Method for producing saccharifying enzymes

Abstract

To provide a method for producing diastatic enzyme in which high viscosity caused by is suppressed in a saccharification process of biomass.SOLUTION: A method for producing biomass diastatic enzyme includes culturing a recombinant filamentous fungus into which either one or both of a polygalacturonase gene selected from a gene group shown by the following (a), and a pectin lyase gene selected from a gene group shown by (b) is introduced. (a) PgaB gene, PgaI gene, PgaII gene, (b) PelD gene, PelF gene.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing a biomass saccharifying enzyme. [Background technology]

[0002] Due to the depletion of fossil fuels used for energy and materials, as well as environmental issues such as global warming, technological development to replace fossil fuels with renewable resources is underway around the world. Among these, there is a demand for advanced utilization of cellulosic biomass from the perspective of carbon neutrality, and there are high hopes for utilizing industrial waste.

[0003] Cassava grown in Africa and Asia is primarily used in Asia, where starch is extracted and made into tapioca starch. The cassava residue left after tapioca starch extraction has a moisture content of 70-90% and is used as livestock feed after drying, but not all of the residue is used, and the remainder is discarded. However, cassava residue contains large amounts of starch and cellulose, and is a glucose-rich biomass, so there is hope for advanced utilization by converting it into sugar. Meanwhile, cassava residue is known to contain a lot of pectin in addition to starch and cellulose. Pectin is a natural polysaccharide, a polymer found primarily in cell walls, which gives it high viscosity.

[0004] To convert the cellulose in cassava residue into sugar, a saccharification process using biomass saccharification enzymes such as cellulase is required, but the high viscosity of pectin affects the efficiency of sugar conversion, which is an issue. Furthermore, cassava residue contains hemicellulose and lignin in addition to starch, cellulose, and pectin, so multiple enzymes such as amylase, cellulase, pectinase, and hemicellulase are essential for saccharification.

[0005] Non-Patent Document 1 discloses that an enzyme mixture containing cellulase, hemicellulase, and pectinase derived from Aspergillus aculeatus reduces the viscosity of cassava roots, chips, and pulp, and Patent Document 1 also discloses that an enzyme preparation containing cellulase and pectinase can reduce the moisture content and material viscosity in cassava residue and increase the starch yield. However, because saccharifying biomass using a mixture of various enzymes is very costly, these techniques are not practical for large-scale saccharification of cassava residue, and there is a need for a technology that can produce saccharifying enzymes inexpensively. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Chinese Patent Application Publication No. 101285058 [Non-patent literature]

[0007] [Non-Patent Document 1] Aphisit Poonsrisawat et al., Process Biochemistry 49(2014)1950-1957 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention relates to a method for producing a saccharifying enzyme that suppresses high viscosity caused by pectin in the saccharification process of biomass. [Means for solving the problem]

[0009] The present inventors have discovered that by culturing a recombinant filamentous fungus into which a gene encoding a specific pectin-degrading enzyme (pectinase) has been introduced, it is possible to efficiently produce a saccharifying enzyme that exhibits an excellent inhibitory effect on the high viscosity of biomass caused by pectin.

[0010] That is, the present invention relates to the following 1) and 2). 1) A method for producing a biomass saccharifying enzyme, comprising culturing a recombinant filamentous fungus into which either or both of a polygalacturonase gene selected from the gene group shown in (a) below and a pectin lyase gene selected from the gene group shown in (b) below have been introduced. (a) PgaB gene, PgaI gene, PgaII gene (b) PelD gene, PelF gene, 2) A method for saccharifying biomass, comprising culturing a recombinant filamentous fungus into which either or both of a polygalacturonase gene selected from the gene group shown in (a) below and a pectin lyase gene selected from the gene group shown in (b) below have been introduced, and using the culture obtained as a biomass saccharifying agent. (a) PgaB gene, PgaI gene, PgaII gene (b) PelD gene, PelF gene, [Effects of the Invention]

[0011] According to the present invention, it is possible to inexpensively produce a saccharifying enzyme that suppresses the increase in viscosity of the slurry during the saccharification treatment of pectic biomass and enables efficient saccharification treatment. DETAILED DESCRIPTION OF THE INVENTION

[0012] (1.Definition) In the present invention, the identity of an amino acid sequence or a nucleotide sequence is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, the identity is calculated by performing analysis using the homology analysis (Search homology) program of the genetic information processing software GENETYCS Ver. 12, with the unit size to compare (ktup) set to 2.

[0013] In the present invention, "at least 90% identity" with respect to an amino acid sequence or a nucleotide sequence means identity of 90% or more, preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, still more preferably 98% or more, and even more preferably 99% or more. Examples of amino acid sequences or nucleotide sequences that are at least 90% identical to a certain amino acid sequence or nucleotide sequence include amino acid sequences in which one or more amino acids have been deleted, substituted, added, or inserted, and nucleotide sequences in which one or more nucleotides have been deleted, substituted, added, or inserted. Here, "an amino acid sequence in which one or more amino acids have been deleted, substituted, added, or inserted" refers to an amino acid sequence in which 1 to 10, preferably 1 to 8, more preferably 1 to 5, and even more preferably 1 to 3 amino acids have been deleted, substituted, added, or inserted. Furthermore, "a nucleotide sequence in which one or more nucleotides have been deleted, substituted, added, or inserted" refers to a nucleotide sequence in which 1 to 30, preferably 1 to 24, more preferably 1 to 15, and even more preferably 1 to 9 nucleotides have been deleted, substituted, added, or inserted. As used herein, "addition" of an amino acid or nucleotide includes addition of an amino acid or nucleotide to one or both ends of a sequence.

[0014] In the present invention, "upstream" and "downstream" of a gene refer to upstream and downstream in the transcription direction of the gene. For example, "a gene located downstream of a promoter" refers to a gene located downstream of a DNA sequence. "Upstream" means that a gene is present on the 3' side of the promoter on the DNA sense strand, and "upstream" of a gene means the region on the 5' side of the gene on the DNA sense strand.

[0015] In the present invention, "operably linked" between a regulatory region and a gene means that the gene and regulatory region are linked in such a way that the gene can be expressed under the control of the regulatory region. Procedures for "operably linking" a gene and a regulatory region are well known to those skilled in the art.

[0016] In the present invention, the term "corresponding gene" refers to a gene encoding an enzyme that catalyzes a predetermined reaction in a certain microorganism, and a gene encoding an enzyme that catalyzes the same or a similar reaction in another microorganism. A certain gene and its corresponding gene may have the same nucleotide sequence or different nucleotide sequences, but it is preferable that the nucleotide sequence identity between a certain gene and its corresponding gene is at least 90%.

[0017] (2. Recombinant filamentous fungi and their production) The recombinant filamentous fungus of the present invention is a filamentous fungus into which a specific pectinase gene has been introduced, specifically, a polygalacturonase gene selected from the gene group shown in (a) below and / or a pectin lyase gene selected from the gene group shown in (b) below. (a) PgaB gene, PgaI gene, PgaII gene (b) PelD gene and PelF gene

[0018] "Polygalacturonase" is an enzyme (EC3.2.1.15) that hydrolyzes the α-1,4-glycosidic bond of D-galacturonic acid that constitutes pectic acid, and the polygalacturonase gene of the present invention includes one or more genes selected from the PgaB gene, PgaI gene, and PgaII gene. Such polygalacturonase genes are preferably derived from filamentous fungi, and suitable examples thereof include those derived from microorganisms of the genus Aspergillus (e.g., Aspergillus niger, Aspergillus japonicus, Aspergillus aculeatus), microorganisms of the genus Rhizopus (e.g., Rhizopus oryzae, Rhizopus delemar), microorganisms of the genus Talaromyces (e.g., Talaromyces cellulolyticus), and microorganisms of the genus Penicillium (e.g., Penicillium roqueforti). Among these, the PgaB gene of Aspergillus niger or a gene corresponding to the PgaB gene, the PgaI gene or a gene corresponding to the PgaI gene, the PgaII gene or a gene corresponding to the PgaII gene, and the like are more preferred.

[0019] The PgaB gene of Aspergillus niger is a gene registered in the NCBI public database under the accession number XM_025597756. Specific examples of the PgaB gene of Aspergillus niger or genes corresponding to the PgaB gene include the following 1) to 3). 1) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 2) A polynucleotide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 1 and encoding a protein having polygalacturonase activity. 3) An amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 2 A polynucleotide encoding a protein having polygalacturonase activity,

[0020] Genes equivalent to the PgaB gene of Aspergillus niger are preferably genes derived from other filamentous fungi that encode polygalacturonase, such as the glycoside hydrolase family 28 protein of Aspergillus aculeatus ATCC 16872 (accession number XM_020201835), the putative extracellular endo-polygalacturonase of Aspergillus japonicus (Aspergillus japonicus CBS 114.51) (accession number XM_025671777), and the CAZyme family GH28 of Penicillium roqueforti (accession number XM_039076692).

[0021] The PgaI gene of Aspergillus niger is registered in the NCBI public database under the accession number XM_025596768. Specific examples of the PgaI gene of Aspergillus niger or genes equivalent to the PgaI gene include the following 4) to 6). 4) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3 5) A polynucleotide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 3 and encoding a protein having polygalacturonase activity. 6) A polynucleotide encoding a protein having polygalacturonase activity and consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 4.

[0022] Genes equivalent to the PgaI gene of Aspergillus niger are preferably genes derived from other filamentous fungi that encode polygalacturonase, such as the putative endopolygalacturonase II of Aspergillus aculeatus [Aspergillus aculeatinus CBS 121060] (accession number XM_025643861) and the putative endopolygalacturonase II of Aspergillus japonicus [Aspergillus japonicus CBS 114.51] (accession number XM_025668037).

[0023] The PgaII gene of Aspergillus niger is registered in the NCBI public database under the accession number XM_025602343.1. Specific examples of the PgaII gene of Aspergillus niger or genes equivalent to the PgaII gene include the following 7) to 9). 7) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5 8) A polynucleotide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 5 and encoding a protein having polygalacturonase activity. 9) A polynucleotide encoding a protein having polygalacturonase activity and consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 6.

[0024] The gene corresponding to the PgaII gene of Aspergillus niger is preferably a gene derived from another filamentous fungus encoding polygalacturonase, such as the PgaII gene of Aspergillus aculeatus ATCC 161646. Examples include the 872 uncharacterized protein (ASPACDRAFT_41690) (accession number XM_020200977.1) and Aspergillus japonicus CBS 114.51 putative endopolygalacturonase II (BO86DRAFT_431298) (accession number XM_025675761.1).

[0025] "Pectin lyase" is an enzyme (EC 4.2.2.10) that catalyzes the reaction of converting pectin into oligosaccharides having an unsaturated bond between the C-4 and C-5 terminal residues. The pectin lyase gene of the present invention includes one or more genes selected from the PelD gene and the PelF gene.

[0026] Such pectin lyase genes are preferably derived from filamentous fungi, such as those derived from Aspergillus microorganisms (e.g., Aspergillus niger, Aspergillus japonicus, Aspergillus ludhuensis), Penicillium microorganisms (e.g., Penicillium digitatum, Penicillium solitum), and Talaromyces microorganisms (e.g., Talaromyces rugulosus, Talaromyces cellulolyticus). Among these, the PelD gene of Aspergillus niger or a gene corresponding to the PelD gene is more preferred.

[0027] The Aspergillus niger PelD gene is registered in the NCBI database under accession number XM_025604039.1. Examples of the Aspergillus niger PelD gene or genes equivalent to the PelD gene include the following 10) to 12). 10) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 7 11) A polynucleotide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 7 and encoding a protein having pectin lyase activity. 12) A polynucleotide encoding a protein having pectin lyase activity and consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 8.

[0028] The gene equivalent to the PelD gene of Aspergillus niger is preferably a gene derived from another filamentous fungus that encodes a pectin lyase, such as the Aspergillus luchuensis uncharacterized protein (AKAW2_50059S) (accession number XM_041689835.1) of Aspergillus luchuensis or the Aspergillus japonicus PelD gene of Aspergillus japonicus. japonicus CBS 114.51 pectin lyase pelD (BO86DRAFT_402188) (accession number XM_025673839.1), Penicillium digitatum The strain Pd01 PL1 mRNA, complete cds (accession number JX298853.1) is one such example.

[0029] The Aspergillus niger PelF gene is registered in the NCBI database under accession number XM_001401024.2. Examples of the Aspergillus niger PelF gene or genes equivalent to the PelF gene include the following 13) to 15). 13) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 9 14) A nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO:9. A polynucleotide encoding a protein having pectin lyase activity and consisting of a nucleotide sequence 15) A polynucleotide encoding a protein having an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 10 and having pectin lyase activity.

[0030] The gene equivalent to the PelF gene of Aspergillus niger is preferably a gene derived from another filamentous fungus that encodes a pectin lyase, such as Aspergillus costaricaensis CBS 115574 pectin lyase A (BO79DRAFT_237612) (accession number XM_025684723.1) from Aspergillus costaricaensis, or Aspergillus japonicus. japonicus CBS 114.51 pectin lyase 1 (BO86DRAFT_306335) (XM_025667691.1) (accession number XM_025673839.1), and Penicillium griseofulvum pectin lyase fold / virulence factor (PGRI_010780) (accession number XM_040788791.1).

[0031] The pectinase gene to be introduced may be either or both of the polygalacturonase gene and the pectin lyase gene, but it is preferable to introduce both the polygalacturonase gene and the pectin lyase gene in terms of the viscosity-reducing effect on the biomass.

[0032] The polygalacturonase gene and / or pectin lyase gene of the present invention can be introduced into filamentous fungi, for example, by introducing a vector or DNA fragment containing the above-mentioned polynucleotide into the host filamentous fungus (parent strain). Here, the vector containing the polynucleotide of the present invention is an expression vector, preferably an expression vector capable of introducing the polynucleotide into a host filamentous fungus and expressing the polynucleotide in the host. The vector preferably contains the polynucleotide of the present invention and a control region operably linked thereto. The vector may be a vector capable of autonomous replication and replication outside a chromosome, such as a plasmid, or may be a vector that is integrated into a chromosome.

[0033] Specific examples of vectors include pBluescript II SK(-) (Stratagene), pUC vectors such as pUC18 / 19 and pUC118 / 119 (Takara Bio), pET vectors (Takara Bio), pGEX vectors (GE Healthcare), pCold vectors (Takara Bio), pHY300PLK (Takara Bio), and pUB110 (Mckenzie, T. et al., 1986, Plasmid 15(2):93-103), pBR322 (Takara Bio), pRS403 (Stratagene), pMW218 / 219 (Nippon Gene), pRI-based vectors such as pRI909 / 910 (Takara Bio), pBI-based vectors (Clontech), IN3-based vectors (Implanta Innovations), pPTR1 / 2 (Takara Bio), pDJB2 (DJ Ballance et al., Gene, 36, 321-331, 1985), pAB4-1 (van Hartingsveldt W et al., Mol Gen Genet, 206, 71-75, 1987), pLeu4 (MIG Roncero et al., Gene, 84, 335-343, 1989), pPyr225 (CDSkory et al., Mol Genet Genomics, 268, 397-406, 2002), pFG1 (Gruber, F. et al., Curr Ge net,18,447-451,1990).

[0034] Examples of DNA fragments containing the polynucleotide of the present invention include PCR-amplified DNA fragments and restriction enzyme-cleaved DNA fragments. Preferably, the DNA fragment may be an expression cassette containing the polynucleotide of the present invention and a control region operably linked thereto.

[0035] The control region contained in the vector or DNA fragment is a sequence for expressing the gene of the present invention in a host into which the vector or DNA fragment has been introduced, and examples thereof include expression regulatory regions such as promoters and terminators, and replication origins. The type of control region can be appropriately selected depending on the type of host cell into which the vector or DNA fragment is introduced. If necessary, the vector or DNA fragment may further contain a selection marker such as an antibiotic resistance gene or an amino acid synthesis-related gene.

[0036] Preferably, the regulatory region contained in the vector or DNA fragment is operably linked upstream of the polygalacturonase gene and / or pectin lyase gene and has the function of constitutively expressing or overexpressing the downstream gene (so-called strong regulatory region). Examples of such strong regulatory regions include the cbh1 promoter, cbh2 promoter, egl1 promoter, xyn1 promoter, xyn2 promoter, and xyn3 promoter of Trichoderma reesei. Further examples of strong regulatory regions include, but are not limited to, regulatory regions of rRNA operons, regulatory regions of genes encoding ribosomal proteins, and the like.

[0037] The target polynucleotide and control region contained in the vector or DNA fragment may be introduced into the host nucleus or into the host genome. Alternatively, the target polynucleotide contained in the vector or DNA fragment may be directly introduced into the host genome and operably linked to a high-expression promoter on the genome. A method for introducing a polynucleotide into a genome includes homologous recombination.

[0038] To introduce the vector or DNA fragment into the above-mentioned host cells, common transformation methods such as electroporation, transformation, transfection, conjugation, protoplast method, particle gun method, Agrobacterium method, etc. can be used.

[0039] Recombinant filamentous fungi into which a vector or DNA fragment of interest has been introduced can be selected using a selection marker. For example, if the selection marker is an antibiotic resistance gene, transformed cells into which a vector or DNA fragment of interest has been introduced can be selected by culturing the cells in a medium containing the antibiotic. Furthermore, if the selection marker is an amino acid synthesis-related gene, the gene can be introduced into an amino acid-requiring filamentous fungal strain, and the presence or absence of the amino acid requirement can be used as an indicator to select filamentous fungal strains into which a vector or DNA fragment of interest has been introduced. Alternatively, introduction of a vector or DNA fragment of interest can be confirmed by examining the DNA sequence of the recombinant strain by PCR or other methods.

[0040] By the above procedure, a recombinant filamentous fungus can be prepared in which the polygalacturonase gene and / or pectin lyase gene of the present invention has been introduced into a filamentous fungal strain. The recombinant filamentous fungus of the present invention has the ability to produce cellulase and pectinase.

[0041] The filamentous fungus used as a parent strain in the present invention is not limited as long as it is a fungus that produces cellulase as a biomass saccharification enzyme and is a filamentous fungus of a different genus or species from that of the polygalacturonase gene or pectin lyase gene to be introduced, and may be of the phylum Eumycota or Oomycota. Examples of the filamentous fungi include those of the genus Trichoderma, Aspergillus, Penicillium, Neurospora, Fusarium, Chrysosporium, Humicola, Emericella, Hypocrea, Acremonium, Chrysosporium, Myceliophthora, Piromyces, Talaromyces, Thermoascus, and Thielavia, with the genus Trichoderma being preferred.

[0042] Examples of the filamentous fungi of the genus Trichoderma include Trichoderma reesei, Trichoderma longibrachiatum, Trichoderma harzianum, Trichoderma koningii, and Trichoderma viride, with Trichoderma reesei being preferred, and Trichoderma reesei strain PCD-10 (FERM P-8172) and Trichoderma reesei strain PC-3-7 (ATCC66589) being more preferred.

[0043] The parent filamentous fungus may be a wild-type strain, a strain artificially bred from the wild-type strain, or a mutant strain (variant) or mutant in which the nucleotide sequence in its genome has been substituted, added, deleted, or modified.

[0044] Suitable examples of the recombinant filamentous fungus of the present invention include recombinant filamentous fungi obtained by introducing the Aspergillus niger PgaB gene or a gene corresponding to the PgaB gene, or the Aspergillus niger PelD gene or a gene corresponding to the PelD gene into Trichoderma reesei strain PC-3-7 (ATCC66589) or a mutant thereof, and recombinant filamentous fungi obtained by introducing both the Aspergillus niger PgaB gene or a gene corresponding to the PgaB gene and the Aspergillus niger PelD gene or a gene corresponding to the PelD gene. Specific examples of such recombinant filamentous fungi include the strains disclosed in the Examples described below.

[0045] The recombinant filamentous fungal strain of the present invention thus constructed has increased intracellular expression of pectinase compared to the parent strain, and therefore, when biomass containing cellulose and pectin is saccharified using this strain, the viscosity of the slurry caused by pectin is reduced compared to when saccharification is performed using the parent strain. Specifically, the viscosity of the slurry obtained by saccharifying cassava residue using the recombinant filamentous fungal strain of the present invention can be reduced by 50% or more, preferably 60% or more, and more preferably 70% or more compared to when saccharification is performed using the parent strain. The viscosity may be measured, for example, with a Brookfield viscometer at a solids concentration of 10-20% by mass and at 50°C. Therefore, by using the recombinant filamentous fungus of the present invention, it is possible to suppress the increase in viscosity and increase the saccharification efficiency in the saccharification treatment of biomass such as cassava, which is rich in pectin.

[0046] (3. Production of biomass saccharification enzymes) Biomass saccharification enzymes can be produced by culturing the above-mentioned recombinant filamentous fungus in the presence of a cellulase inducer, producing and accumulating enzymes including cellulase and pectinase in the culture, and collecting the enzymes from the culture.

[0047] Here, the term "cellulase inducer" is not limited as long as it is a substance that induces cellulase production in cellulase-producing filamentous fungi, but examples include compounds selected from cellulose; sophorose; and cellooligosaccharides such as cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose.

[0048] Here, cellulose includes polymers in which glucose is polymerized via β-1,4-glucosidic bonds and derivatives thereof. The degree of polymerization of glucose is not particularly limited. Examples of derivatives include carboxymethylated, aldehyde-modified, or esterified derivatives. Furthermore, cellulose may be a glycoside β-glucoside, lignocellulose, which is a complex with lignin and / or hemicellulose, or a complex with pectin or the like. Cellulose may be crystalline cellulose or amorphous cellulose.

[0049] The cellulase inducer can be added in any manner, such as all at once (batch method), in portions (fed-batch method), or continuously (feed method). The amount of cellulase inducer added to the medium may be any amount that can induce the filamentous fungus of the present invention to produce cellulase and pectinase, and although this amount varies depending on the addition method, the total amount relative to the medium is preferably 0.1% by mass or more, more preferably 0.5% by mass or more, more preferably 1% by mass or more, and preferably 40% by mass or less, more preferably 35% by mass or less, and more preferably 30% by mass or less. The total amount is also preferably 0.1 to 40% by mass, more preferably 0.5 to 35% by mass, and more preferably 1 to 30% by mass. When added all at once, the amount added is preferably 0.1% by mass or more, more preferably 0.5% by mass or more, more preferably 1% by mass or more, and is preferably 16% by mass or less, more preferably 14% by mass or less, more preferably 12% by mass or less, and is preferably 0.1 to 16% by mass, more preferably 0.5 to 14% by mass, more preferably 1 to 12% by mass.

[0050] The medium used in the method of the present invention may be either a synthetic medium or a natural medium, as long as it contains nutrients necessary for the growth of the filamentous fungus of the present invention and the production of cellulase and pectinase, such as a carbon source, a nitrogen source, inorganic salts, and vitamins.

[0051] Any carbon source can be used as long as it can be utilized by the recombinant filamentous fungus of the present invention. Specific examples include the cellulase inducers described above, as well as carbohydrates such as glucose and fructose, alcohols such as ethanol and glycerol, and organic acids such as acetic acid. These can be used alone or in combination. Carbohydrates such as glucose and fructose are desirable carbon sources, with glucose being more preferred. The amount of glucose added is preferably 0.1% by mass or more, more preferably 0.5% by mass or more, and more preferably 2.5% by mass or more, and is preferably 15% by mass or less, more preferably 10% by mass or less, and more preferably 5% by mass or less, based on the medium. The amount is preferably 0.1 to 15% by mass, more preferably 0.5 to 10% by mass, and more preferably 2.5 to 5% by mass. The mass ratio of the cellulase inducer to glucose in the medium is preferably 10:1 to 1:1, and more preferably 4:1 to 2:1.

[0052] Examples of nitrogen sources include ammonia, ammonium salts such as ammonium sulfate, nitrogen compounds such as amines, and natural nitrogen sources such as peptone and soybean hydrolysate.

[0053] Examples of inorganic salts include potassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, and potassium carbonate.

[0054] Examples of vitamins include biotin, thiamine, etc. Furthermore, substances required for the growth of the recombinant filamentous fungus of the present invention can be added as needed.

[0055] The culture is preferably carried out under aerobic conditions such as shaking culture or aeration and agitation culture. The culture temperature is preferably 10°C or higher, more preferably 20°C or higher, more preferably 25°C or higher, and preferably 50°C or lower, more preferably 42°C or lower, more preferably 35°C or lower. The culture temperature is preferably 10 to 50°C, more preferably 20 to 42°C, more preferably 25 to 35°C. The pH during cultivation is 3 to 9, preferably 4 to 5. The cultivation time is 10 hours to 10 days, preferably 2 to 7 days.

[0056] After the cultivation is completed, the culture is recovered and, if necessary, the cells are disrupted by ultrasonication, pressure, or the like. After solid-liquid separation by filtration, centrifugation, or the like, saccharifying enzymes including cellulase and pectinase can be obtained by an appropriate combination of ultrafiltration, salting out, dialysis, chromatography, and the like. The degree of separation and purification is not particularly limited. The culture supernatant or a crudely separated and purified product thereof can also be used as a biomass saccharifying enzyme.

[0057] "Cellulase" is a general term for enzymes that decompose cellulose, and includes endoglucanases (EC 3.2.1.4), which cleave cellulose from the inside of the molecule; exoglucanases (cellobiohydrolases, EC 3.2.1.91), which decompose cellulose from the reducing or non-reducing end and release cellobiose; and β-glucosidases (EC 3.2.1.21). Furthermore, "pectinase" is a general term for a group of enzymes that decompose pectic substances (polysaccharides consisting of α-1,4 bonds of D-galacturonic acid), and includes polygalacturonase, pectin lyase, and pectin esterase. However, polygalacturonase and pectin lyase are preferred as saccharifying enzymes of the present invention.

[0058] (4. Biomass Saccharification) By using the recombinant filamentous fungus of the present invention, biomass can be decomposed and saccharified to produce monosaccharides, and such a method can be carried out using known techniques. That is, the culture obtained by culturing the recombinant filamentous fungus of the present invention described above in the presence of a cellulase inducer is used as a biomass saccharifying agent, and by coexisting this with biomass in an aqueous medium and heating while stirring or shaking, the biomass can be saccharified to produce monosaccharides. In the present invention, examples of biomass include cellulose- and pectin-containing substances, such as cassava, cassava residue, corn, corn hulls, apple residue, and citrus peel, and preferably cassava residue. In the saccharification of biomass, the pH and temperature of the reaction solution may be within ranges that do not inactivate cellulase and pectinase. Generally, when the reaction is carried out at normal pressure, the temperature is 5 to 95°C and the pH is in the range of 1 to 11. The biomass saccharification process may be carried out in a batch or continuous manner.

[0059] The present invention also includes the following as exemplary embodiments, however, the present invention is not limited to these embodiments. <1> A method for producing a biomass saccharifying enzyme, comprising culturing a recombinant filamentous fungus into which either or both of a polygalacturonase gene selected from the gene group shown in (a) below and a pectin lyase gene selected from the gene group shown in (b) below have been introduced. (a) PgaB gene, PgaI gene, PgaII gene (b) PelD gene and PelF gene <2> the polygalacturonase gene is the PgaB gene of Aspergillus niger or a gene corresponding to the PgaB gene, and the pectin lyase gene is PelD of Aspergillus niger or a gene corresponding to the PelD gene; <1> The method described below. <3> The biomass is a biomass containing cellulose and pectin; <1> or <2> The method described below. <4> The biomass is cassava, <1> or <2> The method described below. <5> The filamentous fungus is a cellulase-producing filamentous fungus <1> ~ <4> A method according to any one of the preceding claims. <6> The filamentous fungus is a Trichoderma fungus, <1> ~ <5> A method according to any one of the preceding claims. <7> The filamentous fungus is Trichoderma reesei, <6> The method described below. <8> A method for saccharifying biomass, comprising culturing a recombinant filamentous fungus into which either or both of a polygalacturonase gene selected from the gene group shown in (a) below and a pectin lyase gene selected from the gene group shown in (b) below have been introduced, and using a culture obtained by culturing the recombinant filamentous fungus as a biomass saccharifying agent. (a) PgaB gene, PgaI gene, PgaII gene (b) PelD gene and PelF gene <9> Prevents the slurry from becoming too viscous. <8> The method described below. <10> The filamentous fungus is a cellulase-producing filamentous fungus <8> or <9> A method according to any one of the preceding claims. <11> The filamentous fungus is a Trichoderma fungus, <8> ~ <10> A method according to any one of the preceding claims. <12> The filamentous fungus is Trichoderma reesei, <11> The method described below. <13> The PgaB gene of Aspergillus niger or a gene corresponding to the PgaB gene is any one of the following 1) to 3): <1> ~ <12> A method according to any one of the preceding claims. 1) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 2) A polynucleotide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 1 and encoding a protein having polygalacturonase activity. 3) A polynucleotide that encodes a protein having polygalacturonase activity and that has an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 2. <14> The PgaI gene of Aspergillus niger or a gene corresponding to the PgaI gene is any one of the following 4) to 6): <1> ~ <12> A method according to any one of the preceding claims. 4) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3 5) A polynucleotide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 3 and encoding a protein having polygalacturonase activity. 6) A polynucleotide encoding a protein having polygalacturonase activity and consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 4. <15> The PgaII gene of Aspergillus niger or a gene corresponding to the PgaII gene is any one of the following 7) to 9): <1> ~ <12> A method according to any one of the preceding claims. 7) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5 8) A polynucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 5, which encodes a protein having polygalacturonase activity. nucleotide 9) A polynucleotide encoding a protein having polygalacturonase activity and consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 6. <16> The PelD gene of Aspergillus niger or a gene corresponding to the PelD gene is any one of the following 10) to 12): <1> ~ <12> A method according to any one of the preceding claims. 10) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 7 11) A polynucleotide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 7 and encoding a protein having pectin lyase activity. 12) A polynucleotide encoding a protein having pectin lyase activity and consisting of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 8. <17> The PelF gene of Aspergillus niger or a gene corresponding to the PelF gene is any one of the following 13) to 15): <1> ~ <12> A method according to any one of the preceding claims. 13) A polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 9 14) A polynucleotide consisting of a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 9 and encoding a protein having pectin lyase activity. 15) A polynucleotide encoding a protein having an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 10 and having pectin lyase activity. [Example]

[0060] Example 1: Production of biomass saccharification enzyme (1) cDNA synthesis of Aspergillus niger NBRC105649 strain Aspergillus niger NBRC105649 strain was purchased from the National Institute of Technology and Evaluation, Biotechnology Center. NBRC105649 strain was grown in pectin medium (1% pectin from citrus (Wako Pure Chemical Industries), 0.14% (NH4)2SO4, 0.2% KH2PO4, 0.03% CaCl2·2H2O, 0.03% The bacteria were inoculated into a 50 mM tartrate buffer (pH 4.0) containing MgSO4 7H2O, 0.1% Bacto Polypepton, 0.05% Bacto Yeast extract, 0.1% Tween 80, and 0.1% Trace element 2. The composition of Trace element 2 was as follows: 6 mg H3BO3, 26 mg (NH4)6Mo7O 24Distilled water was added to a 100 mL solution of 100 mg FeCl3·6H2O, 40 mg CuSO4·5H2O, 8 mg MnCl2·4H2O, and 200 mg ZnCl2. The mixture was shaken at 28°C for 3 days, and the cultured cells were harvested using Miracloth (Millipore), frozen in liquid nitrogen, and disrupted using a Multi-Bead Shocker (Yasui Kikai). A metal cone was used as the grinding medium, and the cells were disrupted at 1700 rpm for 30 seconds. RNA was extracted from the disrupted cells using the RNeasy Mini Kit (Qiagen) according to the protocol. The RNA solution was then subjected to SuperScript III First-Strand Synthesis System for RT-PCR (Thermo Fisher Scientific) to identify Aspergillus niger strains. cDNA of the NBRC105649 strain was synthesized.

[0061] (2) Construction of vectors for expressing PgaI, PgaII, PgaB, PelD, and PelF A plasmid in which the region from upstream to downstream of the xyn2 gene (SEQ ID NO: 11) derived from Trichoderma reesei was inserted into the HincII restriction enzyme cleavage site of pUC118 (Takara Bio) Fragment (A) was amplified by PCR using the genomic DNA of Trichoderma reesei as a template and Fw primer 1 (SEQ ID NO: 12) and Rv primer 1 (SEQ ID NO: 13) shown in Table 1. Fragment (B) was amplified by PCR using Trichoderma reesei genomic DNA as a template and Fw primer 2 (SEQ ID NO: 14) and Rv primer 2 (SEQ ID NO: 15) shown in Table 1. The resulting DNA fragments (A) and (B) were treated according to the protocol of the In-Fusion HD Cloning Kit (Takara Bio) to construct the plasmid pUCf-xyn2, in which the cbh1 terminator was ligated to the xyn2 gene. Furthermore, fragment (C) was amplified by PCR using pUCf-xyn2 as a template and Fw primer 3 (sequence number 16) and Rv primer 3 (sequence number 17) shown in Table 1, and fragment (D) was amplified by PCR using Trichoderma reesei genomic DNA as a template and Fw primer 4 (sequence number 18) and Rv primer 4 (sequence number 19) shown in Table 1. These fragments were then similarly treated with the In-Fusion HD Cloning Kit to obtain plasmid pUCf-xyn2-pyr4. Next, using pUCf-xyn2-pyr4 as a template, PCR was performed using Fw primer 5 (sequence number 20) and Rv primer 5 (sequence number 21) shown in Table 1 to obtain a fragment (E), and using Trichoderma reesei genomic DNA as a template, PCR was performed using Fw primer 6 (sequence number 22) and Rv primer 6 (sequence number 23) shown in Table 1 to obtain a fragment (F). These fragments were then similarly treated with the In-Fusion HD Cloning Kit to obtain the plasmid pUCf-xyn2-pyr4rec. Fragment (G) was amplified by PCR using pUCf-xyn2-pyr4rec as a template and Fw primer 7 (SEQ ID NO: 24) and Rv primer 7 (SEQ ID NO: 25) shown in Table 1. Furthermore, fragments (H) to (L) were amplified by PCR using cDNA from the Aspergillus niger NBRC105649 strain as a template and Fw primer 8 (SEQ ID NO: 26) and Rv primer 8 (SEQ ID NO: 27), Fw primer 9 (SEQ ID NO: 28) and Rv primer 9 (SEQ ID NO: 29), Fw primer 10 (SEQ ID NO: 30) and Rv primer 10 (SEQ ID NO: 31), Fw primer 11 (SEQ ID NO: 32) and Rv primer 11 (SEQ ID NO: 33), and Fw primer 12 (SEQ ID NO: 34) and Rv primer 12 (SEQ ID NO: 35) shown in Table 1. Fragment (G) and fragments (H) to (L) were treated with the In-Fusion HD Cloning Kit to obtain expression vectors for PgaI, PgaII, PgaB, PelD, and PelF, respectively: pUCf-Pxyn2-pgaI-pyr4rec, pUCf-Pxyn2-pgaII-pyr4rec, pUCf-Pxyn2-pgaB-pyr4rec, pUCf-Pxyn2-pelD-pyr4rec, and pUCf-Pxyn2-pelF-pyr4rec.

[0062] [Table 1]

[0063] (3) Preparation of transformants Trichoderma reesei strain E1AB1 (Enzyme and Microbial Technology Volume 82, The vector constructed in (1) above was transformed into a uracil-auxotrophic strain of Escherichia coli (January 2016, Pages 89-95). Transformation was performed using the protoplast PEG method. Transformants were selected on selective medium (2% glucose, 1.1 M sorbitol, 2% agar, 0.2% KH2PO4 (pH 5.5), 0.06% CaCl2·2H2O, 0.06% CsCl2, 0.06% MgSO4·7H2O, 0.5% (NH4)2SO4, 0.1% Trace element 1; all percentages are w / v%). Trace element 1 consisted of 0.5 g FeSO4·7H2O, 0.2 g CoCl2, 0.16 g MnSO4·H2O, and 0.14 g ZnSO4·7H2O, diluted to 100 mL with distilled water. The selected transformants were stabilized by subculture, and then strains that stably retained the target gene were further selected by colony PCR.

[0064] (4) Cultivation of transformants Avicel medium (1% Avicel (Sigma-Aldrich), 0.14% (NH4)2SO4, 0.2% KH2PO4, 0.03% CaCl2·2H2O, 0.03% MgSO4) 2 × 10 spores of the strain selected in (3) above were added to 4·7H2O, 0.1% Bacto Polypepton, 0.05% Bacto Yeast extract, 0.1% Tween 80, 0.1% Trace element 2, 50 mM tartaric acid buffer (pH 4.0; all percentages are w / v%). 5 The bacteria were inoculated at a concentration of 1000 cells / mL and cultured at 28°C for 5 days with shaking. The composition of Trace element 2 is as follows: 6 mg H3BO3, 26 mg (NH4)6Mo7O 24 100mg FeCl3·6H2O, 40mg CuSO4·5H2O, 8mg MnCl2·4H2O, and 200mg ZnCl2 were diluted with distilled water to a total volume of 100mL. The resulting cultures were centrifuged and then filtered to obtain the culture supernatants. The resulting culture supernatants were used as saccharification enzyme solutions for the study.

[0065] Example 2: Saccharification of biomass (1) The protein concentration of the saccharification enzyme solution was quantified by the Bradford method. Using the Quick Start Protein Assay (BioRad), which is based on the Bradford method, the protein concentration of the supernatant was calculated based on a calibration curve using bovine gamma globulin as the standard protein.

[0066] (2) Cassava residue was used as biomass. Dried cassava residue was milled in 100g portions using an Absolute Mill (ABS-W, manufactured by Osaka Chemical Co., Ltd.) at speed control 10 for 30 seconds. Composition analysis of the cassava residue was performed by Tokai Techno Co., Ltd. and Japan Food Analysis Center. The composition analysis results (wt%) were as follows: Glucose 63.1% Xylose 5.2% Arabinose 2.2% Galactose 6.5% Lignin 7.4% Galacturonic acid 7.0% Rhamnose 0.7%

[0067] (3) A saccharification test was conducted using the saccharification enzyme solution obtained in Example 1, in which the six pectinases were expressed, and crushed cassava residue as a substrate. 40 g of crushed cassava residue was weighed dry and placed in a 500 mL baffled flask. 0.07 mg of α-amylase (Sigma-Aldrich, α-Amylase from Bacillus licheniformis Type XII-A) and milli-Q water were added to a substrate concentration of 15 wt%. The flask was then thoroughly shaken by hand. The baffled flask was sealed with a cotton stopper, covered with aluminum foil, and autoclaved (TOMY LSX-700) at 90°C for 2 hours (gelatinization). The mixture was then left at room temperature until the temperature reached approximately 60°C, and then shaken at 210 rpm for 1 hour in a shaker (PRECI PRXY-30-R-3F) set at 50°C. Thereafter, 2.0 mg protein of glucoamylase (Sigma-Aldrich, Amyloglucosidase from Aspergillus niger) and 8 mg protein of the pectinase-expressing enzyme were added to the cassava residue slurry, and the mixture was reacted at 50°C and 200 rpm for 24 hours.

[0068] (4) Measurement of slurry viscosity The viscosity of the slurry after saccharification enzyme treatment of cassava residue was measured using a B-type viscometer VISCOMETER TVB-10 (manufactured by Toki Sangyo Co., Ltd.) at a rotation speed of 60 rpm and a rotation time of 20 min. The viscosity values ​​were measured under conditions of 180 seconds and 50°C, and the values ​​obtained for the six pectinase-expressing enzymes and the enzymes that did not express pectinase were compared. As a result, the viscosity values ​​obtained for the enzymes that expressed pectinase were generally higher than those for the enzymes that did not express pectinase. The viscosity was reduced, and the viscosity was reduced most significantly when the enzyme was co-expressed with a heterologous pectinase.

[0069] [Table 2]

[0070] <Comparative Example: Preparation of Pectinase> (1) cDNA synthesis of Aspergillus niger NBRC105649 strain The A. niger NBRC105649 strain was purchased from the National Institute of Technology and Evaluation, Biotechnology Center. The NBRC105649 strain was inoculated into pectin medium (1% pectin from citrus (Wako Pure Chemical Industries), 0.14% (NH4)2SO4, 0.2% KH2PO4, 0.03% CaCl2·2H2O, 0.03% MgSO4·7H2O, 0.1% Bacto Peptone, 0.05% Bacto Yeast Extract, 0.1% trace element, 50 mM tartrate buffer (pH 4.0). After shaking at 28°C for 3 days, the cultured cells were harvested using Miracloth (Millipore), frozen in liquid nitrogen, and disrupted using a Multi-Beads Shocker (Yasui Kikai). A metal cone was used as the grinding medium, and the cells were disrupted at 1700 rpm for 30 seconds. RNA was extracted from the disrupted cells using the RNeasy Mini Kit (Qiagen) according to the protocol. The RNA solution was then analyzed using the SuperScript III First-Strand Synthesis System for cDNA of the NBRC105649 strain was synthesized by RT-PCR (Thermo Fisher Scientific).

[0071] (2) Artificial synthesis of DNA derived from Fusarium oxysporum Based on publicly available DNA information, the pgx1 gene derived from F. oxysporum was artificially synthesized. Pgx1 is an exopolygalacturonase, a type of pectinase, and its amino acid sequence is shown in SEQ ID NO:41. The Pgx1 gene consists of the nucleotide sequence shown in SEQ ID NO:40.

[0072] (3) Construction of expression vectors for PgxC, RgxC, Pgx1, PgaB, and PelD Fragment (A) was amplified by PCR using the Pichia expression vector pD912 (ATUM) as a template and Fw primer 13 (SEQ ID NO: 42) and Rv primer 13 (SEQ ID NO: 43) shown in Table 3. Fragment (B) was amplified by PCR using the cDNA of the NBRC105649 strain as a template and Fw primer 14 (SEQ ID NO: 44) and Rv primer 14 (SEQ ID NO: 45) shown in Table 1. The resulting DNA fragments (A) and (B) were treated according to the protocol of the In-Fusion HD Cloning Kit (Takara Bio), and the ligated plasmid pD912-PgxC was constructed. Fragment (C) was amplified by PCR using cDNA of the NBRC105649 strain as a template and Fw primer 15 (SEQ ID NO: 46) and Rv primer 15 (SEQ ID NO: 47) shown in Table 3. The resulting DNA fragments (A) and (C) were treated according to the protocol of the In-Fusion HD Cloning Kit, and the ligated plasmid pD912-RgxC was constructed. Using the artificially synthesized pgx1 gene derived from F. oxysporum as a template, P was generated using Fw primer 16 (SEQ ID NO: 48) and Rv primer 16 (SEQ ID NO: 49) shown in Table 3. The resulting fragment (D) was amplified by PCR. The resulting DNA fragments (A) and (D) were treated according to the protocol of the In-Fusion HD Cloning Kit, and the ligated plasmid pD912-Pgx1 was constructed. Fragment (E) was amplified by PCR using cDNA of the NBRC105649 strain as a template and Fw primer 17 (SEQ ID NO: 50) and Rv primer 18 (SEQ ID NO: 51) shown in Table 3. The resulting DNA fragments (A) and (E) were treated according to the protocol of the In-Fusion HD Cloning Kit, and the ligated plasmid pD912-PgaB was constructed. Fragment (D) was amplified by PCR using cDNA of the NBRC105649 strain as a template and Fw primer 18 (SEQ ID NO: 52) and Rv primer 18 (SEQ ID NO: 53) shown in Table 3. The resulting DNA fragments (A) and (D) were treated according to the protocol of the In-Fusion HD Cloning Kit, and the ligated plasmid pD912-PelD was constructed.

[0073] The above-mentioned PgxC is an exopolygalacturonase, a type of pectinase, and its amino acid sequence is shown in SEQ ID NO:37, and the PgxC gene consists of the nucleotide sequence shown in SEQ ID NO:36. Furthermore, RgxC is an exorhamnogalacturonan hydrolase, a type of pectinase, and its amino acid sequence is shown in SEQ ID NO: 39. The RgxC gene consists of the nucleotide sequence shown in SEQ ID NO: 38.

[0074] [Table 3]

[0075] (4) Preparation of transformants (3) Each target gene expression vector prepared in (3) was transformed into Pichia pastoris PPS-90102 (ATUM) according to the ATUM protocol. It was.

[0076] (5) Cultivation of transformants P. pastoris was cultured as follows: Each transformant was inoculated into 1% BMD medium (0.2 M potassium phosphate (pH 6.0), 13.4 g / L Yeast Nitrogen Base, 0.4 mg / L biotin, 1.1% glucose) and cultured with shaking at 28°C. Once the bacterial cells had grown to a certain mass, methanol was added to induce expression. After 3–5 days, the resulting cultures were checked for enzyme expression levels by SDS-PAGE. The cultures were then centrifuged and filtered to obtain the culture supernatants. The resulting culture supernatants were concentrated and used as enzyme solutions for the various pectinases (PgxC, RgxC, Pgx1, PgaB, and PelD).

[0077] <Comparative Test Example 1> Cassava residue was weighed out to a dry weight of 4 g into a 100 mL baffled Erlenmeyer flask, and α-amylase (Sigma-Aldrich, α-Amylase from Bacillus licheniformis Type XII-A) was added at 0.002 mg protein / g substrate, and milli-Q water was added to a substrate concentration of 15 wt%. The baffled flask was then thoroughly mixed, sealed with a cotton stopper, and covered with aluminum foil. The mixture was then gelatinized at 90°C for 2 hours (autoclave, TOMY LSX-700). The mixture was then cooled to 50°C in a shaker (PRXY-30-R-3F, ​​manufactured by Preci) set to 50°C. Glucoamylase (Amyloglucosidase from Aspergillus niger, manufactured by Sigma-Aldrich) was added at 0.05 mg protein / g substrate, and the saccharifying enzyme obtained by culturing the E1AB1 strain according to Example 1 was added at a total concentration of 0.2 mg protein / g substrate. The mixture was allowed to react at 50°C and 200 rpm for 18 hours. The viscosity was then measured using a tuning fork vibrating viscometer (SV-10H, manufactured by A&D Co., Ltd.). The slurry did not become liquid after saccharification, making it impossible to measure its viscosity.

[0078] <Comparative Test Example 2> The same study as in Test Example 1 was carried out, except that the saccharifying enzyme derived from the E1AB1 strain and PgxC were added to the gelatinized substrate at 0.14 mg-protein / g-substrate and 0.06 mg-protein / g-substrate, respectively. The slurry after saccharification did not become liquid, and the viscosity could not be measured.

[0079] <Comparative Test Example 3> The same study as in Test Example 1 was carried out, except that the saccharifying enzyme derived from the E1AB1 strain and RgxC were added to the gelatinized substrate at 0.14 mg-protein / g-substrate and 0.06 mg-protein / g-substrate, respectively. The slurry after saccharification did not become liquid, and the viscosity could not be measured.

[0080] <Comparative Test Example 4> The same study as in Test Example 1 was carried out, except that the saccharifying enzyme derived from the E1AB1 strain and Pgx1 were added to the gelatinized substrate at 0.14 mg-protein / g-substrate and 0.06 mg-protein / g-substrate, respectively. The slurry after saccharification did not become liquid, and the viscosity could not be measured.

[0081] <Reference test example 1> The same study as in Test Example 1 was conducted, except that the saccharifying enzyme derived from the E1AB1 strain and PgaB were added to the gelatinized substrate at 0.18 mg-protein / g-substrate and 0.02 mg-protein / g-substrate, respectively. The viscosity of the slurry after saccharification was 84 mPa s.

[0082] <Reference test example 2> The same study as in Test Example 1 was conducted, except that the saccharifying enzyme derived from the E1AB1 strain and PelD were added to the gelatinized substrate at 0.14 mg protein / g substrate and 0.06 mg protein / g substrate, respectively. The viscosity of the slurry after saccharification was 459 mPa s. These results suggest that PgaB and PelD are effective in reducing viscosity.

Claims

1. A method for producing a cassava saccharifying enzyme, comprising culturing a recombinant filamentous fungus into which both a polygalacturonase gene selected from the gene group shown in (a) below and a pectin lyase gene selected from the gene group shown in (b) below have been introduced. (a) PgaB gene, PgaI gene, PgaII gene (b) PelD gene, PelF gene

2. 2. The method of claim 1, wherein the polygalacturonase gene is the PgaB gene of Aspergillus niger or a gene corresponding to the PgaB gene, and the pectin lyase gene is PelD of Aspergillus niger or a gene corresponding to the PelD gene.

3. The method according to claim 1, wherein the filamentous fungus is a cellulase-producing filamentous fungus.

4. The method according to any one of claims 1 to 3, wherein the filamentous fungus is a filamentous fungus of the genus Trichoderma.

5. The method of claim 4, wherein the filamentous fungus is Trichoderma reesei.

6. A method for saccharifying cassava, comprising culturing a recombinant filamentous fungus into which both a polygalacturonase gene selected from the gene group shown in (a) below and a pectin lyase gene selected from the gene group shown in (b) below have been introduced, and using the resulting culture as a cassava saccharifying agent. (a) PgaB gene, PgaI gene, PgaII gene (b) PelD gene, PelF gene

7. The method according to claim 6, wherein the viscosity of the slurry is prevented from increasing.

8. The method according to claim 6, wherein the filamentous fungus is a cellulase-producing filamentous fungus.

9. The method according to any one of claims 6 to 8, wherein the filamentous fungus is a filamentous fungus of the genus Trichoderma.

10. The method of claim 9, wherein the filamentous fungus is Trichoderma reesei.

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