). Moreover, as a cellulase, the beta-glucosidase classified into GH3 is mentioned especially.

Cellulase activity can be measured, for example, by a known technique. The endoglucanase activity and cellobiohydrolase activity can be determined, for example, by performing an enzymatic reaction using cellulose such as microcrystalline cellulose (Avicel) or carboxymethyl cellulose (CMC) as a substrate and using the amount of reducing sugar produced as an index. The amount of reducing sugar can be measured, for example, by a known method such as a dinitrosalicylic acid (DNS) method or a Somogie Nelson method. The cellobiohydrolase activity can be determined using, for example, the amount of pNP (p-nitrophenol) produced by carrying out an enzymatic reaction using pNP-β-D-Cellobioside as a substrate. The cellobiohydrolase activity can be determined, for example, by performing an enzymatic reaction using pNP-β-D-Glucose as a substrate and using the amount of pNP produced as an index.

Examples of the cellulase include fungal and bacterial cellulases. Examples of fungal cellulases include Trichoderma fungi such as Trichoderma reesei, Talaromyces (Acremonium) fungi such as Talaromyces cellulolyticus (former name: Acremonium cellulolyticus), and Phanerochaete fungal cellulases such as Phanerochaete chrysosporium. Examples of bacterial cellulase include cellulases of bacteria belonging to the genus Ruminiclostridium (Clostridium) such as Ruminiclostridium thermocellum (former name: Clostridium thermocellum). Specific examples of cellulases include β-glucosidase Bgl3A, endoglucanases Eg5A, Eg5X1, Eg5X2, and Eg7B, and cellobiohydrolases Cbh1 and Cbh2. Specifically, Bgl3A may be a β-glucosidase classified as GH3. These cellulases can be found in fungi such as Talaromyces cellulolyticus. The amino acid sequences of various cellulases and the base sequences of genes encoding them can be obtained from public databases such as NCBI. The amino acid sequences of Talaromyces cellulolyticus Cbh1, Cbh2, Bgl3A, Eg5A, Eg5X1, Eg5X2, and Eg7B are shown in SEQ ID NOs: 9 to 1.
5 respectively. In the amino acid sequence of Bgl3A shown in SEQ ID NO: 11, the amino acid sequence at positions 1 to 18 is a signal peptide, the amino acid sequence at positions 25 to 601 is a catalytic domain, and the amino acid sequence at positions 651 to 721 is a fibronectin type III-like domain ( Fibronectin type III-like domain), the amino acid sequence of positions 732 to 776 is the linker region, positions 780 to 807
The amino acid sequence at the position corresponds to CBM. In addition, the nucleotide sequences of cDNAs of genes encoding Calhmyces cellulolyticus Cbh1, Cbh2, Bgl3A, Eg5A, Eg5X1, Eg5X2, and Eg7B are shown in SEQ ID NOs: 25 to 31, respectively.

  That is, the cellulase may be, for example, a protein having the amino acid sequence shown in SEQ ID NOs: 9 to 15 or the amino acid sequence of cellulase disclosed in the above-mentioned database or literature. Similarly, the cellulase-encoding gene (also referred to as “cellulase gene”) is, for example, the base sequence shown in SEQ ID NO: 25 to 31, or the base sequence of the cellulase gene disclosed in the above database or literature, or the base sequence of its cDNA It may be a gene having In addition, the expression “having an (amino acid or base) sequence” includes the case of “including the (amino acid or base) sequence” and the case of “consisting of the (amino acid or base) sequence”.

As long as the original function is maintained, the cellulase is a variant of the cellulase exemplified above (for example, a protein having the amino acid sequence shown in SEQ ID NOs: 9 to 15 or other cellulase amino acid sequences disclosed in the above-mentioned database or literature). There may be. Similarly, as long as the original function is maintained, the cellulase gene may be the above-exemplified cellulase gene (for example, the nucleotide sequence shown in SEQ ID NOs: 25 to 31 or the nucleotide sequence of the cellulase gene disclosed in the above database or literature) It may be a variant of a gene having the base sequence of the cDNA. Such a variant in which the original function is maintained may be referred to as a “conservative variant”. In the present invention, the protein specified by the above protein name and the gene encoding the same include the conservative variant in addition to the above exemplified protein and the gene encoding the same, respectively. That is, for example, the term “Bgl3A” is intended to encompass a conservative variant thereof in addition to the above-exemplified Bgl3A (ie, Bgl3A from Talaromyces cellulolyticus). Examples of conservative variants include cellulases exemplified above, homologues of genes encoding them, and artificially modified variants.

“The original function is maintained” means that the variant of the gene or protein has a function (activity or property) corresponding to the function (activity or property) of the original gene or protein. That is, “the original function is maintained” means that in the case of cellulase, the variant of the protein has cellulase activity (endoglucanase activity, cellobiohydrolase activity, β-glucosidase activity, etc.). . In addition, "the original function is maintained"
The term “cellulase gene” means that a variant of the gene encodes a protein in which the original function is maintained (that is, a protein having cellulase activity).

  Examples of cellulase homologs include proteins obtained from public databases by BLAST search or FASTA search using the amino acid sequence of the cellulase as a query sequence. The homologue of the cellulase gene can be obtained, for example, by PCR using a chromosome of various organisms as a template and an oligonucleotide prepared based on the base sequence of the cellulase gene as a primer.

  Hereinafter, cellulase and conservative variants of cellulase gene will be exemplified.

  As long as the original function is maintained, the cellulase has an amino acid sequence in which one or several amino acids at one or several positions are substituted, deleted, inserted or added in the amino acid sequence of the cellulase. It may be a protein. The above “one or several” varies depending on the position and type of the amino acid residue in the three-dimensional structure of the protein. Preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 5, particularly preferably 1 to 3.

One or several amino acid substitutions, deletions, insertions or additions described above are conservative mutations in which the function of the protein is maintained normally. A typical conservative mutation is a conservative substitution. Conservative substitution means that when the substitution site is an aromatic amino acid, Phe, Trp, Tyr,
When the substitution site is a hydrophobic amino acid, it is between Leu, Ile, and Val, when it is a polar amino acid, it is between Gln and Asn, and when it is a basic amino acid, it is between Lys, Arg, and His. In the case of an acidic amino acid, it is a mutation that substitutes between Asp and Glu. In the case of an amino acid having a hydroxyl group, it is a mutation that substitutes between Ser and Thr. Specifically, substitutions considered as conservative substitutions include substitution from Ala to Ser or Thr, substitution from Arg to Gln, His or Lys, substitution from Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Gly, Asn, Gln, Lys or Asp Substitution, Gly to Pro substitution, His to Asn, Lys, Gln, Arg or Tyr substitution, Ile to Leu, Met, Val or Phe substitution, Leu to Ile, Met, Val or Phe substitution, Substitution from Lys to Asn, Glu, Gln, His or Arg, substitution from Met to Ile, Leu, Val or Phe, substitution from Phe to Trp, Tyr, Met, Ile or Leu, Ser to Thr or Ala Substitution, substitution from Thr to Ser or Ala, substitution from Trp to Phe or Tyr, substitution from Tyr to His, Phe or Trp, and substitution from Val to Met, Ile or Leu. In addition, amino acid substitutions, deletions, insertions, additions, or inversions as described above include naturally occurring mutations (mutants or variants) such as those based on individual differences or species differences in the organism from which the protein is derived. Also included by

  In addition, as long as the original function is maintained, the cellulase is, for example, 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% with respect to the whole amino acid sequence of the cellulase. More preferably, it may be a protein having an amino acid sequence having a homology of 97% or more, particularly preferably 99% or more. In the present specification, “homology” refers to “identity”.

Cellulase hybridizes under stringent conditions with a probe that can be prepared from the base sequence of the cellulase gene, for example, a complementary sequence to the whole or a part of the base sequence, as long as the original function is maintained. It may be a protein encoded by DNA. Such a probe can be prepared, for example, by PCR using an oligonucleotide prepared based on the base sequence as a primer and a DNA fragment containing the base sequence as a template. “Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, DNA with high homology
DNAs having homology of 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, further preferably 97% or more, and particularly preferably 99% or more. 60 ° C, 1 x SSC, 0.1% SDS, which is a condition that hybridizes and DNAs with lower homology do not hybridize with each other, or washing conditions for normal Southern hybridization
, Preferably 60 ° C., 0.1 × SSC, 0.1% SDS, more preferably 68 ° C., 0.1 × SSC, 0.1% SDS. be able to. For example, when using a DNA fragment having a length of about 300 bp as a probe,
Hybridization washing conditions include 50 ° C., 2 × SSC, and 0.1% SDS.

  In addition, since the degeneracy of codons varies depending on the host, the cellulase gene may be obtained by replacing any codon with an equivalent codon. That is, the cellulase gene may be a variant of the cellulase gene exemplified above due to codon degeneracy. For example, the cellulase gene may be modified to have an optimal codon depending on the codon usage frequency of the host to be used.

  The percentage sequence identity between two sequences can be determined, for example, using a mathematical algorithm. Non-limiting examples of such mathematical algorithms include Myers and Miller (1988) CABIOS 4:11 17 algorithm, Smith et al (1981) Adv. Appl. Math. 2: 482 local homology algorithm, Needleman and Wunsch. (1970) J. Mol. Biol. 48: 443 453 homology alignment algorithm, Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85: 2444 A method to search for similarities of 2448, Karlin and Altschul (1993) A modified algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 872264, as described in Proc. Natl. Acad. Sci. USA 90: 5873 5877.

  A program based on these mathematical algorithms can be used to perform sequence comparison (alignment) to determine sequence identity. The program can be appropriately executed by a computer. Such programs include, but are not limited to, the PC / Gene program CLUSTAL (available from Intelligents, Mountain View, Calif.), The ALIGN program (Version 2.0), and the Wisconsin Genetics Software Package, Version 8 (Genetics Computer Group (GCG), 575 Science Drive, available from Madison, Wis., USA) GAP, BESTFIT, BLAST, FASTA, and TFASTA. Alignment using these programs can be performed using initial parameters, for example. For the CLUSTAL program, HigGlns et al. (1988) Gene 73: 237 244 (1988), HigGlns et al. (1989) CABIOS 5: 151 153, Corpet et al. (1988) Nucleic Acids Res. 16: 10881 90, Huang et al. (1992) CABIOS 8: 155 65 and Pearson et al. (1994) Meth. Mol. Biol. 24: 307 331.

In order to obtain a nucleotide sequence having homology with the nucleotide sequence encoding the protein of interest, specifically, for example, a BLAST nucleotide search can be performed with the BLASTN program, score = 100, word length = 12. In order to obtain an amino acid sequence having homology with the protein of interest, specifically, for example, BLAST protein search, BLASTX program,
This can be done with score = 50 and word length = 3. Please refer to http://www.ncbi.nlm.nih.gov for BLAST nucleotide search and BLAST protein search. In addition, Gapped BLAST (BLAST 2.0) can be used to obtain an alignment with a gap added for the purpose of comparison. PSI-BLAST (BLAST 2.0) can also be used to perform iterative searches that detect distant relationships between sequences. For Gapped BLAST and PSI-BLAST, see Altschul et al. (1997) Nucleic Acids Res. 25: 3389. When utilizing BLAST, Gapped BLAST, or PSI-BLAST, for example, the initial parameters of each program (eg, BLASTN for nucleotide sequences, BLASTX for amino acid sequences) can be used. The alignment may be performed manually.

  Sequence identity between two sequences is calculated as the percentage of residues that match between the two sequences when the two sequences are aligned for maximum matching.

<1-2> Xylanase “Xylanase” is a general term for enzymes involved in the degradation of xylan. Specifically, “xylanase” refers to a protein having an activity of catalyzing a reaction of hydrolyzing β-1,4 bonds in xylan (EC 3.2.1.8). This activity is also referred to as “xylanase activity”. Xylanase activity can be measured, for example, by a known method. The xylanase activity can be determined, for example, by performing an enzymatic reaction using xylan as a substrate and using the amount of reducing sugar produced as an index.

  The xylanase that can be referred to for specifying the chimeric enzyme is not particularly limited as long as it has CBM.

  Examples of xylanases include those classified into glycoside hydrolase families 3, 5, 8, 9, 10, 11, 12, 16, 26, 30, 43, 44, 51, 62, or 98 ( http://www.cazy.org/).

Examples of xylanases include fungal and bacterial xylanases. Examples of fungal xylanases include Trichoderma fungi such as Trichoderma reesei, Talaromyces (Acremonium) fungi such as Talaromyces cellulolyticus (former name: Acremonium cellulolyticus), and Phanerochaete fungi xylanases such as Phanerochaete chrysosporium. Examples of bacterial xylanases include xylanases of bacteria belonging to the genus Ruminiclostridium (Clostridium) such as Ruminiclostridium thermocellum (former name: Clostridium thermocellum). Specific examples of the xylanase include Xyl10A (also referred to as Xyn10A). Xyl10A can be found in fungi such as Talaromyces cellulolyticus. The amino acid sequences of various xylanases and the base sequences of genes encoding them can be obtained from public databases such as NCBI. The amino acid sequence of Xal10A of Talaromyces cellulolyticus is shown in SEQ ID NO: 16. In the amino acid sequence shown in SEQ ID NO: 16, the amino acid sequence at positions 1 to 19 is the signal peptide, the amino acid sequence at positions 20 to 335 is the catalytic domain, the amino acid sequence at positions 336 to 372 is the linker region, and positions 375 to 403. The amino acid sequence corresponds to CBM. In addition, the nucleotide sequence of cDNA of the gene encoding Xal10A of Talaromyces cellulolyticus is shown in SEQ ID NO: 32.

That is, the xylanase may be, for example, a protein having the amino acid sequence shown in SEQ ID NO: 16 or the amino acid sequence of xylanase disclosed in the above database or literature. Similarly, a gene encoding xylanase (also referred to as “xylanase gene”) has, for example, the base sequence shown in SEQ ID NO: 32, the base sequence of the xylanase gene disclosed in the above database or literature, or the base sequence of its cDNA. It can be a gene. The xylanase may be a conservative variant of the xylanase exemplified above (for example, a protein having the amino acid sequence shown in SEQ ID NO: 16 or other amino acid sequence of xylanase disclosed in the above database or literature). Similarly, the xylanase gene is a conservative of the above-exemplified xylanase gene (for example, the gene having the nucleotide sequence shown in SEQ ID NO: 32 or the nucleotide sequence of the xylanase gene disclosed in the above database or literature or the nucleotide sequence of its cDNA). It may be a variant. Regarding the xylanase and the conservative variant of the xylanase gene, the description of the cellulase and the conservative variant of the cellulase gene can be applied mutatis mutandis. “The original function is maintained” means that, in the case of xylanase, a protein variant has xylanase activity.

<1-3> Chimeric Enzyme The chimeric enzyme has a xylanase CBM and is configured to have cellulase activity. The chimeric enzyme may be, for example, a protein having the same amino acid sequence as the cellulase exemplified above or a conservative variant thereof, or a part thereof, except that it has a xylanase CBM. The “part of the amino acid sequence” is not particularly limited as long as the chimeric enzyme has a desired cellulase activity. “Part of the amino acid sequence” includes a catalytic domain of cellulase. That is, the chimeric enzyme may have a cellulase catalytic domain. That is, the chimeric enzyme may specifically be a protein having a xylanase CBM and a cellulase catalytic domain. A chimeric enzyme may have cellulase activity by having a catalytic domain of cellulase.

  Examples of the cellulase catalytic domain include the cellulase catalytic domain exemplified above. Specific examples of the cellulase catalytic domain include the amino acid sequence at positions 25 to 601 in SEQ ID NO: 11. That is, the catalytic domain of cellulase may have, for example, the amino acid sequence from position 25 to position 601 of SEQ ID NO: 11. Examples of the cellulase catalytic domain also include conservative variants of the cellulase catalytic domain exemplified above (for example, the amino acid sequence at positions 25 to 601 of SEQ ID NO: 11). That is, the term “cellulase catalytic domain” is not limited to the catalytic domain of natural cellulases, but also includes conservative variants thereof. Regarding the conservative variant of the catalytic domain of cellulase, the description of the conservative variant of cellulase can be applied mutatis mutandis. The cellulase catalytic domain has, for example, an amino acid sequence in which one or several amino acids at one or several positions are substituted, deleted, inserted, or added in the amino acid sequence of the cellulase catalytic domain exemplified above. It may be a thing. The cellulase catalytic domain is, for example, 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, based on the entire amino acid sequence of the cellulase catalytic domain exemplified above. More preferably, it may have an amino acid sequence having an identity of 97% or more, particularly preferably 99% or more. “The original function is maintained” means that, in the cellulase catalytic domain, a variant of the domain may have cellulase activity, specifically, a chimeric enzyme having the domain. May have cellulase activity.

Examples of xylanase CBMs include CBM family 1, 2, 3, 4, 5, 6, 9, 10, 11, 13, 15, 22, 35, 36, 37, 49, 59, 60, 64, or Those classified into 72 are listed (http://www.cazy.org/). Examples of the xylanase CBM include those classified into CBM family 1 (CBM1). Many of the fungal xylanase CBMs are CBM1. For example, the Cyl of Xyl10A can be CBM1. Examples of the xylanase CBM include the xylanase CBMs exemplified above. Specific examples of the xylanase CBM include the amino acid sequence at positions 375 to 403 of SEQ ID NO: 16. That is, the xylanase CBM may have, for example, the amino acid sequence of positions 375 to 403 of SEQ ID NO: 16. Examples of the xylanase CBM include conservative variants of the above-exemplified xylanase CBM (for example, the amino acid sequence at positions 375 to 403 of SEQ ID NO: 16). That is, the term “xylanase CBM” is not limited to the CBM of natural xylanase, but also includes conservative variants thereof. Regarding the conservative variant of CBM of xylanase, the description of the conservative variant of cellulase can be applied mutatis mutandis. The xylanase CBM has, for example, an amino acid sequence in which one or several amino acids at one or several positions are substituted, deleted, inserted or added in the amino acid sequence of the xylanase CBM exemplified above. There may be. The xylanase CBM is, for example, 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, and more preferably, with respect to the entire amino acid sequence of the xylanase CBM exemplified above. May have an amino acid sequence having an identity of 97% or more, particularly preferably 99% or more. In addition, in the xylanase CBM, “the original function is maintained” may mean that the variant of the module has a binding ability to a cellulosic substrate. It may be that the chimeric enzyme has a binding ability to a cellulosic substrate. Further, the xylanase CBM may have a higher binding ability to the cellulosic substrate as compared to the native CBM of the original cellulase. “Original cellulase” refers to a cellulase from which a chimeric enzyme is derived, and more specifically, may be a cellulase from which the catalytic domain of cellulase contained in the chimeric enzyme is derived. The presence and extent of the binding ability to the cellulosic substrate is determined by reacting the protein having CBM with the cellulosic substrate and using the presence or absence and the extent of the change in the activity of the protein in the reaction supernatant before and after the reaction as an index. Can do. Specifically, the presence or absence and the degree of binding ability to the cellulosic substrate can be determined by the technique described in the Examples. Examples of the cellulose substrate that can be used for the evaluation of the binding ability to the cellulose substrate include water-insoluble cellulose substrates. Specifically, for example, microcrystalline cellulose (Avicel), filter paper, rice straw, sugar cane. Bagasse is mentioned.

  The chimeric enzyme may have only one xylanase CBM, or may have two or more. The number of CBM of xylanase contained in the chimeric enzyme may be, for example, 1 or more, 2 or more, or 3 or more, 4 or less, 3 or less, or 2 or less, They may be a combination that does not contradict them. The number of CBMs of the xylanase contained in the chimeric enzyme may be preferably 1 to 2, more preferably 1. When the chimeric enzyme has two or more xylanase CBMs, the structures of these CBMs can be set independently.

  The position of the xylanase CBM in the chimeric enzyme is not particularly limited as long as the function of the chimeric enzyme is not impaired. The position of the CBM of the xylanase in the chimeric enzyme may be outside the cellulase catalytic domain or inside the cellulase catalytic domain. That is, in the chimeric enzyme, the xylanase CBM may be present on the N-terminal side of the cellulase catalytic domain, and specifically, may be present on the N-terminus. When the chimeric enzyme has a signal peptide, the xylanase CBM may exist, for example, between the signal peptide and the catalytic domain of cellulase in the chimeric enzyme. In the chimeric enzyme, the CBM of xylanase may be present on the C-terminal side of the cellulase catalytic domain, and specifically may be present on the C-terminus. In the chimeric enzyme, the xylanase CBM may be present, for example, inside the catalytic domain of cellulase. In other words, the cellulase catalytic domain of the protein of the present invention may be a cellulase catalytic domain into which a xylanase CBM is inserted. When the chimeric enzyme has two or more xylanase CBMs, the positions of the CBMs in the chimeric enzyme can be set independently. For example, the CBMs may or may not be in tandem in the chimeric enzyme.

  The chimeric enzyme may or may not have a CBM other than the xylanase CBM. Examples of CBMs other than xylanase CBMs include CBMs of carbohydrate hydrolases other than xylanase, such as CBM inherent to the original cellulase, and variants thereof. Regarding variants, the description of conservative variants of cellulase can be applied mutatis mutandis. That is, if the original cellulase has a unique CBM, the chimeric enzyme may have, for example, a xylanase CBM in addition to or in place of the original cellulase's unique CBM.

  By having the xylanase CBM, the chimeric enzyme improves the binding ability to the cellulosic substrate as compared to the case where the xylanase CBM is not present. Examples of “without xylanase CBM” include a case without CBM at all or a case with only CBM unique to the original cellulase. That is, the chimeric enzyme may have improved binding ability to a cellulosic substrate, for example, by having a xylanase CBM instead of the original CBM of the cellulase.

In the chimeric enzyme, the CBM of xylanase and the other region (for example, the catalytic domain of cellulase) may or may not be directly linked. In the chimeric enzyme, the CBM of xylanase and the other region may be linked via, for example, a linker region (also simply referred to as “linker”). That is, the chimeric enzyme may have a linker region. Specifically, the chimeric enzyme may have a linker region between the xylanase CBM and other regions. More specifically, the chimeric enzyme may have a linker region between the xylanase CBM and the cellulase catalytic domain. The linker region is not particularly limited as long as the function of the chimeric enzyme is not impaired. Examples of the linker region include amino acid sequences that do not correspond to the catalytic domain of cellulase or CBM. In addition, examples of the linker region include amino acid sequences that do not correspond to the signal peptide, the catalytic domain of carbohydrate hydrolase, the fibronectin type III-like domain, or CBM. The linker region includes an amino acid sequence between a catalytic domain and a CBM in a carbohydrate hydrolase such as cellulase or xylanase, and an amino acid between a fibronectin type III-like domain and a CBM in a carbohydrate hydrolase such as cellulase or xylanase. Array,
Their partial sequences and their variants are mentioned. Regarding variants, the description of conservative variants of cellulase can be applied mutatis mutandis. For example, when the original cellulase has a unique linker region, the unique linker region may be used as it is or after being appropriately modified. For example, a part of the intrinsic linker region of the original cellulase may be deleted, and the remainder may be used as the linker region. Further, for example, a part or all of the intrinsic linker region of the original cellulase may be substituted with part or all of the linker region of another carbohydrate hydrolase such as xylanase. Specific examples of the linker region of the carbohydrate hydrolase include the amino acid sequence at positions 732 to 776 in SEQ ID NO: 11 and the amino acid sequence at positions 336 to 372 in SEQ ID NO: 16. The length of the linker region is not particularly limited, and may be, for example, 10 residues or more, 20 residues or more, 30 residues or more, 40 residues or more, 50 residues or more, 200 residues or less, It may be 150 residues or less, 100 residues or less, 80 residues or less, 60 residues or less, or 40 residues or less, or a non-conflicting combination thereof. The length of the linker region may be, for example, 20 to 60 residues.

The chimeric enzyme may have any other region as long as the function of the chimeric enzyme is not impaired. The other region may or may not be a region found in a carbohydrate hydrolase such as cellulase or xylanase. Examples of other regions include a signal peptide (also referred to as a signal sequence), a fibronectin type III-like domain, a peptide tag, and a protease recognition sequence.

The signal peptide is not particularly limited as long as it functions in a host that expresses the chimeric enzyme. Examples of the signal peptide include signal peptides of carbohydrate hydrolases such as cellulase and xylanase, and variants thereof. Regarding variants, the description of conservative variants of cellulase can be applied mutatis mutandis. For example, when the original cellulase has a specific signal peptide, the specific signal peptide may be used as it is or after being appropriately modified. Specific examples of the carbohydrate hydrolase signal peptide include the amino acid sequence at positions 1 to 18 in SEQ ID NO: 11 and the amino acid sequence at positions 1 to 19 in SEQ ID NO: 16. Examples of signal peptides that function in bacteria such as coryneform bacteria include signal peptides recognized by the Sec-type secretory pathway and signal peptides recognized by the Tat-type secretory pathway. Specific examples of signal peptides recognized by the Tat secretion pathway include E. coli TorA signal sequence, E. coli SufI signal sequence, Bacillus subtilis PhoD signal sequence, Bacillus subtilis LipA signal sequence, Arthrobacter globiformis An IMD signal sequence is mentioned (WO2013 / 118544). In the chimeric enzyme, the signal peptide may be present at the N-terminus. The signal peptide can be used for secretory production of a chimeric enzyme, for example. When a chimeric enzyme is secreted and produced using a signal peptide, the signal peptide is usually cleaved during secretion, and a chimeric enzyme that does not have a signal peptide can be secreted outside the cell. That is, “a chimeric enzyme has a signal peptide” means that the chimeric enzyme is expressed in a form having a signal peptide, and it does not matter whether the mature protein of the chimeric enzyme has a signal peptide.

Examples of fibronectin type III-like domains include fibronectin type III-like domains of carbohydrate hydrolases such as cellulase and xylanase, and variants thereof. Regarding variants, the description of conservative variants of cellulase can be applied mutatis mutandis. Specific examples of the fibronectin type III-like domain of saccharide hydrolase include, for example, the amino acid sequence at positions 651 to 721 of SEQ ID NO: 11. The position of the fibronectin type III-like domain in the chimeric enzyme is not particularly limited. In the chimeric enzyme, the fibronectin type III-like domain may be present, for example, on the N-terminal side or the C-terminal side of the catalytic domain of cellulase. In the chimeric enzyme, the fibronectin type III-like domain may be present, for example, inside the catalytic domain of cellulase.
For example, the amino acid sequence including the catalytic domain of cellulase and the fibronectin type III-like domain on the C-terminal side thereof includes the amino acid sequence at positions 25 to 721 of SEQ ID NO: 11. That is, the chimeric enzyme may include, for example, the amino acid sequence at positions 25 to 721 of SEQ ID NO: 11 or a variant thereof.

Specific peptide tags include His tags, FLAG tags, GST tags, Myc tags, MBP (maltose binding protein), CBP (cellulose binding protein), TRX (Thioredoxin), GFP (green fluorescent protein), HRP (horseradish) peroxidase), ALP (Alkaline Phosphatase), and antibody Fc region. An example of a His tag is a 6xHis tag. The peptide tag can be used, for example, for detection and purification of the expressed chimeric enzyme.

Specific examples of protease recognition sequences include Factor Xa protease recognition sequences and proTEV protease recognition sequences. A protease recognition sequence can be used, for example, to cleave the expressed chimeric enzyme. Specifically, for example, when a chimeric enzyme is expressed as a fusion protein with a peptide tag, a protease recognition sequence is introduced into the junction between the chimeric enzyme and the peptide tag, so that the protease can be used from the expressed chimeric enzyme. By cleaving the peptide tag, a chimeric enzyme having no peptide tag can be obtained.

  The components of the chimeric enzyme (eg, xylanase CBM, cellulase catalytic domain, linker region, and other regions) may or may not be derived from the same organism. For example, the CBM of xylanase and the catalytic domain of cellulase may be derived from the same organism or may be derived from different organisms.

Specific examples of the chimeric enzyme include Bgl3A-10LC and Bgl3A-10C used in the Examples, and conservative variants thereof. The amino acid sequences of Bgl3A-10LC and Bgl3A-10C are shown in SEQ ID NOs: 34 and 36, respectively. In addition, examples of base sequences of genes encoding Bgl3A-10LC and Bgl3A-10C are shown in SEQ ID NOs: 33 and 35, respectively. That is, the chimeric enzyme may have, for example, the amino acid sequence of SEQ ID NO: 34 or 36, or the amino acid sequence of a conservative variant thereof. Regarding the conservative variant of the chimeric enzyme, the description of the conservative variant of cellulase can be applied mutatis mutandis.

  The chimeric enzyme gene is not particularly limited as long as it encodes a chimeric enzyme. In the present invention, the term “gene” is not limited to DNA as long as it encodes a target protein, and may include any polynucleotide. That is, “chimeric enzyme gene” may mean any polynucleotide encoding a chimeric enzyme. The chimeric enzyme gene may be DNA, RNA, or a combination thereof. The chimeric enzyme gene may be single-stranded or double-stranded. The chimeric enzyme gene may be single-stranded DNA or single-stranded RNA. The chimeric enzyme gene may be double-stranded DNA, double-stranded RNA, or a hybrid strand composed of a DNA strand and an RNA strand. A chimeric enzyme gene may contain both DNA and RNA residues in a single polynucleotide chain. When the chimeric enzyme gene contains RNA, the description regarding DNA such as the exemplified base sequence may be appropriately read according to RNA. The mode of the chimeric enzyme gene can be appropriately selected according to various conditions such as the usage mode.

<2> Production of Chimeric Enzyme A chimeric enzyme can be produced by expressing the gene in a host having the chimeric enzyme gene. A host having a chimeric enzyme gene can be obtained by introducing the gene into an appropriate host. Note that “introducing a chimeric enzyme gene into a host” is not limited to the case where the entire gene is introduced into a host, but a gene such as a cellulase gene or a xylanase gene on the host chromosome is encoded as a chimeric enzyme. It is also included to modify. A host having a chimeric enzyme gene is also referred to as a “host having a chimeric enzyme”.

  A chimeric enzyme can also be produced by expressing a chimeric enzyme gene in a cell-free protein synthesis system.

The chimeric enzyme gene can be obtained, for example, by modifying a gene such as a cellulase gene or xylanase gene so as to encode the chimeric enzyme. The content of the modification can be appropriately selected according to various conditions such as the configuration of the gene of the modification source and the configuration of the chimeric enzyme. For example, a chimeric enzyme gene can be obtained by introducing (for example, substituting or inserting) a base sequence encoding a region containing the xylanase CBM into the cellulase gene. Specifically, for example, by substituting the base sequence encoding the region containing CBM in the cellulase gene with the base sequence encoding the region containing CBM of xylanase, or at the desired position in the cellulase gene, A chimeric enzyme gene can be obtained by inserting a base sequence encoding a region containing CBM. The gene of the modification source can be obtained, for example, by cloning from a nucleic acid such as genomic DNA or cDNA of an organism having the same gene, or by chemical synthesis. The gene can be modified by a known method. For example, a target mutation can be introduced into a target site of DNA by site-specific mutagenesis. That is, the coding region of a gene can be modified, for example, by site-directed mutagenesis such that the amino acid residue at a particular site of the encoded protein contains a substitution, deletion, insertion, or addition. As a site-specific mutation method, a method using PCR (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton press (1989); Carter, P., Meth. In Enzymol., 154, 382 (1987)) and methods using phage (Kramer, W. and Frits, HJ, Meth. In Enzymol., 154,
350 (1987); Kunkel, TA et al., Meth. In Enzymol., 154, 367 (1987)). The chimeric enzyme gene can also be obtained, for example, by chemical synthesis (Gene, 60 (1), 115-127 (1987)).

  The host is not particularly limited as long as it can express a functioning chimeric enzyme. Examples of the host include bacteria, fungi, plant cells, insect cells, and animal cells. Preferred hosts include microorganisms such as bacteria and fungi.

Examples of bacteria include gram negative bacteria and gram positive bacteria. Examples of gram-negative bacteria include bacteria belonging to the genus Escherichia and Enterobacter.
Examples include bacteria belonging to the family Enterobacteriaceae such as genus bacteria and Pantoea bacteria. Examples of Gram-positive bacteria include coryneform bacteria such as Bacillus genus bacteria, Corynebacterium genus bacteria, and actinomycetes. Examples of bacteria belonging to the genus Escherichia include Escherichia coli. Examples of coryneform bacteria include Corynebacterium glutamicum and Corynebacterium ammoniagenes (Corynebacterium stationis). Specific examples of Escherichia coli include, for example, Escherichia coli K-12 strains such as W3110 strain (ATCC 27325) and MG1655 strain (ATCC 47076); Escherichia coli K5 strain (ATCC 23506); BL21 (DE3) strain And Escherichia coli B strains such as the BLR (DE3) strain, which is a recA-strain thereof, and derivatives thereof.

Examples of fungi include yeast and filamentous fungi. Specific examples of the fungi include Talaromyces (Acremonium) fungi such as Talaromyces cellulolyticus (former name: Acremonium cellulolyticus). Specific examples of Talaromyces cellulolyticus include Talaromyces cellulolyticus S6-25 strain (NITE BP-01685; WO2015 / 093467), Talaromyces cellulolyticus Y-94 strain (FERM BP-5826), and derivatives thereof. On August 8, 2013, the S6-25 strain was transferred to the Patent Microorganism Depositary Center (Postal Code 292-0818, Kisarazu Kazusa Kamashi 2-2-8 122, Chiba, Japan) on August 8, 2013 The original deposit was made, and on November 15, 2013, the deposit was transferred to an international deposit under the Budapest Treaty, and the deposit number NITE BP-01685 was assigned. Y-94 was established on January 12, 1983 at the Institute of Biotechnology, National Institute of Technology, Ministry of International Trade and Industry (currently the National Institute for Product Evaluation Technology Patent Biological Depositary, ZIP Code: 292-0818, Address : Deposited in Kazusa Kamashitsu 2-5-8 Room 120, Kisarazu City, Chiba, Japan), transferred to an international deposit based on the Budapest Treaty on February 19, 1997, and was given the accession number FERM BP-5826 ing.

These strains can be sold, for example, from the American Type Culture Collection (address 12301 Parklawn Drive, Rockville, Maryland 20852 PO Box 1549, Manassas, VA 20108, United States of America). That is, a registration number corresponding to each strain is given, and it is possible to receive a sale using this registration number (see http://www.atcc.org/). The registration number corresponding to each strain is described in the catalog of American Type Culture Collection. Moreover, these strains can be obtained from, for example, a depository institution where each strain is deposited. The BL21 (DE3) strain is available, for example, from Life Technologies (product number C6000-03). The BLR (DE3) strain is available, for example, from Merck Millipore (product number 69053).

  The technique for introducing the chimeric enzyme gene into the host is not particularly limited. In the host, the chimeric enzyme gene only needs to be retained so that it can be expressed under the control of a promoter that functions in the host. In the host, the chimeric enzyme gene may be present on a vector that autonomously replicates outside the chromosome, such as a plasmid, or may be introduced on the chromosome. The host may have only one copy of the chimeric enzyme gene or may have two or more copies. The host may have only one type of chimeric enzyme gene or may have two or more types of chimeric enzyme genes.

The promoter for expressing the chimeric enzyme gene is not particularly limited as long as it functions in the host. The “promoter that functions in the host” refers to a promoter having promoter activity in the host. The promoter may be a host-derived promoter or a heterologous promoter. The promoter may be a promoter specific to a gene to be modified such as a cellulase gene or a xylanase gene, or may be a promoter of another gene. The promoter may be a stronger promoter than the native promoter of the gene to be modified. Examples of strong promoters that function in Enterobacteriaceae bacteria such as Escherichia coli include, for example, T7 promoter, trp promoter, trc promoter, lac promoter, tac promoter, tet promoter, araBAD promoter, rpoH promoter, PR promoter, and PL promoter is mentioned. In addition, as a powerful promoter that functions in coryneform bacteria, an artificially modified P54-6 promoter (Appl. Microbiol. Biotechnolo., 53, 674-679 (2000))
Pta, aceA, aceB, adh, amyE promoter that can be induced with acetic acid, ethanol, pyruvate, etc. in coryneform bacteria, cspB, SOD, tuf (EF-Tu), which are powerful promoters with high expression in coryneform bacteria ) Promoter (Journal of Biotechnology 104 (2003) 311-323, Appl Environ Microbiol. 2005 Dec; 71 (12): 8587-96.), Lac promoter, tac promoter, trc promoter. Examples of promoters that function in fungi such as Talaromyces cellulolyticus include glaA promoters and promoters of genes encoding carbohydrate hydrolases such as cellulase genes and xylanase genes. Moreover, as a promoter, you may acquire and utilize the highly active type of a conventional promoter by using various reporter genes. For example, the activity of the promoter can be increased by bringing the −35 and −10 regions in the promoter region closer to the consensus sequence (International Publication No. 00/18935). Highly active promoters include various tac
Like promoter (Katashkina JI et al. Russian Federation Patent application 2006134574) and pnlp8 promoter (WO2010 / 027045). Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)).

Further, a terminator for terminating transcription can be arranged downstream of the chimeric enzyme gene. The terminator is not particularly limited as long as it functions in the host. The terminator may be a host-derived terminator or a heterologous terminator. The terminator may be a specific terminator of a gene that is a modification source such as a cellulase gene or a xylanase gene, or may be a terminator of another gene. Specific examples of the terminator include T7 terminator, T4 terminator, fd phage terminator, tet terminator, and trpA terminator.

A chimeric enzyme gene can be introduced into a host using a vector containing the gene, for example. A vector containing a chimeric enzyme gene is also referred to as an expression vector or a recombinant vector of the chimeric enzyme gene. An expression vector for a chimeric enzyme gene can be constructed, for example, by linking a DNA fragment containing the chimeric enzyme gene with a vector that functions in the host. By transforming the host with the expression vector of the chimeric enzyme gene, a transformant introduced with the vector can be obtained, that is, the gene can be introduced into the host. As the vector, a vector capable of autonomous replication in a host cell can be used. The vector is preferably a multicopy vector. Further, the vector preferably has a marker such as an antibiotic resistance gene in order to select a transformant. Moreover, the vector may be equipped with a promoter or terminator for expressing the inserted gene. The vector may be, for example, a vector derived from a bacterial plasmid, a vector derived from a yeast plasmid, a vector derived from a bacteriophage, a cosmid, or a phagemid. As vectors capable of autonomous replication in bacteria of the Enterobacteriaceae family such as Escherichia coli, specifically, for example, pUC19, pUC18, pHSG299, pHSG399, pHSG398, pBR322, pSTV29 (all available from Takara Bio Inc.), pACYC184, pMW219 (Nippon Gene), pTrc99A (Pharmacia), pPROK vector (Clontech), pKK233-2 (Clontech), pET vector (Novagen), pQE vector (Qiagen), pCold TF D
NA (TaKaRa), pACYC vectors, and wide host range vectors RSF1010. As a vector capable of autonomous replication in coryneform bacteria, specifically, for example, pHM1519 (Agric. Biol. Chem.,
48, 2901-2903 (1984)); pAM330 (Agric. Biol. Chem., 48, 2901-2903 (1984)); a plasmid having a drug resistance gene improved from these; described in JP-A-3-210184 Plasmid pCRY30; plasmids pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE and pCRY3KX described in JP-A-2-72876 and US Pat. No. 5,185,262; plasmids pCRY2 and pCRY3 described in JP-A-1-91686; PAJ655, pAJ611 and pAJ1844 described in Kaikai 58-192900; pCG1 described in JP-A-57-134500; pCG2 described in JP-A-58-35197; JP-A-57-183799 PCG4 and pCG11 described in JP-A-10-215883; pVK7 described in JP-A-10-215883;
Examples include pVC7 described in JP-A-9-070291. When constructing an expression vector, for example, a chimeric enzyme gene containing a unique promoter region may be incorporated into the vector as it is, or the chimeric enzyme coding region may be incorporated downstream into the promoter and then incorporated into the vector. Of course, the coding region of the chimeric enzyme may be incorporated downstream of the promoter originally provided on the vector.

  Vectors, promoters, and terminators that can be used in various microorganisms are described in detail in, for example, “Basic Course of Microbiology 8 Genetic Engineering, Kyoritsu Shuppan, 1987”, and these can be used.

The chimeric enzyme gene can be introduced, for example, onto the host chromosome. Introduction of a gene into a chromosome can be performed, for example, using homologous recombination (Miller, JH Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Examples of gene introduction methods using homologous recombination include the Red-driven integration method (Datsenko, KA, and Wanner, BL Proc. Natl. Acad. Sci. US A. 97: 6640-6645). (2000)), etc., a method using a linear DNA, a method using a plasmid containing a temperature-sensitive replication origin, a method using a plasmid capable of conjugation transfer, a method using a suicide vector that does not have a replication origin and functions in a host, A transduction method using a phage can be mentioned. Only one copy of the gene may be introduced, or two copies or more may be introduced. For example, multiple copies of a gene can be introduced into a chromosome by performing homologous recombination with a sequence having multiple copies on the chromosome as a target. Examples of sequences having multiple copies on a chromosome include repetitive DNA sequences and inverted repeats present at both ends of a transposon. Alternatively, homologous recombination may be performed by targeting an appropriate sequence on a chromosome such as a gene unnecessary for production of the target substance. The gene can also be randomly introduced onto the chromosome using transposon or Mini-Mu (Japanese Patent Laid-Open No. 2-109985, US Pat. No. 5,882,888, EP805867B1). When introducing a gene into a chromosome, for example, a chimeric enzyme gene containing a unique promoter region may be incorporated into the chromosome as it is, and the coding region of the chimeric enzyme is bound downstream of the promoter as described above and then inserted into the chromosome. The coding region of the chimeric enzyme may be incorporated downstream of the promoter originally present on the chromosome.

  The introduction of a gene onto a chromosome can be attributed to, for example, Southern hybridization using a probe having a base sequence complementary to all or part of the gene, or a primer prepared based on the base sequence of the gene. Can be confirmed by PCR.

The transformation method is not particularly limited, and a conventionally known method can be used. As a transformation method, for example, a method of increasing the permeability of DNA by treating a recipient cell with calcium chloride as reported for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol.
Biol. 1970, 53, 159-162), as described for Bacillus subtilis, a method of preparing competent cells from proliferating cells and introducing DNA (Duncan, CH, Wilson, GA and Young, FE., 1997. Gene 1: 153-167). In addition, as a transformation method, recombinant DNA is prepared by transforming DNA-receptive cells, such as those known for Bacillus subtilis, actinomycetes, and yeast, into a protoplast or spheroplast state that easily incorporates recombinant DNA. (Shang, S. and Choen, SN, 1979. Mol. Gen. Genet. 168: 111-115; Bibb, MJ, Ward, JM and Hopwood, OA 1978. Nature 274: 398- 400; Hinnen, A., Hicks, JB and Fink, GR 1978. Proc. Natl. Acad. Sci. USA 75: 1929-1933). As a transformation method, an electric pulse method (Japanese Patent Laid-Open No. 2-207791) as reported for coryneform bacteria can also be used.

  The host for expressing the chimeric enzyme gene may or may not have a modification source gene such as a cellulase gene or a xylanase gene.

The host may have any property as long as it can express a functioning chimeric enzyme. The host may be modified, for example, to reduce the activity of the CreA protein (WO2015 / 093467
). More specifically, the host may be modified so that, for example, the expression of the creA gene is decreased, or may be modified so that the creA gene is disrupted. The creA gene is a gene encoding a transcription factor involved in catabolite repression. The creA gene is known to be involved in cellulase expression in filamentous fungi (Mol Gen Genet. 1996 Jun 24; 251 (4): 451-60, Biosci Biotechnol Biochem. 1998 Dec; 62 (12 ): 2364-70). By modifying the host so that the activity of the CreA protein is reduced, the cellulase production ability of the host can be improved.

  The creA gene can be found in fungi such as Talaromyces cellulolyticus. The base sequence of the creA gene of Talaromyces cellulolyticus is shown in SEQ ID NO: 2. The creA gene and the CreA protein encoded thereby may be a conservative variant of the creA gene exemplified above (for example, a gene having the base sequence shown in SEQ ID NO: 2) and the CreA protein encoded thereby. For conservative variants of the creA gene and CreA protein, the description of the conservative variant of cellulase can be applied mutatis mutandis. The phrase “the original function is maintained” may mean that, in the CreA protein, the variant of the protein has a function as a transcription factor involved in catabolite repression.

  Hereinafter, a technique for reducing the activity of a protein such as CreA protein will be described.

  “Protein activity is decreased” means that the activity per cell of the protein is decreased as compared to an unmodified strain, and includes the case where the activity is completely lost. As used herein, “unmodified strain” refers to a control strain that has not been modified so that the activity of the target protein is reduced. Non-modified strains include wild strains and parent strains. Specifically, “the activity of the protein is decreased” means that the number of molecules per cell of the protein is decreased and / or the function per molecule of the protein compared to the unmodified strain. Means that it is decreasing. In other words, “activity” in the case of “decrease in protein activity” means not only the catalytic activity of the protein but also the transcription amount (mRNA amount) or translation amount (protein amount) of the gene encoding the protein. May be. Note that “the number of molecules per cell of the protein is decreased” includes a case where the protein does not exist at all. Moreover, “the function per molecule of the protein is reduced” includes the case where the function per molecule of the protein is completely lost. The degree of the decrease in protein activity is not particularly limited as long as the activity of the protein is decreased as compared with the unmodified strain. The activity of the protein may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of an unmodified strain.

The modification that decreases the activity of the protein can be achieved, for example, by decreasing the expression of a gene encoding the protein. “Gene expression decreases” means that the expression level of the gene per cell decreases as compared to an unmodified strain such as a wild strain or a parent strain. “Gene expression decreases” specifically means that the amount of gene transcription (mRNA amount) decreases and / or the amount of gene translation (protein amount) decreases. Good. “Gene expression decreases” includes the case where the gene is not expressed at all. In addition, “the expression of the gene is reduced” is also referred to as “the expression of the gene is weakened”. Gene expression may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of an unmodified strain.

The decrease in gene expression may be due to, for example, a decrease in transcription efficiency, a decrease in translation efficiency, or a combination thereof. For example, gene expression can be reduced by altering expression regulatory sequences such as the promoter of the gene, Shine-Dalgarno (SD) sequence (also called ribosome binding site (RBS)), spacer region between RBS and start codon. Can be achieved. In the case of modifying the expression control sequence, the expression control sequence is preferably modified by 1 base or more, more preferably 2 bases or more, particularly preferably 3 bases or more. Further, part or all of the expression regulatory sequence may be deleted. In addition, reduction of gene expression can be achieved, for example, by manipulating factors involved in expression control. Factors involved in expression control include small molecules (such as inducers and inhibitors) involved in transcription and translation control, proteins (such as transcription factors), nucleic acids (such as siRNA), and the like. Also,
Reduction of gene expression can also be achieved, for example, by introducing a mutation that reduces gene expression into the coding region of the gene. For example, gene expression can be reduced by replacing codons in the coding region of the gene with synonymous codons that are used less frequently in the host. In addition, for example, gene expression itself may be reduced by gene disruption as described below.

  Moreover, the modification that decreases the activity of the protein can be achieved, for example, by destroying a gene encoding the protein. “Gene is disrupted” means that the gene is modified so that it does not produce a normally functioning protein. “Does not produce a protein that functions normally” includes the case where no protein is produced from the same gene, or the case where a protein whose function (activity or property) per molecule is reduced or lost is produced from the same gene. It is.

  Gene disruption can be achieved, for example, by deleting part or all of the coding region of the gene on the chromosome. Furthermore, the entire gene including the sequences before and after the gene on the chromosome may be deleted. The region to be deleted may be any region such as an N-terminal region, an internal region, or a C-terminal region as long as a decrease in protein activity can be achieved. Usually, the longer region to be deleted can surely inactivate the gene. Moreover, it is preferable that the reading frames of the sequences before and after the region to be deleted do not match.

  In addition, gene disruption is, for example, introducing an amino acid substitution (missense mutation) into a coding region of a gene on a chromosome, introducing a stop codon (nonsense mutation), or adding or deleting 1 to 2 bases. It can also be achieved by introducing a frameshift mutation (Journal of Biological Chemistry 272: 8611-8617 (1997), Proceedings of the National Academy of Sciences, USA 95 5511-5515 (1998), Journal of Biological Chemistry 26 116, 20833-20839 (1991)).

In addition, gene disruption can be achieved, for example, by inserting another sequence into the coding region of the gene on the chromosome. The insertion site may be any region of the gene, but the longer the inserted sequence, the more reliably the gene can be inactivated. Moreover, it is preferable that the reading frames of sequences before and after the insertion site do not match. The other sequence is not particularly limited as long as it reduces or eliminates the activity of the encoded protein, and examples thereof include marker genes such as antibiotic resistance genes and genes useful for the production of target substances.

To modify a gene on a chromosome as described above, for example, a deletion type gene modified so as not to produce a normally functioning protein is prepared, and a host is transformed with a recombinant DNA containing the deletion type gene. This can be accomplished by replacing the wild-type gene on the chromosome with the deletion-type gene by converting and causing homologous recombination between the deletion-type gene and the wild-type gene on the chromosome. At this time, the recombinant DNA can be easily manipulated by including a marker gene in accordance with a trait such as auxotrophy of the host. Deletion-type genes include genes that have lost all or part of the gene, genes that have been introduced with gene nonsense mutations that have introduced missense mutations, genes that have introduced frameshift mutations, insertions of transposon, marker genes, etc. A gene into which a sequence has been introduced may be mentioned. Even if the protein encoded by the deletion-type gene is produced, it has a three-dimensional structure different from that of the wild-type protein, and its function is reduced or lost. Gene disruption by gene replacement using such homologous recombination has already been established, and a method called “Red-driven integration” (Datsenko, KA, and Wanner, BL Proc. Natl. Acad. Sci. US A. 97: 6640-6645 (2000)), Red-driven integration method and excision system derived from λ phage (Cho, EH, Gumport, RI, Gardner, JFJ Bacteriol. 184: 5200-5203 (2002) ) And the like (see WO2005 / 010175), a method using linear DNA, a method using a plasmid containing a temperature-sensitive replication origin, a method using a plasmid capable of conjugation transfer, replication functioning in the host There is a method using a suicidal vector having no origin (US Pat. No. 6,303,383, Japanese Patent Laid-Open No. 05-007491).

Further, the modification that decreases the activity of the protein may be performed by, for example, a mutation treatment. Mutation treatment includes X-ray irradiation, ultraviolet irradiation, and N-methyl-N ′.
-Treatment with mutating agents such as nitro-N-nitrosoguanidine (MNNG), ethyl methane sulfonate (EMS), and methyl methane sulfonate (MMS).

  The decrease in protein activity can be confirmed by measuring the activity of the protein. The activity of CreA protein can be measured, for example, by measuring the degree of catabolite repression. The degree of catabolite repression can be measured, for example, by measuring cellulase production under culture conditions containing glucose as a carbon source. That is, the decrease in the activity of the CreA protein can be confirmed specifically using, for example, an improvement in cellulase production under culture conditions containing glucose as a carbon source as an index.

  The decrease in the activity of the protein can also be confirmed by confirming that the expression of the gene encoding the protein has decreased. The decrease in gene expression can be confirmed by confirming that the transcription amount of the gene has decreased, or confirming that the amount of protein expressed from the gene has decreased.

Confirmation that the amount of gene transcription has been reduced can be confirmed by comparing the amount of mRNA transcribed from the same gene with an unmodified strain. Examples of methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR and the like (Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). The amount of mRNA may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% compared to the unmodified strain.

Confirmation that the amount of protein has decreased can be performed by Western blotting using an antibody (Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). The amount of protein is, for example, 50% compared to the unmodified strain
Hereinafter, it may be reduced to 20% or less, 10% or less, 5% or less, or 0%.

  Whether or not a gene has been destroyed can be confirmed by determining part or all of the base sequence, restriction enzyme map, full length, etc. of the gene according to the means used for the destruction.

  The chimeric enzyme can be expressed by culturing a host having the chimeric enzyme gene in a medium. At that time, expression of the chimeric enzyme gene can be induced as necessary. The conditions for inducing gene expression can be appropriately selected according to various conditions such as the configuration of the gene expression system.

The medium and culture conditions are not particularly limited as long as the host having the chimeric enzyme gene can be grown and the chimeric enzyme is produced. The medium and culture conditions can be appropriately set according to various conditions such as the type of host. The culture can be carried out under normal conditions using, for example, a normal medium in which microorganisms such as bacteria and fungi are used for culture. For specific medium composition and culture conditions for culturing bacteria, for example, medium compositions and culture conditions used for production of various substances using bacteria such as E. coli and coryneform bacteria can be referred to. . In addition, as for the specific medium composition and culture conditions for culturing fungi, for example, the medium composition described in Talaromyces cellulolyticus reports (JP 2003-135052, JP 2008-271826, JP 2008-271927, etc.) And culture conditions, or culture medium compositions and culture conditions used for culturing other cellulase-producing microorganisms such as Trichoderma reesei.

  As the medium, for example, a medium containing a carbon source, a nitrogen source, a phosphate source, a sulfur source, and other components selected from various organic components and inorganic components as necessary can be used. A person skilled in the art can appropriately set the type and concentration of the medium components.

  The carbon source is not particularly limited as long as a host having a chimeric enzyme gene can be used. Specific examples of carbon sources include glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, waste molasses, starch hydrolysate, biomass hydrolyzate, and other sugars, acetic acid, fumaric acid, citric acid, Examples thereof include organic acids such as succinic acid and malic acid, alcohols such as glycerol, crude glycerol and ethanol, and fatty acids. Examples of the carbon source also include cellulosic substrates. Specific examples of the cellulosic substrate include microcrystalline cellulose (Avicel), filter paper, waste paper, pulp, wood, rice straw, straw, rice husk, rice bran, wheat bran, sugar cane bagasse, coffee lees, and tea lees. . Suitable commercially available cellulosic substrates include Solcaflock (International Fiber Corp, North Tonawanda, NY, U.S.A). As the carbon source, one type of carbon source may be used, or two or more types of carbon sources may be used in combination.

  Specific examples of the nitrogen source include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, organic nitrogen sources such as peptone, yeast extract, meat extract, corn steep liquor, and soy protein degradation product, ammonia, and urea. Can be mentioned. As the nitrogen source, one kind of nitrogen source may be used, or two or more kinds of nitrogen sources may be used in combination.

  Specific examples of the phosphoric acid source include phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphoric acid. As the phosphoric acid source, one type of phosphoric acid source may be used, or two or more types of phosphoric acid sources may be used in combination.

Specific examples of the sulfur source include inorganic sulfur compounds such as sulfate, thiosulfate, and sulfite, and sulfur-containing amino acids such as cysteine, cystine, and glutathione. As the sulfur source, one kind of sulfur source may be used, or two or more kinds of sulfur sources may be used in combination.

Other various organic and inorganic components include, for example, inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium and calcium; vitamin B1, vitamin B2, vitamin B6 and nicotine Examples include vitamins such as acid, nicotinamide, and vitamin B12; amino acids; nucleic acids; and organic components such as peptone, casamino acid, yeast extract, and soybean protein degradation products containing these. As other various organic components and inorganic components, one component may be used, or two or more components may be used in combination.

The culture can be performed aerobically, for example, by aeration culture or shaking culture using a liquid medium. The culture temperature may be, for example, 15 to 43 ° C. The pH during the culture may be, for example, 5-9. The culture period may be, for example, 2 hours to 20 days. The culture can be performed by batch culture, fed-batch culture, continuous culture, or a combination thereof. Moreover, you may implement culture | cultivation by dividing into preculture and main culture. For example, pre-culture may be performed on a solid medium such as an agar medium, and main culture may be performed on a liquid medium.

  By culturing a host having the chimeric enzyme gene as described above, a culture containing the chimeric enzyme can be obtained. The chimeric enzyme can accumulate, for example, in the host cell and / or in the medium. “Cells” may be appropriately read as “cells” depending on the type of host.

  The production of the chimeric enzyme can be confirmed, for example, by measuring the cellulase activity of an appropriate fraction such as a culture supernatant.

  The chimeric enzyme may be used as it is contained in a culture or the like, or may be appropriately separated and purified from the culture or the like and used as a crude enzyme fraction or a purified enzyme.

  That is, for example, when the chimeric enzyme accumulates in the host cell, the chimeric enzyme can be recovered by appropriately crushing, dissolving, or extracting the cell. The cells can be recovered from the culture by centrifugation or the like. Cell disruption, lysis, extraction or the like can be performed by a known method. Examples of such a method include ultrasonic crushing method, dynomill method, bead crushing, French press crushing, and lysozyme treatment. One of these methods may be used alone, or two or more thereof may be used in appropriate combination. For example, when the chimeric enzyme accumulates in the medium, the culture supernatant can be obtained by centrifugation or the like, and the chimeric enzyme can be recovered from the culture supernatant.

  Purification of the chimeric enzyme can be performed by a known method used for purification of the enzyme. Examples of such methods include ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration chromatography, and isoelectric point precipitation. One of these methods may be used alone, or two or more thereof may be used in appropriate combination. Purification of the chimeric enzyme can be performed to a desired degree.

  The purified chimeric enzyme can be used as a “chimeric enzyme” for uses such as cellulose degradation. The chimeric enzyme may be used in a free state, or may be used in the state of an immobilized enzyme immobilized on a solid phase such as a resin.

Further, the present invention is not limited to a purified chimeric enzyme, and any fraction containing the chimeric enzyme may be used as a “chimeric enzyme” for applications such as cellulose degradation. The fraction containing the chimeric enzyme is not particularly limited as long as it is contained so that the chimeric enzyme can act on cellulose. Such fractions include, for example, cultures, culture supernatants, treated cells (crushed materials, lysates, extracts (cell-free extracts), etc.), and partially purified products (crude products). And combinations thereof. Any of these fractions may be used alone or together with the purified chimeric enzyme.

  In addition to the chimeric enzyme, other enzymes such as other cellulases and hemicellulases such as xylanase, xylobiase (β-xylosidase), and arabinofuranosidase can also be produced and accumulated in the culture. The chimeric enzyme may be recovered as a mixture with such other enzymes, or may be recovered separately from such other enzymes.

  The collected chimeric enzyme may be appropriately formulated. The dosage form is not particularly limited, and can be appropriately set according to various conditions such as usage of the chimeric enzyme. Examples of the dosage form include solutions, suspensions, powders, tablets, pills, and capsules. In formulation, for example, excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, flavoring agents, fragrances, diluents, surfactants and other pharmacologically acceptable additives. Can be used.

<3> Use of Chimeric Enzyme The chimeric enzyme can be used, for example, for the decomposition of cellulose. Specifically, for example, a saccharified solution containing a cellulose degradation product such as glucose can be obtained by saccharifying a cellulose component contained in a cellulose-based substrate using a chimeric enzyme. That is, this invention provides the saccharification method of a cellulosic substrate including treating a cellulosic substrate with a chimeric enzyme. One aspect of the method is a method for producing a saccharified product, which comprises treating a cellulosic substrate with a chimeric enzyme.

  An example of the cellulosic substrate is plant biomass. Examples of plant biomass include woody biomass and herbaceous biomass. Specific examples of plant biomass include rice straw, straw, rice husk, sugar cane bagasse, oil palm empty fruit bunch, corn stover, corn cob, switchgrass, Eliansus, napiergrass, and waste wood. Examples of plant biomass include waste discharged from a production process of products made from plant biomass such as papermaking process waste, and discarded products made from plant biomass such as waste paper.

  Cellulose-based substrates such as plant biomass may be subjected to saccharification treatment as they are, or after being appropriately subjected to pretreatment. That is, the method for producing the target substance may include subjecting the cellulosic substrate to pretreatment before saccharification treatment. Examples of the pretreatment method include hydrothermal decomposition treatment, acid treatment, alkali treatment, solvent treatment, cavitation treatment, supercritical water treatment, steaming, explosion, and pulverization. Among these, hydrothermal decomposition treatment is preferable. These pretreatments may be used alone or in combination. For example, the cellulosic substrate may be pulverized to 5 mm or less and subjected to hydrothermal decomposition treatment.

The hydrothermal decomposition treatment can be performed using, for example, pressurized hot water of preferably 175 to 240 ° C, more preferably 200 to 230 ° C. Cellulosic substrates such as plant biomass are generally composed of components such as cellulose, hemicellulose, and lignin. The hemicellulose component is about 140 ° C or higher, the cellulose is about 230 ° C or higher, and the lignin component is about 140 ° C. It dissolves as described above. Therefore, in order to sufficiently separate the cellulose component from other components, it is preferable to perform the hydrothermal decomposition treatment at a temperature in the above range. The reaction pressure of the hydrothermal decomposition treatment is preferably set to a pressure that is 0.1 to 0.5 MPa higher than the saturated vapor pressure of water at each temperature so that the reaction system is in a pressurized hot water state. The reaction time of the hydrothermal decomposition treatment is, for example, usually 20 minutes or less, preferably 3 to 15 minutes. The hydrothermal decomposition treatment may be performed once, or may be performed twice or more. When the hydrothermal decomposition treatment is performed twice or more, the implementation conditions for each hydrothermal decomposition treatment may or may not be the same. The hydrothermal decomposition treatment as described above can be performed by bringing a cellulosic substrate into contact with pressurized hot water. Such a process can be performed using, for example, an apparatus described in Japanese Patent No. 4436429, Japanese Patent No. 4524351, or Japanese Patent No. 4427583. By the hydrothermal decomposition treatment of the cellulosic substrate, the lignin component and the hemicellulose component are transferred from the cellulosic substrate to hot water, and the cellulose component remains as a solid content. After the hydrothermal decomposition treatment, hot water and solids may be separated and saccharified if necessary.

  The acid treatment can be performed by bringing a cellulosic substrate into contact with an acid. Examples of the acid used for the acid treatment include inorganic acids and organic acids. Examples of inorganic acids include sulfuric acid, nitric acid, and hydrochloric acid. Of these, sulfuric acid is preferred. The acid concentration in the acid treatment may be, for example, 0.1 to 15% by weight, preferably 0.5 to 5% by weight. The reaction temperature of the acid treatment may be, for example, 100 to 300 ° C, preferably 120 to 250 ° C. The reaction time of the acid treatment may be, for example, 1 second to 60 minutes. The number of acid treatments is not particularly limited, and the acid treatment may be performed once, or may be performed twice or more. When the acid treatment is performed twice or more, the conditions for performing the acid treatment each time may or may not be the same. In acid treatment, the hemicellulose component is generally hydrolyzed first. Therefore, by acid treatment, for example, a liquid fraction containing a large amount of xylose derived from hemicellulose and a solid fraction containing a large amount of cellulose component can be obtained. After the acid treatment, saccharification may be performed by performing a treatment such as neutralization or solid-liquid separation if necessary. Neutralization can be carried out using a suitable alkali. Examples of the alkali used for neutralization include monovalent alkalis such as ammonia, sodium hydroxide, and potassium hydroxide, and divalent or higher alkalis such as calcium hydroxide. Among them, for example, from the viewpoint of preventing salt precipitation, a monovalent alkali may be preferable.

  The alkali treatment can be performed by contacting the cellulosic substrate with an alkali. Examples of the alkali used for the alkali treatment include sodium hydroxide, calcium hydroxide, and ammonia. The alkali concentration in the alkali treatment may be, for example, 0.1 to 60% by weight. The reaction temperature of the alkali treatment may be, for example, 100 to 200 ° C, preferably 110 to 180 ° C. Examples of the treatment conditions when ammonia is used include the conditions described in JP2008-161125A and JP2008-535664A. The number of alkali treatments is not particularly limited, and the alkali treatment may be performed once, or may be performed twice or more. When the alkali treatment is performed twice or more, the execution conditions of the alkali treatment each time may or may not be the same. After the alkali treatment, neutralization or solid-liquid separation may be performed as necessary to carry out saccharification. Neutralization can be carried out using a suitable acid. Examples of the acid used for neutralization include monovalent acids such as nitric acid and hydrochloric acid, and divalent or higher acids such as sulfuric acid and phosphoric acid. Among these, for example, from the viewpoint of preventing salt precipitation, a monovalent acid may be preferable.

The saccharification reaction can be performed in an appropriate aqueous solvent such as water or a buffer solution. The reaction conditions can be appropriately set with reference to, for example, known reaction conditions for saccharifying enzymes such as cellulase or based on preliminary experiments. The reaction temperature may be, for example, usually 5 to 95 ° C. The pH may typically be 1-11, for example. The enzyme amount may be, for example, 0.001 to 10 g per 1 g of the substrate solid amount. The reaction time may usually be 12 to 144 hours, for example. The enzyme reaction may be performed by standing or may be performed with stirring. In the saccharification reaction, the chimeric enzyme may be used alone or in combination with other saccharifying enzymes.

  Chimeric enzymes and other saccharifying enzymes may be recovered and reused. For example, a saccharifying enzyme can be recovered by being bound to a cellulosic substrate such as plant biomass (Japanese Patent Laid-Open No. 2010-098951, WO2016 / 043281).

Specifically, a cellulosic substrate can be saccharified while reusing a chimeric enzyme or other saccharifying enzyme by a method including the following steps (A) to (C) (WO2016 / 043281):
(A) a step of contacting a first saccharified solution containing a free saccharifying enzyme obtained by enzymatic saccharification of the first cellulosic substrate with a second cellulosic substrate;
(B) a step of recovering the second cellulosic substrate after the step (A),
(C) A step of enzymatically saccharifying the second cellulosic substrate after the step (B) to obtain a second saccharified solution.

  That is, by bringing the first saccharified solution into contact with the second cellulosic substrate, free saccharifying enzymes (such as free chimeric enzymes) in the first saccharified solution adhere to the second cellulosic substrate. . Next, the second cellulosic substrate to which the saccharifying enzyme (chimeric enzyme, etc.) is collected is recovered and used as the saccharifying enzyme substrate and saccharifying enzyme source, so that the saccharifying enzyme (chimeric enzyme, etc.) can be efficiently recycled. The cellulosic substrate can be efficiently enzymatically saccharified.

  The saccharified solution thus obtained can be used, for example, as a carbon source for culturing microorganisms. In one aspect, for example, the target substance can be produced by culturing a microorganism having the ability to produce a desired target substance such as an L-amino acid using the saccharified solution thus obtained as a carbon source. . The saccharified solution can be used as it is, or appropriately subjected to a treatment such as concentration, dilution, drying, fractionation, purification, etc., and then used as a carbon source for culturing microorganisms. For example, components such as glucose produced by saccharification may be separated and purified to a desired degree and used as a carbon source for culturing microorganisms.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to the examples.

(1) Preparation of enzyme solution and hydrothermal bagasse (1-1) Construction of Talaromyces cellulolyticus F09ΔcreA strain
Using Talaromyces cellulolyticus S6-25 strain (NITE BP-01685, hereinafter referred to as S6-25 strain) as a parent strain, a parent strain T. cellulolyticus F09 strain for gene recombination was constructed according to the following procedure.

S6-25 strain 5 g / L Solkafloc (International Fiber Corp, North Tonawanda, NY, USA), 24 g / L KH ₂ PO ₄ , 5 g / L (NH ₄ ) ₂ SO ₄ , 2 g / L Urea, 1.2 g / L MgSO ₄・ 7H ₂ O, 0.01 g / L ZnSO ₄・ 7H ₂ O, 0.01 g / L MnSO ₄・ 5H ₂ O, 0.01 g / L CuSO ₄・ 5H ₂ O, 20 g / L The medium containing Bacto agar was inoculated and cultured at 30 ° C. One agar disk punched out with a straw near the end of the colony formed on the agar medium, 40 g / L solka floc, 10 g / L Bacto peptone, 6 g / L KNO ₃ , 2 g / L Urea, 1.6 Inoculate a medium (pH 4.0) containing g / L KCl, 1.2 g / L MgSO ₄ · 7H ₂ O, 12 g / L KH ₂ PO ₄ and swirl culture at 30 ° C and 220 rpm for 10-11 days It was. Next, 5 mL of the culture solution was filtered with a glass filter (pore size: 40 to 100 μm) to remove the remaining solka floc and long mycelia. The resulting filtrate mycelia were collected by centrifugation (3 min at 5000 rpm) and the resulting mycelia _{0.1% Tween80,0.05% MgSO 4 · 7H} 2 O, suspended in a solution containing 0.5% NaCl, suspended The washing operation of centrifuging the suspension (5000 rpm for 3 minutes) was performed twice. Dilute appropriately so that the turbidity (OD 660 nm) of the cell suspension after washing becomes 1.0, dispense 1 mL into a Petri dish (bottom diameter: about 35 mm), and use a 15 W germicidal lamp. And irradiated with ultraviolet rays. The cell suspension after irradiation is diluted as appropriate, and a minimal medium (10 g / L glucose, 10 mM NH ₄ Cl, 10 g / L 5-fluoroorotic acid, 1 g / L Uracil, 1 g / L Uridine, 10 mM KH ₂ PO ₄ , 7 mM KCl, 2 mM MgSO ₄ , 0.06 mg / LH ₃ BO ₃ , 0.26 mg / L (NH ₄ ) ₆ Mo ₇ O ₂₄・ 4H ₂ O, 1 mg / L FeCl ₃・ 6H ₂ O, 0.4 mg / L CuSO ₄ .5H ₂ O, 0.08 mg / L MnCl ₂ , 2 mg / L ZnCl ₂ , 20 g / L Bacto Agar) and cultured at 30 ° C. 5-fluoroorotic acid is an intermediate analog of the Uracil biosynthetic pathway and is toxic to strains in which the Uracil biosynthetic pathway functions normally. Therefore, by selecting with a medium containing 5-fluoroorotic acid, it is possible to obtain a strain that has entered the Uracil biosynthetic pathway and has become nonfunctional. Mutant strains grown in a medium containing 5-fluoroorotic acid are transferred to a minimal medium containing 1 g / L Uracil and 1 g / L Uridine and a minimal medium not containing these, and then 1 g / L Uracil and 1 g / L. Uridine
The strain that grew only on the minimal medium containing was designated F09 strain. In strains exhibiting resistance to 5-fluoroorotic acid, there is a high possibility that the pyrF gene or the pyrG gene is mutated and no longer functions. Therefore, nucleotide sequences of these gene regions on the F09 strain genomic DNA were determined. As a result, in the F09 strain, the 629th base of the pyrF gene (SEQ ID NO: 1) was changed from A to G, and it was speculated that this mutation caused the pyrF gene product to stop functioning.

Next, using the T. cellulolyticus F09 strain as a parent strain, the creA gene (SEQ ID NO: 2) was disrupted by the following procedure to construct a T. cellulolyticus F09ΔcreA strain. The creA gene is a gene encoding a transcription factor involved in catabolite repression. The creA gene is known to be involved in cellulase expression in filamentous fungi (Mol Gen Genet. 1996 Jun 24; 251 (4): 451-60, Biosci Biotechnol Biochem. 1998 Dec; 62 (12 ): 2364-70). Disruption of the creA gene can improve the ability of T. cellulolyticus to produce cellulase (WO2015 / 093467).

First, a creA disrupting DNA fragment having a base sequence linked in the order of a creA gene upstream region, a pyrF gene marker, and a creA gene downstream region of T. cellulolyticus was prepared according to the following procedure. PCR using the genomic DNA of T. cellulolyticus Y-94 strain (FERM BP-5826) as a template and primers (SEQ ID NOs: 3 and 4), using the upstream region of the creA gene and primers (SEQ ID NOs: 5 and 6) The downstream region of the creA gene was amplified by PCR. In addition, the entire region of the pyrF gene (including promoter and terminator) was amplified by PCR using primers (SEQ ID NOs: 7 and 8) using the genomic DNA of T. cellulolyticus strain Y-94 (FERM BP-5826) as a template. . The PCR product was purified using Wizard SV Gel and PCR Clean-Up System (Promega). The purified PCR product was incorporated into the pUC plasmid attached to the kit using In-Fusion HD Cloning Kit (Takara Bio) and ligated. E. coli JM109 was transformed with the reaction product, and colonies were formed by culturing overnight at 37 ° C. on LB agar medium (containing 100 mg / L ampicillin). A pUC-creA :: pyrF plasmid incorporating a DNA fragment for creA disruption was obtained from the resulting transformant using Wizard Plus Miniprep System (Promega). The creA disruption DNA fragment was amplified by PCR using the pUC-creA :: pyrF plasmid as a template and primers (SEQ ID NOs: 3 and 6), and concentrated and purified by ethanol precipitation.

Next, the F09 strain was inoculated into a medium containing 12 g / L Potato Dextrose Broth (Difco) and 20 g / L Bacto Agar (Difco) and cultured at 30 ° C. One agar disk obtained by punching the vicinity of the end of the colony formed on the agar medium with a straw was inoculated into a medium containing 24 g / L Potato Dextrose Broth, and rotated and cultured at 30 ° C. and 220 rpm for 2 days. Collect the cells by centrifugation (5000 rpm, 5 minutes), add 30 mL of an aqueous solution (pH 6.0) containing 10 g / L Yatalase (Takara Bio), 10 mM KH ₂ PO ₄ , and 0.8 M NaCl, and shake. The reaction was carried out at 30 ° C. for 2 hours to digest the cell wall and protoplast it. After removing the residue with a glass filter, the protoplasts are collected by centrifugation (2000 rpm, 10 minutes) and suspended in 1 mL with Tris-HCl buffer (pH 7.5) containing 1.2 M Sorbitol and 10 mM CaCl _2. A cloudy protoplast solution was prepared. Add 10 μg of purified creA DNA fragment and 50 μL of Tris-HCl buffer solution (pH 7.5) containing 400 g / L PEG4000 and 10 mM CaCl ₂ to 200 μL protoplast solution, and let stand on ice for 30 minutes . Thereafter, Tris-HCl buffer (pH 7.5) containing 1 mL of 400 g / L PEG4000 and 10 mM CaCl ₂ was added and mixed, and the mixture was allowed to stand at room temperature for 15 minutes for transformation. Protoplasts recovered by centrifugation (2000 rpm, 10 minutes) were inoculated on a minimal medium containing 1 M Sucrose and cultured at 30 ° C. for 7 days to select a strain with complemented Uracil requirement. The emerged colonies were inoculated into a minimal medium and cultured at 30 ° C. for 4 days. After confirming that the creA gene region was replaced with the pyrF gene, the F09ΔcreA strain was obtained.

(1-2) Preparation of enzyme solution
The F09ΔcreA strain was inoculated into a medium containing 12 g / L Potato Dextrose Broth (Difco) and 20 g / L Bacto Agar (Difco) and cultured at 30 ° C. One agar disk obtained by punching the vicinity of the end of the colony formed on the agar medium with a straw, 20 mL of 20 g / L glucose, 24 g / L KH ₂ PO ₄ , 5 g / L (NH ₄ ) ₂ SO ₄ , 2 g / L Urea, 1.2 g / L MgSO ₄・ 7H ₂ O, 0.01 g / L ZnSO ₄・ 7H ₂ O, 0.01 g / L MnSO ₄・ 5H ₂ O, 0.01 g / L CuSO ₄・A liquid medium containing 5H ₂ O, 1 g / L corn steep liquor (C4648, SIGMA) and 1 g / L Tween 80 was inoculated, and pre-cultured by rotating culture at 30 ° C. and 220 rpm for 5 days. Next, 15 mL of the preculture was added to 300 mL of 15 g / L glucose, 12 g / L KH ₂ PO ₄ , 10 g / L (NH ₄ ) ₂ SO ₄ , 1.2 g / L in a jar fermenter. MgSO ₄・ 7H ₂ O, 0.01 g / L ZnSO ₄・ 7H ₂ O, 0.01 g / L MnSO ₄・ 5H ₂ O, 0.01 g / L CuSO ₄・ 5H ₂ O, 5 g / L Corn steep liquor, 1 g / L Tween 80, 0.5 mL / L Inoculated into liquid medium containing Dis home GD (NOF), culture temperature 30 ° C, aeration volume 1/2 vvm, and dissolved oxygen concentration against saturation concentration by stirring Control over 5%,
While controlling the culture pH to 5 using ammonia gas, fed-batch culture for 78 hours was performed. The composition of the feed solution was 360 g / L Glucose, 60 g / L Cellobiose, and 0.5 mL / L Dis Home GD. The fed solution was continuously fed from 22 hours after the start of the culture. The culture solution was sampled 72 hours after the start of the culture, and centrifuged (15000 rpm for 5 minutes) to obtain a supernatant. The obtained supernatant was used as an enzyme solution.

(1-3) Preparation of Hydrothermal Bagasse Bagasse was pulverized using a cutting mill (SM100, Retsch, screen 0.75 mm). After bagasse was mixed in water at 7% (w / v) and immersed overnight, hydrothermal treatment was performed with a batch-type small hydrothermal treatment device (070-07013, OM Lab Tech). Hydrothermal treatment was performed at 200 ° C. for 15 minutes with a constant stirring speed of 200 rpm. Hydrothermal slurry after hydrothermal treatment is filter paper (5C, Advantec)
Solid and liquid were separated by suction filtration using and washed with water. The obtained solid content was designated as hydrothermal bagasse.

(2) Evaluation of the binding ability of carbohydrate hydrolases in the enzyme solution to hydrothermal bagasse
5% (w / v) equivalent hydrothermal bagasse and 20% (v / v) equivalent enzyme solution (culture supernatant of T. cellulolyticus F09ΔcreA strain in 50 mM sodium citrate buffer (pH 5.1) ) And add 24 at 50 ° C
The reaction was carried out with shaking. Next, 5% (w / v) equivalent hydrothermal bagasse was added and mixed well, then centrifuged (15000 rpm for 5 minutes) to obtain a supernatant, which was used as a sample after enzyme recovery. In addition, 20% (v / v) equivalent enzyme solution (culture supernatant of T. cellulolyticus F09ΔcreA strain) was added to 50 mM sodium citrate buffer (pH 5.1) as a sample before saccharification reaction.

Take 40 μg of these two samples as protein, treat with 200 ng / μL Trypsin at 37 ° C. for 18 hours, dissolve in 0.01% TFA / 2% ACN solution, and add 0.5 μg / μL of analysis sample. Prepared. These analysis samples were subjected to proteome analysis by LC-MS to identify proteins from the detected peptides, and simultaneously evaluate the abundance ratio of each protein from the number of peaks obtained at that time. For each specific protein, the ratio of the abundance of the protein in the sample after enzyme recovery was calculated when the abundance of the protein in the sample before the saccharification reaction was 100%. The lower the ratio, the higher the bonding rate to the hydrothermal bagasse. Cbh1 (SEQ ID NO: 9), Cbh2 (SEQ ID NO: 10), Bgl3A (SEQ ID NO: 11), Eg5A (SEQ ID NO: 12), Eg5X1 (SEQ ID NO: 13), Eg5X2 (SEQ ID NO: 14), Eg7B (SEQ ID NO: 15), Xyl10A The result of (SEQ ID NO: 16) is shown in FIG. From these results, it was shown that Cbh2, Eg5X2, and Xyl10A CBMs have a higher binding capacity to hydrothermally treated bagasse than Cbh1, Bgl3A, Eg5A, Eg5X1, and Eg7B CBMs.

(3) Improvement of binding ability of Bgl3A derived from T. cellulolyticus to cellulosic substrate (3-1) Construction of chimeric enzyme production strain As shown in Fig. 2, a region containing CBM present at the C-terminus of Bgl3A of T. cellulolyticus T. cel
Two types of chimeric enzymes (Bgl3A-10LC and Bgl3A-10C) were designed by substitution in the region containing CBM of Xyl10A of lulolyticus. In order to construct strains that secrete and express these chimeric enzymes, wild-type Bgl3A (Bgl3Awt), and Bgl3A (Bgl3AΔLC) lacking the linker region and CBM, respectively, four expression plasmids pANC239, pANC247 were constructed by the following method. PANC248 and pANC244 were constructed.

The bgl3A gene was amplified by PCR using the genomic DNA of T. cellulolyticus strain Y-94 (FERM BP-5826) as a template and primers (SEQ ID NOs: 17 and 18). The PCR product was purified using Min Elute PCR Purification Kit (QIAGEN). The purified PCR product and the product obtained by cleaving the pANC202 plasmid (J Ind Microbiol Biotechnol. 2013 Aug; 40 (8): 823-30) with restriction enzymes EcoRV and SbfI were ligated using DNA Ligation Kit (Takara). . E. coli DH5α was transformed with the ligation product, and colonies were formed by culturing overnight at 37 ° C. on LB agar medium (containing 100 mg / L ampicillin). The pANC211 plasmid was obtained from the obtained transformant using QIAprep Spin Miniprep kit (QIAGEN). pANC211 was transformed into T. cellulolyticus YP-4 strain (Biosci Biotechnol Biochem. 2012; 76 (2): 245-9). The YP-4 strain is a uracil-requiring strain of the T. cellulolyticus Y-94 strain (FERM BP-5826) (AMB Express. 2013; 3: 73.). RNA was purified from the obtained transformant using FAST RNA Pro Blue Kit (Qbiogene) and RNeasy Mini Kit (QIAGEN). CDNA was prepared from the obtained RNA using M-MLV reverse transcriptase (Takara), and the cDNA of the bgl3A gene was amplified by PCR using the cDNA as a template and primers (SEQ ID NOs: 17 and 18). The PCR product was purified using Min Elute PCR Purification Kit (QIAGEN). The purified PCR product and the product obtained by cleaving the pANC202 plasmid (J Ind Microbiol Biotechnol. 2013 Aug; 40 (8): 823-30) with restriction enzymes EcoRV and SbfI were ligated using DNA Ligation Kit (Takara). . E. coli DH5α was transformed with the ligation product, and colonies were formed by culturing overnight at 37 ° C. on LB agar medium (containing 100 mg / L ampicillin). An expression plasmid pANC239 of wild type Bgl3A (Bgl3Awt) was obtained from the obtained transformant using QIAprep Spin Miniprep kit (QIAGEN). The nucleotide sequence of the bgl3A gene cDNA and the amino acid sequence of wild-type Bgl3A mounted on the plasmid are shown in SEQ ID NOs: 27 and 11, respectively.

Next, the bgl3A gene lacking the linker region and the portion encoding the CBM region was amplified by PCR using pANC239 as a template and primers (SEQ ID NOs: 17 and 20). PCR product is HpaI
After digestion with SbfI, purification was performed using Min Elute PCR Purification Kit (QIAGEN). The purified PCR product and the product obtained by cleaving the pANC202 plasmid (J Ind Microbiol Biotechnol. 2013 Aug; 40 (8): 823-30) with restriction enzymes EcoRV and SbfI were ligated using DNA Ligation Kit (Takara). . E. coli DH5α was transformed with the ligation product, and colonies were formed by culturing overnight at 37 ° C. on LB agar medium (containing 100 mg / L ampicillin). The pANC108 plasmid was obtained from the obtained transformant using QIAprep Spin Miniprep kit (QIAGEN). Separately, pANC208 plasmid (Protein Expr. Purif. 2014; 94: 40-45)
The DNA fragment encoding the linker region and CBM of the Xyl10A gene of T. cellulolyticus was amplified by PCR using as a template and primers (SEQ ID NOs: 21 and 22). The PCR product was cleaved with SbfI and then purified using Min Elute PCR Purification Kit (QIAGEN). The purified PCR product and the product obtained by cleaving the pANC108 plasmid with SfoI and SbfI were ligated using a DNA Ligation Kit (Takara). E. coli DH5α was transformed with the ligation product, and colonies were formed by culturing overnight at 37 ° C. on LB agar medium (containing 100 mg / L ampicillin). An expression plasmid pANC247 of the chimeric enzyme Bgl3A-10LC was obtained from the obtained transformant using QIAprep Spin Miniprep kit (QIAGEN). The nucleotide sequence of the gene encoding Bgl3A-10LC and the amino acid sequence of Bgl3A-10LC mounted on the plasmid are shown in SEQ ID NOs: 33 and 34, respectively.

In addition, the bgl3A gene lacking a part of the linker region and a part encoding the CBM region was amplified by PCR using pANC239 as a template and primers (SEQ ID NOs: 17 and 23). The PCR product was cleaved with HpaI and SbfI and then purified using the Min Elute PCR Purification Kit (QIAGEN). The purified PCR product and the product obtained by cleaving the pANC202 plasmid (J Ind Microbiol Biotechnol. 2013 Aug; 40 (8): 823-30) with restriction enzymes EcoRV and SbfI
Ligation was performed using Kit (Takara). E. coli DH5α was transformed with the ligation product, and colonies were formed by culturing overnight at 37 ° C. on LB agar medium (containing 100 mg / L ampicillin). The pANC110 plasmid was obtained from the obtained transformant using QIAprep Spin Miniprep kit (QIAGEN). Separately, CBM of Xyl10A gene of T. cellulolyticus CF-2612 strain is encoded by PCR using pANC208 plasmid (Protein Expr. Purif. 2014; 94: 40-45) as a template and primers (SEQ ID NOs: 24 and 22). The DNA fragment was amplified. PCR product is SbfI
After cleaving, purification was performed using Min Elute PCR Purification Kit (QIAGEN). The purified PCR product and the product obtained by cleaving the pANC110 plasmid with SfoI and SbfI were ligated using a DNA Ligation Kit (Takara). E. coli DH5α was transformed with the ligation product, and colonies were formed by culturing overnight at 37 ° C. on LB agar medium (containing 100 mg / L ampicillin). Bgl3A-10C expression plasmid pANC248 was obtained from the resulting transformant using QIAprep Spin Miniprep kit (QIAGEN). The nucleotide sequence of the gene encoding Bgl3A-10C and the amino acid sequence of Bgl3A-10C mounted on the plasmid are represented by SEQ ID NOs: 35 and 36, respectively.
Respectively.

Further, the bgl3A gene lacking the linker region and the portion encoding the CBM region was amplified by PCR using pANC239 as a template and primers (SEQ ID NOs: 17 and 19). The PCR product was ligated with the product obtained by cleaving pANC202 with restriction enzymes EcoRV and SbfI. E. coli DH5α was transformed with the ligation product, and colonies were formed by culturing overnight at 37 ° C. on LB agar medium (containing 100 mg / L ampicillin). Bgl3AΔLC expression plasmid pANC244 was obtained from the resulting transformant using QIAprep Spin Miniprep kit (QIAGEN). The nucleotide sequence of the gene encoding Bgl3AΔLC and the amino acid sequence of Bgl3AΔLC mounted on the plasmid are shown in SEQ ID NOs: 37 and 38, respectively.

Each bgl3A gene carried in pANC239, pANC247, pANC248, and pANC244 is expressed under the control of the glaA promoter. Expression of the gene from the glaA promoter can be induced by starch.

  pANC239, pANC247, pANC248 and pANC244 were transformed into T. cellulolyticus YP-4 strain (Biosci Biotechnol Biochem. 2012; 76 (2): 245-9) to obtain Y239 strain, Y247 strain, Y248 strain and Y244 strain did.

(3-2) Expression of chimeric enzyme
Y239 strain, Y247 strain, Y248 strain, and Y244 strain were inoculated in a medium containing 12 g / L Potato Dextrose Broth (Difco) and 20 g / L Bacto Agar (Difco), respectively, and cultured at 30 ° C. One agar disk obtained by punching the vicinity of the end of the colony formed on the agar medium with a straw was added to 10 mL of 20 g / L solubilized starch, 24 g / L KH ₂ PO ₄ , 1 g in a flask. / L Tween 80, 5 g / L (NH ₄ ) ₂ SO ₄ , 1.2 g / L MgSO ₄ · 7H ₂ O, inoculated in medium containing 0.8 g / L urea, 5 at 30 ° C, 220 rpm
Culturing was carried out by swirling culture for days.

(3-3) Purification of chimeric enzyme After the culture, the culture solution was centrifuged to obtain a supernatant. It was confirmed by SDS-PAGE and chromatography that each Bgl3A was contained in the obtained culture supernatant. These culture supernatants were desalted using HiPrep 26/10 desalting chromatography (GE Healthcare) and then subjected to Resource Q anion exchange chromatography (GE Healthcare) and Resource ISO hydrophobic column chromatography (GE Healthcare). Each Bgl3A was purified until it was detected in a single band by SDS-PAGE.

(3-4) Measurement of activity of chimeric enzyme The activity of each purified enzyme (purified Bgl3A) was measured using pNP-β-D-Glucose as a substrate. 1
The enzyme activity of U was defined as the enzyme activity that produces 1 μmol of pNP (p-nitrophenol) per minute. As a result, all of Bgl3A-10LC, Bgl3A-10C, and Bgl3AΔLC showed specific activities substantially equivalent to Bgl3Awt (Table 1). This result shows that substitution or deletion of CBM or linker has little effect on the function of the catalytic domain of Bgl3A.

(3-5) Evaluation of binding ability of chimeric enzyme to cellulosic substrate The evaluation of the binding ability of each purified enzyme (purified Bgl3A) to the cellulosic substrate was performed using 50 mM Na acetate.
The substrate was 2% Avicel suspended in (pH 5.0), 3% disc mill pulverized rice straw, and 2% hydrothermally treated bagasse. 10 μL of 1 mg / mL of each purified enzyme was added to 1 mL of each substrate suspension, incubated at 45 ° C. for 2 hours, and then Bgl3A activity remaining in the supernatant was measured using pNP-β-D-Glucose . As a control, add 10 μL of 1 mg / mL of each purified enzyme to 1 mL of the supernatant of each substrate suspension, incubate at 30 ° C for 2 hours, and then measure the remaining Bgl3A activity in the solution with pNP-β-D-Glucose It measured using. The activity of the supernatant after reaction with the cellulosic substrate was determined as the relative activity when the activity obtained in the control was taken as 100. It shows that the ratio couple | bonded with the cellulosic substrate is so high that the said relative activity is low. The results are shown in Table 2. Bgl3A (Bgl3Awt, Bgl3A-10LC, Bgl3A-10C) holding CBM bound at a high ratio in the order of bagasse>avicell> rice straw. On the other hand, Bgl3AΔLC not retaining CBM hardly bound to any cellulosic substrate. Therefore, it was found that the binding of Bgl3A to the cellulosic substrate depends on CBM. In addition, Bgl3A-10LC and Bgl3A-10C introduced with CBM of xylanase (Xyl10A) are about 2.2 times as much for Avicel, about 2.5 times as much for rice straw, and Bagas as compared to Bgl3Awt, which holds the intrinsic CBM of cellulase The binding ability was 1.6 to 2.8 times higher. These results indicate that Cyl of Xyl10A contributes to high binding ability to cellulosic substrates.

(3-6) Evaluation of saccharification ability of the chimeric enzyme for cellulosic substrates
Add 5% (w / v) equivalent hydrothermal bagasse, culture supernatant of T. cellulolyticus F09ΔcreA strain (Example (2)) and purified Bgl3A to 50 mM sodium citrate buffer (pH 5.1) The reaction was carried out with shaking at 50 ° C. for 24 hours. The amount of T. cellulolyticus culture supernatant added was 5.92 mg-protein / g-bagasse TS, and the amount of each purified Bgl3A added was 0.592 mg-protein / g-bagasse TS. After 24 hours of the reaction, the supernatant was collected, and the glucose concentration was measured with Biotech Analyzer AS-310 (Sakura Seye). The results are shown in Table 3. Compared with the group without addition of Bgl3A (group with only T. cellulolyticus culture supernatant added), the concentration of produced glucose increased in each group with purified Bgl3A (group with T. cellulolyticus culture supernatant and each purified Bgl3A added). However, there was no difference in the concentration of glucose produced in each purified Bgl3A addition interval. This result shows that the saccharification ability for cellulosic substrates is not changed by substitution or deletion of CBM. The results also show that the ability of Bgl3A to bind to a cellulosic substrate has not improved the ability to saccharify the cellulosic substrate. From the above, it was shown that a chimeric enzyme having a high binding ability to a cellulosic substrate advantageous for enzyme recovery can be constructed without reducing the saccharification ability to the cellulosic substrate.

<Explanation of Sequence Listing>
SEQ ID NO: 1: nucleotide sequence of pyrF gene of Talaromyces cellulolyticus SEQ ID NO: 2: nucleotide sequence of creA gene of Talaromyces cellulolyticus SEQ ID NO: 3-8: primer SEQ ID NO: 9: amino acid sequence of Cbh1 protein of Talaromyces cellulolyticus SEQ ID NO: 10: Talaromyces cellulolyticus Cbh2 protein amino acid sequence SEQ ID NO: 11: Talaromyces cellulolyticus Bgl3A protein amino acid sequence SEQ ID NO: 12: Talaromyces cellulolyticus Eg5A protein amino acid sequence SEQ ID NO: 13: Talaromyces cellulolyticus Eg5X1 protein amino acid sequence SEQ ID NO: 14: Talaromyces cellulolyticus Eg5X2 protein Amino acid sequence of SEQ ID NO: 15: amino acid sequence of Eg7B protein of Talaromyces cellulolyticus SEQ ID NO: 16: amino acid sequence of Xyl10A protein of Talaromyces cellulolyticus SEQ ID NO: 17-24: primer SEQ ID NO: 25: Talaromyces cellulolyti cus cbh1 gene base sequence number 26: Talaromyces cellulolyticus cbh2 gene base sequence number 27: Talaromyces cellulolyticus bgl3A gene base sequence number 28: Talaromyces cellulolyticus eg5A gene base sequence number 29: Talaromyces cellulolyticus eg5X1 gene base sequence SEQ ID NO: 30: Talaromyces cellulolyticus eg5X2 gene base sequence SEQ ID NO: 31: Talaromyces cellulolyticus eg7B gene base sequence SEQ ID NO: 32: Talaromyces cellulolyticus xyl10A gene base sequence SEQ ID NO: 33: encodes Bgl3A-10LC SEQ ID NO: 34: amino acid sequence of Bgl3A-10LC SEQ ID NO: 35: nucleotide sequence of the gene encoding Bgl3A-10C SEQ ID NO: 36: amino acid sequence of Bgl3A-10C SEQ ID NO: 37: gene encoding Bgl3A-ΔLC SEQ ID NO: 38 amino acid sequence of Bgl3A-ΔLC

Claims (19)

  A protein having a carbohydrate binding module of xylanase and having cellulase activity.   The protein according to claim 1, which is a protein having a carbohydrate binding module of xylanase and a catalytic domain of cellulase.   The protein according to claim 1 or 2, wherein the carbohydrate binding module is a carbohydrate binding module classified into Family 1 of carbohydrate binding modules.   The protein according to any one of claims 1 to 3, wherein the carbohydrate binding module is a carbohydrate binding module of Xyl10A. The protein according to any one of claims 1 to 4, wherein the carbohydrate binding module comprises the amino acid sequence described in (a), (b), or (c) below:
(A) the amino acid sequence of positions 375 to 403 of SEQ ID NO: 16;
(B) an amino acid sequence having a binding ability to a cellulosic substrate, including substitution, deletion, insertion, or addition of 1 to 5 amino acid residues in the amino acid sequence at positions 375 to 403 of SEQ ID NO: 16;
(C) an amino acid sequence having 90% or more identity to the amino acid sequence at positions 375 to 403 of SEQ ID NO: 16 and having an ability to bind to a cellulosic substrate.   The protein according to any one of claims 2 to 5, wherein the catalytic domain is a catalytic domain of β-glucosidase.   The protein according to any one of claims 2 to 6, wherein the catalytic domain is a catalytic domain of a cellulase classified into family 3 of glycoside hydrolases. The protein according to any one of claims 2 to 7, wherein the catalytic domain is a catalytic domain of Bgl3A. The protein according to any one of claims 2 to 8, wherein the catalytic domain comprises an amino acid sequence described in the following (a), (b), or (c):
(A) the amino acid sequence of positions 25 to 601 of SEQ ID NO: 11;
(B) an amino acid sequence having cellulase activity, including substitution, deletion, insertion, or addition of 1 to 10 amino acid residues in the amino acid sequence at positions 25 to 601 of SEQ ID NO: 11;
(C) an amino acid sequence having cellulase activity having 90% or more identity to the amino acid sequence at positions 25 to 601 of SEQ ID NO: 11.   The protein according to any one of claims 2 to 9, which has a linker region between the carbohydrate binding module and the catalytic domain. The protein according to any one of claims 2 to 10, which has a fibronectin type III-like domain.   The protein according to any one of claims 1 to 11, which does not have an inherent carbohydrate binding module of cellulase from which the catalytic domain is derived. The protein according to any one of claims 1 to 12, which is the protein according to the following (a), (b), or (c):
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 34 or 36;
(B) a protein having an amino acid sequence containing substitution, deletion, insertion or addition of 1 to 10 amino acid residues and having cellulase activity in the amino acid sequence shown in SEQ ID NO: 34 or 36;
(C) A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 34 or 36 and having cellulase activity.   A gene encoding the protein according to any one of claims 1 to 13.   A vector carrying the gene according to claim 14.   A host comprising the gene according to claim 14.   17. A host according to claim 16, which is a bacterium or a fungus.   A method for producing a saccharified product, comprising treating a cellulosic substrate with the protein according to any one of claims 1 to 13.   The method of claim 18, wherein the cellulosic substrate is plant biomass.