Cellulase composition and method for producing sugar solution

Abstract

A sugar solution having low viscosity or capable of reducing turbidity due to solid-liquid separation can be obtained by enzymatically treating a biomass containing cellulose and starch with a cellulase composition containing a cellulase composition derived from filamentous bacteria of the genus Talaromyces and a cellulase composition derived from filamentous bacteria of the genus Trichoderma.

Description

Technical Field

[0001] The present invention relates to a cellulase composition containing a cellulase composition derived from a filamentous fungus of the genus Talaromyces as a main constituent, and a method for producing a sugar solution using the cellulase composition. Background Art

[0002] The fermentation production process of chemicals using saccharides as raw materials is used in the production of various industrial raw materials. As the saccharides that become the fermentation raw materials, at present, starch derived from edible raw materials such as sugarcane and sugar beet is used industrially. However, considering the sharp rise in the price of edible raw materials caused by the increase in the world population in the future, or the ethical aspect of competition with food, the construction of a process for obtaining a sugar solution from inedible biomass has become a problem.

[0003] As a method for producing saccharides using inedible biomass as a raw material, Patent Document 1 discloses a method of filtering a sugar solution obtained by hydrolyzing biomass containing cellulose with an enzyme through pressure filtration, microfiltration membrane, ultrafiltration membrane, nanofiltration membrane, reverse osmosis membrane, etc., performing solid-liquid separation, and concentrating the obtained saccharides.

[0004] As a microorganism that produces an enzyme for hydrolyzing biomass containing cellulose, for example, as disclosed in Non-Patent Document 1, it is known that filamentous fungi of the genus Talaromyces have the ability to produce a cellulase classified as a glucoamylase. In addition, as disclosed in Non-Patent Document 2, it is known that filamentous fungi of the genus Trichoderma have the same ability.

[0005] In addition, a mixed (cocktail) enzyme obtained by culturing multiple microorganisms separately and mixing the obtained multiple enzymes has been developed according to the purpose. For example, Patent Document 2 discloses a cellulase preparation for silage preparation for reducing juice discharge, which is obtained by mixing two enzymes, an enzyme obtained by culturing filamentous fungi of the genus Talaromyces and an enzyme obtained by culturing filamentous fungi of the genus Trichoderma.

[0006] Prior Art Documents

[0007] Patent Documents

[0008] Patent Document 1: International Publication No. 2019 / 189650

[0009] Patent Document 2: Japanese Patent Laid-Open No. 9-238679

[0010] Non-Patent Documents

[0011] Non-Patent Document 1: Identification and characterization of core cellulolytic enzymes from Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) critical for hydrolysis of lignocellulosic biomass, Biotechnology for Biofuels and Bioproducts, Volume 7, 15, 2014

[0012] Non-Patent Document 2: Isolation of a cellulase hyperproducing mutant strain of Trichoderma reesei, Bioresource Technology Reports, Volume 15, 100733, 2021 Summary of the Invention

[0013] Problems to be Solved by the Invention

[0014] The sugar solution obtained by hydrolyzing biomass containing cellulose and starch with cellulase in biomass containing cellulose has the following problems: due to its high viscosity, defects are likely to occur during transportation to the subsequent solid-liquid separation process and during solid-liquid separation. In addition, since the turbidity of the filtrate after solid-liquid separation is also high, the membrane is likely to be blocked in the subsequent membrane treatment process. Therefore, the present invention aims to establish a technique capable of reducing the viscosity of the sugar solution obtained by hydrolyzing biomass containing cellulose and starch, and further reducing the turbidity of the filtrate after solid-liquid separation of the sugar solution as a problem.

[0015] Means for Solving the Problems

[0016] As a means for solving the above problems, the present inventors focused on adjusting the composition of cellulase, which is a saccharifying enzyme for hydrolyzing biomass containing cellulose, and conducted in-depth research. As a result, it was found that the above problems can be solved by using a cellulase composition mainly composed of a cellulase composition derived from filamentous fungi of the genus Talaromyces, and thus the present invention was completed.

[0017] That is, the present invention consists of the following (1) to (18).

[0018] (1) A sugar solution obtained by an enzyme treatment step of treating biomass containing cellulose and starch with a saccharifying enzyme, wherein the saccharifying enzyme contains a cellulase composition derived from filamentous fungi of the genus Talaromyces.

[0019] (2) A sugar solution, which is obtained by the following steps, said steps comprising: a step of subjecting biomass containing cellulose and starch to a hydration treatment; and a step of subjecting the hydrated product of the above step to an enzymatic treatment with a saccharifying enzyme, said saccharifying enzyme comprising a cellulase composition derived from a filamentous fungus of the genus Talaromyces.

[0020] (3) The sugar solution according to (1) or (2), wherein the saccharifying enzyme is a saccharifying enzyme comprising a cellulase composition derived from a filamentous fungus of the genus Talaromyces and a cellulase composition derived from a filamentous fungus of the genus Trichoderma.

[0021] (4) The sugar solution according to any one of (1) to (3), wherein the saccharifying enzyme contains a cellulase composition derived from a filamentous fungus of the genus Talaromyces and a cellulase composition derived from a filamentous fungus of the genus Trichoderma, and has enzymatic activities (a) to (c).

[0022] (a) The enzymatic activity for decomposing galactan is 0.5 to 20 U per 1 mg of protein

[0023] (b) The enzymatic activity for decomposing pectin is 2.5 to 40 U per 1 mg of protein

[0024] (c) The enzymatic activity for decomposing crystalline cellulose is 0.5 to 1.5 U per 1 mg of protein

[0025] (5) The sugar solution according to any one of (1) to (4), wherein the step of obtaining the sugar solution comprises a step of performing solid-liquid separation on the saccharifying enzyme-treated product.

[0026] (6) The sugar solution according to any one of (1) to (5), wherein the step of obtaining the sugar solution comprises a step of performing pressure filtration on the saccharifying enzyme-treated product.

[0027] (7) The sugar solution according to any one of (1) to (6), wherein the biomass containing cellulose and starch is cassava residue.

[0028] (8) A cellulase composition, which contains a cellulase composition derived from a filamentous fungus of the genus Talaromyces and a cellulase composition derived from a filamentous fungus of the genus Trichoderma, and has enzymatic activities (a) to (c).

[0029] (a) The enzymatic activity for decomposing galactan is 0.5 to 20 U per 1 mg of protein

[0030] (b) The enzymatic activity for decomposing pectin is 2.5 to 40 U per 1 mg of protein

[0031] (c) The enzymatic activity for decomposing crystalline cellulose is 0.5 to 1.5 U per 1 mg of protein

[0032] (9) The cellulase composition according to (8) has an enzyme activity for decomposing carboxymethyl cellulose of 5 to 15 U per 1 mg of protein.

[0033] (10) The cellulase composition according to (8) or (9) has an enzyme activity for decomposing xylan of 10 to 45 U per 1 mg of protein.

[0034] (11) In the cellulase composition according to any one of (8) to (10), the filamentous fungus of the genus Talaromyces is Talaromyces cellulolyticus.

[0035] (12) In the cellulase composition according to any one of (8) to (11), the filamentous fungus of the genus Trichoderma is Trichoderma reesei.

[0036] (13) A method for producing a cellulase composition, comprising the following steps (i) to (iii).

[0037] (i) A step of culturing a filamentous fungus of the genus Talaromyces in a medium containing a cellulase inducer mainly composed of crushed corn hulls

[0038] (ii) A step of culturing a filamentous fungus of the genus Talaromyces in a medium containing a cellulase inducer mainly composed of lactose

[0039] (iii) A step of mixing the culture solution obtained in (i) and / or (ii) with a cellulase composition derived from a filamentous fungus of the genus Trichoderma to prepare a cellulase composition having the enzyme activities (a) to (c)

[0040] (a) The enzyme activity for decomposing galactan is 0.5 to 20 U per 1 mg of protein

[0041] (b) The enzyme activity for decomposing pectin is 2.5 to 40 U per 1 mg of protein

[0042] (c) The enzyme activity for decomposing crystalline cellulose is 0.5 to 1.5 U per 1 mg of protein

[0043] (14) The method for producing a cellulase composition according to (13) includes a step of culturing a filamentous fungus of the genus Trichoderma as a step of obtaining the cellulase composition derived from the filamentous fungus of the genus Trichoderma.

[0044] (15) A method for producing a sugar solution, which comprises a step of subjecting biomass containing cellulose and starch to enzymatic treatment with a cellulase composition, the cellulase composition containing a cellulase composition derived from a filamentous fungus of the genus Talaromyces and a cellulase composition derived from a filamentous fungus of the genus Trichoderma and having the enzymatic activities of (a) to (c).

[0045] (a) The enzymatic activity for decomposing galactan is 0.5 to 20 U per 1 mg of protein.

[0046] (b) The enzymatic activity for decomposing pectin is 2.5 to 40 U per 1 mg of protein.

[0047] (c) The enzymatic activity for decomposing crystalline cellulose is 0.5 to 1.5 U per 1 mg of protein.

[0048] (16) A sugar solution derived from biomass containing cellulose and starch, which contains a protein derived from a filamentous fungus of the genus Talaromyces and a protein derived from a filamentous fungus of the genus Trichoderma.

[0049] (17) A method for producing a chemical, which uses, as a fermentation raw material, the sugar solution described in any one of (1) to (7), the sugar solution obtained by the method for producing a sugar solution described in (15), or the sugar solution derived from biomass containing cellulose and starch described in (16).

[0050] (18) A method for producing an aviation fuel, which comprises a step of producing a chemical by the method described in (17) and a step of producing an aviation fuel using the chemical obtained in the above step as a raw material.

[0051] Effects of the Invention

[0052] By the present invention, a sugar solution having a low viscosity can be obtained from biomass containing cellulose and starch, or the turbidity of the liquid portion (sugar solution) obtained by solid-liquid separation of the enzymatic treatment product of biomass containing cellulose and starch can be reduced. Detailed Description

[0053] The cellulase composition is characterized by comprising cellulase and hemicellulase having hydrolysis activity of cellulose and hemicellulose. As specific examples of cellulase that can be included in the cellulase composition, endoglucanase (EC 3.2.1.4) that hydrolyzes cellulose from the inside, cellobiohydrolase (EC 3.2.1.91) that hydrolyzes cellulose and cello-oligosaccharides from the end to free cellobiose, β-glucosidase (EC 3.2.1.21) that hydrolyzes cellulose and cello-oligosaccharides from the end to free glucose, etc. As specific examples of hemicellulase that can be included in the cellulase composition, endoxylanase (EC 3.2.1.8) that hydrolyzes xylan from the inside, β-xylosidase (EC 3.2.1.37) that hydrolyzes xylo-oligosaccharides from the end, etc. can be cited.

[0054] In addition, the cellulase composition also includes pectin lyase (EC 4.2.2.10) that decomposes pectin from the inside by elimination reaction, pectinesterase (EC3.1.1.11) that produces pectin acid by deesterifying pectin, endogalacturonase (EC3.2.1.15) that hydrolyzes the polygalacturonic acid main chain constituting pectin acid from the inside, α-galacturonase (EC3.2.1.67) that hydrolyzes polygalacturonic acid and oligogalacturonic acid from the end, endogalactanase (EC 3.2.1.181) that hydrolyzes galactans located on the side chains of pectin from the inside, and β-galactosidase (EC 3.2.1.23) that hydrolyzes galactans and oligogalactoses from the end.

[0055] Since the cellulase composition is a complex of cellulase and hemicellulase, it is difficult to evaluate the above enzyme activity alone, and it is generally evaluated as a composite enzyme activity to a specific substrate. For example, CMCase and microcrystalline cellulase are evaluated as the activities to carboxymethyl cellulose (CMC) and crystalline cellulose as cellulose substrates, respectively, and xylanase, pectinase and galactanase are generally evaluated as the activities to xylan, pectin and galactan as hemicellulose substrates, respectively. The enzyme activity contained in the cellulase composition of the present invention is also specified as the enzyme activity to a specific substrate according to the above-mentioned general evaluation method. In the following description, for convenience, the enzyme activity to carboxymethyl cellulose (CMC) is referred to as CMCase, the enzyme activity to crystalline cellulose is referred to as microcrystalline cellulase, the enzyme activity to xylan is referred to as xylanase, the enzyme activity to pectin is referred to as pectinase, and the enzyme activity to galactan is referred to as galactanase.

[0056] The cellulase composition of the present invention is characterized in that a cellulase composition derived from filamentous fungi of the genus Talaromyces is used as a main constituent, and preferably a cellulase composition derived from filamentous fungi of the genus Talaromyces and a cellulase composition derived from filamentous fungi of the genus Trichoderma are used as raw materials.

[0057] Filamentous fungi of the genus Talaromyces are also known as filamentous fungi of the genus Acremonium. In addition, compared with conventional filamentous fungi of the genus Trichoderma, filamentous fungi of the genus Talaromyces have the characteristics of high heat resistance of the produced enzyme and high β-glucosidase activity. The filamentous fungi of the genus Talaromyces are not limited to wild strains, and mutant strains of filamentous fungi of the genus Talaromyces improved in the ability to produce cellulase can also be preferably used. For example, as a mutant strain of filamentous fungi of the genus Talaromyces, a mutant strain with a reduced viscosity of the culture broth during cultivation or an improved cellulase production ability obtained by subjecting it to mutation treatment using a mutagenic agent for induced mutation, ultraviolet irradiation, etc. can be used. In addition, a recombinant strain with a reduced viscosity of the culture broth during cultivation of the strain using genetic recombination technology, or a recombinant strain with an improved cellulase production ability can be used. In addition, mutant strains obtained by combining the above mutation treatment using a drug, ultraviolet irradiation, etc. with genetic recombination technology can be used.

[0058] The Talaromyces filamentous fungi used in the present invention are not particularly limited as long as they have the above characteristics. Talaromyces marneffei, Talaromyces proteolyticus, Talaromyces stipitatus, Talaromyces rugulosus, Talaromyces pinophilus, Talaromyces amestolkiae, Talaromyces atroroseus, Talaromyces verruculosus, Talaromyces islandicus, Talaromyces wortmannii, Talaromyces funiculosus, Talaromyces purpureogenus, Talaromyces variabilis, Talaromyces stollii, Talaromyces sp., Talaromyces cellulolyticus, Talaromyces emersonii or Talaromyces australis are preferred, and Talaromyces cellulolyticus is more preferred.

[0059] As specific examples of Talaromyces cellulolyticus, the Y-94 strain (FERM BP-5826), TN strain (FERM BP-11452), C1 strain (FERM P-18508), CF-2612 strain (FERM BP-10848) and their derivative strains, which are well-known mutant strains derived from Talaromyces cellulolyticus, can be cited.

[0060] Filamentous fungi of the genus Trichoderma are also known as the genus Hypocrea. In addition, there are substances of filamentous fungi of the genus Trichoderma that show a cellulase accumulation concentration of 100 g / L or more through submerged agitation culture, and about half of the cellulase is known as cellobiohydrolase I. The filamentous fungi of the genus Trichoderma are not limited to wild strains, and mutant strains of filamentous fungi of the genus Trichoderma improved in a manner of enhancing protein production ability can also be preferably used. For example, among the mutant strains of filamentous fungi of the genus Trichoderma, mutant strains with reduced viscosity of the culture broth during cultivation or enhanced protein production ability, which are obtained by subjecting them to mutagenesis treatment such as using a mutagen or ultraviolet irradiation, can be utilized. In addition, recombinant strains with reduced viscosity of the culture broth during cultivation of the strain using genetic recombination technology or recombinant strains with enhanced protein production ability can also be utilized. Furthermore, mutant strains obtained by combining the above mutagenesis treatment using a mutagen, ultraviolet irradiation, etc. with genetic recombination technology can be used.

[0061] The filamentous fungi of the genus Trichoderma used in the present invention are not particularly limited as long as they have the above characteristics, and Trichoderma reesei, Trichoderma viride, Trichoderma atroviride or Trichoderma longibrachiatum are preferred, and Trichoderma reesei is more preferred.

[0062] As a specific example of Trichoderma reesei, Trichoderma parareesei (ATCC MYA-4777) which is derived from the ancestor equivalent to Trichoderma reesei, the well-known mutant strains of Trichoderma reesei such as QM6a strain (NBRC31326), QM9123 strain (ATCC24449), QM9414 strain (NBRC31329), PC-3-7 strain (ATCC66589), QM9123 strain (NBRC31327), RutC-30 strain (ATCC56765), CL-847 strain (Enzyme. Microbiol. Technol., 10, 341-346 (1988)), MCG77 strain (Biotechnol. Bioeng. Symp., 8, 89 (1978)), MCG80 strain (Biotechnol. Bioeng., 12, 451-459 (1982)) and their derivative strains, etc. It should be noted that the QM6a strain, QM9414 strain, and QM9123 strain can be obtained from NBRC (NITE Biological Resource Center), and the PC-3-7 strain and RutC-30 strain can be obtained from ATCC (American Type Culture Collection).

[0063] The cellulase composition of the present invention is characterized in that the galactanase activity, pectinase activity, and microcrystalline cellulase activity per 1 mg of protein contained in the cellulase composition are as described below.

[0064] The galactanase activity per 1 mg of protein is 0.5 to 20 U, preferably 0.5 to 19 U, more preferably 0.9 to 15 U. When the galactanase activity per 1 mg of protein is less than 0.5 U, the turbidity of the filtrate after solid-liquid separation of the enzyme-treated product of biomass containing cellulose becomes high, which is not preferable. In addition, when the galactanase activity per 1 mg of protein exceeds 20 U, the viscosity of the sugar solution becomes high, which is not preferable. The galactanase activity is measured by a method for quantifying reducing sugars using dinitrosalicylic acid (DNS) with galactan from potato as a substrate.

[0065] The pectinase activity per 1 mg of protein is 2.5 to 40 U, ​​preferably 4.0 to 40 U, ​​and more preferably 4.5 to 35 U. When the pectinase activity per 1 mg of protein is less than 2.5 U, the viscosity of the sugar solution or the turbidity of the filtrate after solid-liquid separation becomes high, which is not preferred. In addition, if the pectinase activity per 1 mg of protein exceeds 40 U, ​​the turbidity of the filtrate after solid-liquid separation becomes high, which is not preferred. The pectinase activity is measured by a quantitative method of reducing sugar using DNS using pectin derived from citrus fruits (citrous) as a substrate.

[0066] The microcrystalline cellulase activity per 1 mg of protein is 0.5 to 1.5 U, preferably 0.9 to 1.3 U. When the microcrystalline cellulase activity per 1 mg of protein is outside the range of 0.5 to 1.5 U, the viscosity of the sugar solution or the turbidity of the filtrate after solid-liquid separation becomes high, which is not preferred. The microcrystalline cellulase activity is measured by a quantitative method of reducing sugar using DNS using "Avicel" which is a crystalline cellulose as a substrate.

[0067] Furthermore, the cellulase composition of the present invention preferably has the following CMCase activity and xylanase activity per 1 mg of protein contained in the cellulase composition.

[0068] The CMCase activity per 1 mg of protein is preferably 5 to 15 U, more preferably 9 to 15 U. By setting the CMCase activity within these ranges, the solubilized cellulose, which is a degradation product of crystalline cellulose contained in the cellulose-containing biomass, can be efficiently decomposed. The CMCase activity is measured by a reducing sugar quantitative method using DNS using carboxymethyl cellulose (CMC) as a substrate.

[0069] The xylanase activity per 1 mg of protein is preferably 10 to 45 U, more preferably 20 to 45 U. By setting the xylanase activity to these ranges, the xylan component contained in the cellulose-containing biomass can be decomposed, thereby improving the decomposition efficiency of the cellulose component. The xylanase activity is measured by a reducing sugar quantitative method using DNS using xylan derived from beech as a substrate.

[0070] The protein concentration for calculating the enzyme activity per 1 mg of protein in the cellulase composition of the present invention is measured by the Bradford method using bovine serum albumin as a standard protein.

[0071] The cellulase composition of the present invention can be prepared by appropriately adjusting the mixing ratio of a cellulase composition derived from a filamentous fungus of the genus Talaromyces and a cellulase composition derived from a filamentous fungus of the genus Trichoderma as raw materials so as to achieve the above enzyme activity. As the raw material cellulase compositions derived from a filamentous fungus of the genus Talaromyces and a filamentous fungus of the genus Trichoderma, commercially available cellulase preparations can be mixed and used, or the culture broths of the filamentous fungus of the genus Talaromyces and the filamentous fungus of the genus Trichoderma can be directly mixed and used without removing the cells from the respective culture broths, or a roughly refined cellulase obtained by solid-liquid separation only from the respective culture broths of the filamentous fungus of the genus Talaromyces and the filamentous fungus of the genus Trichoderma can be mixed and used, or a refined cellulase obtained by subjecting the filtrate to membrane and column treatments after solid-liquid separation from the culture broth of the filamentous fungus of the genus Talaromyces and the respective culture broths of the filamentous fungus of the genus Trichoderma can be mixed and used.

[0072] Examples of commercially available cellulase preparations include, as a cellulase preparation derived from a filamentous fungus of the genus Talaromyces, Acremonium cellulase (Meiji Seika Pharma) derived from Talaromyces cellulolyticus, and Filtrolase NL (DSM) derived from Talaromyces emersonii. As a cellulase preparation derived from a filamentous fungus of the genus Trichoderma, examples include cellulase (Meicellase) (Meiji Seika Pharma), Cellulase Onozuka R-10 (Yakult Pharmaceutical Industry), Cellulase Onozuka RS (Yakult Pharmaceutical Industry), Cellulase Onozuka 3S (Yakult Pharmaceutical Industry), Bakezyme Real-X (DSM), Scrase X (Mitsubishi Chemical), Scrase C (Mitsubishi Chemical), Citrase CL (DSM), Celluclast TP25 (HBI), Optimase CX (Danisco Japan), Multifect GC (Danisco Japan), Multifect B (Danisco Japan), GODO-TCF (Contract Alcohol), GODO-TCL (Contract Alcohol), Besserex (Contract Alcohol), and Bakezyme X-CELL (DSM) derived from Trichoderma longibrachiatum, and Celluclast TP25 (HBI), Optimase CX (Danisco Japan), Multifect GC (Danisco Japan), Multifect B (Danisco Japan), GODO-TCF (Contract Alcohol), GODO-TCL (Contract Alcohol), Besserex (Contract Alcohol), and Bakezyme X-CELL (DSM) derived from Trichoderma reesei.

[0073] The culture method of Talaromyces filamentous fungi and Trichoderma filamentous fungi is not particularly limited as long as it can produce a culture solution with cellulase activity. For example, it can be cultured by liquid culture using centrifuge tubes, flasks, jar fermentors, tanks, etc., or solid culture using plates, etc. The filamentous fungi need to be cultured under aerobic conditions. Among these culture methods, submerged culture with aeration and agitation in a fermentor or tank is particularly preferred. The aeration rate is preferably 0.1 - 2.0 vvm, more preferably 0.3 - 1.5 vvm, and particularly preferably 0.5 - 1.0 vvm. The culture temperature is preferably 25 - 35°C, more preferably 25 - 31°C. The pH condition during culture is preferably pH 3.0 - 7.0, more preferably pH 4.0 - 6.0. Regarding the culture time, under the conditions of producing proteins, culture is carried out until an amount of protein that can be recovered accumulates. Usually, it is about 24 - 288 hours, more preferably 36 - 240 hours.

[0074] The composition of the medium for culturing Talaromyces filamentous fungi or Trichoderma filamentous fungi is not particularly limited as long as it can be a medium composition in which Talaromyces filamentous fungi and Trichoderma filamentous fungi can produce cellulase, and a well-known medium composition for Talaromyces filamentous fungi or Trichoderma filamentous fungi can be adopted.

[0075] In addition, it is preferable to add a cellulase inducer to the above-mentioned medium. Specific examples of the cellulase inducer preferably used in the present invention include lactose and cellobiose, but lactose is particularly suitable as the cellulase inducer used in the culture of Talaromyces filamentous fungi.

[0076] In addition, cellulose, xylan, and biomass containing cellulose and / or xylan can also be used as cellulase inducers. Specific examples of the biomass containing cellulose and / or xylan (hereinafter referred to as "cellulase-inducing biomass") include, in addition to plants such as seed plants, ferns, mosses, algae, and aquatic plants, waste building materials, etc. Seed plants are divided into gymnosperms and angiosperms, and both can be preferably used. Angiosperms are further divided into monocotyledonous plants and dicotyledonous plants. Specific examples of monocotyledonous plants include bagasse, switchgrass, elephant grass, saccharum arundinaceum, corn stover, corn cob, corn bran, rice straw, wheat straw, etc., and specific examples of dicotyledonous plants preferably include beet pulp, eucalyptus, oak, birch, cassava, etc.

[0077] Cellulase-induced biomass preferably uses pretreated substances. The pretreatment method of cellulase-induced biomass is not particularly limited, and known methods such as acid treatment, sulfuric acid treatment, dilute sulfuric acid treatment, alkali treatment, hydrothermal treatment, subcritical treatment, micro-crushing treatment, cooking treatment, etc. can be used. As a specific example of the pretreated cellulase-induced biomass, pulp and corn hull powder can be cited. As the cellulase-induced substance used in the cultivation of filamentous fungi of the genus Talaromyces, corn hull powder is preferred. The corn hull powder having a relative particle amount peak in the range of 70 μm or more and 150 μm or less in the volume-based particle size distribution obtained by the measurement method using laser diffraction / scattering method described in International Publication No. 2021 / 235419 is particularly suitable.

[0078] Regarding the concentration of the cellulase-induced substance added to the above-mentioned medium, as the final concentration in the medium, it may be about 1 to 50% by weight, preferably 5 to 30% by weight, more preferably 5 to 25% by weight.

[0079] It should be noted that as a raw material for preparing the cellulase composition of the present invention, when a commercially available cellulase preparation derived from filamentous fungi of the genus Talaromyces is not used, that is, when the cellulase composition of the present invention is prepared using the culture solution of filamentous fungi of the genus Talaromyces as a raw material, it is preferably prepared using (i) a culture solution obtained by culturing filamentous fungi of the genus Talaromyces in a medium containing a cellulase-induced substance mainly composed of corn hull powder, (ii) a culture solution obtained by culturing filamentous fungi of the genus Talaromyces in a medium containing a cellulase-induced substance mainly composed of lactose, or a culture solution of (i) and (ii) as a raw material. Here, the so-called “cellulase-induced substance (mainly composed of corn hull powder or lactose)” means that more than 50% by weight, preferably 70% by weight or more, more preferably 80% by weight or more, further preferably 90% by weight or more, and particularly preferably 100% by weight of the total weight of the cellulase-induced substance is corn hull powder or lactose.

[0080] The cellulase composition of the present invention can be widely used in the hydrolysis of biomass containing cellulose. The so-called biomass containing cellulose is a substance containing at least cellulose or hemicellulose. Specifically, pulp, bagasse, cassava pulp, switchgrass, elephant grass, saccharum arundinaceum, corn straw, corn cob, corn hull, rice straw, wheat straw, beet pulp, eucalyptus, oak, birch, wheat bran, etc. can be cited.

[0081] Since biomass containing cellulose contains impurities such as high-molecular aromatic compounds lignin and hemicellulose, as a pretreatment for enzymatic treatment of the cellulase composition of the present invention, biomass containing cellulose in which lignin and hemicellulose are partially decomposed using an acid, a base, pressurized hot water, etc. can be used as cellulose. The conditions for the saccharification reaction are not particularly limited. The temperature of the saccharification reaction is preferably in the range of 25 to 60°C, more preferably in the range of 30 to 55°C. The time of the saccharification reaction is preferably in the range of 2 to 200 hours. The pH of the saccharification reaction is preferably in the range of pH 3.0 to 7.0, more preferably in the range of pH 4.0 to 6.0. Further, since the pH changes during hydrolysis, it is preferable to add a buffer to the reaction solution or to carry out the reaction while maintaining a constant pH using an acid or a base.

[0082] In addition, the cellulase composition of the present invention is suitable for the hydrolysis of biomass containing cellulose and starch in biomass containing cellulose. Biomass containing cellulose and starch is a substance containing at least cellulose, hemicellulose, and starch. Specifically, tapioca meal, sweet potato pulp, sugar beet pulp, wheat bran, etc. can be mentioned, but tapioca meal is preferred.

[0083] Since biomass containing cellulose and starch contains highly crystalline starch, the reaction efficiency of glucoamylase treatment is low. Therefore, it is preferable to carry out a hydration (gelatinization) treatment before the glucoamylase treatment. The hydration treatment can be carried out by known physical or chemical methods. For example, physical treatments such as heating at 50°C or higher and chemical treatments such as adding an alkaline substance are known. However, if the subsequent enzymatic treatment process is considered, a heating treatment without accompanying a pH change is preferred.

[0084] When hydrolyzing biomass containing cellulose and starch with the cellulase composition of the present invention, it is preferable to further carry out an enzymatic treatment using amylase.

[0085] Examples of amylase include α-amylase (EC 3.2.1.1), β-amylase (EC 3.2.1.2), glucoamylase (EC 3.2.1.3), pullulanase (EC 3.2.1.41), isoamylase (EC 3.2.1.68), etc. As the amylase that can be used in the present invention, it is preferable to have at least glucoamylase activity and / or α-amylase activity.

[0086] Amylase can be used as a glucoamylase for biomass containing cellulose and starch. As the amylase in this case, it is suitable to have glucoamylase activity. As the timing of allowing amylase to act for the saccharification of biomass containing cellulose and starch, it can be simultaneous with the hydrolysis using the cellulase composition or after the hydrolysis using the cellulase composition, but it is preferably simultaneous with the hydrolysis using the cellulase composition.

[0087] Regarding the conditions for enzymatic treatment with amylase for saccharification of biomass containing cellulose and starch, when carried out simultaneously with the cellulase composition of the present invention, it is sufficient to follow the enzymatic treatment conditions of the cellulase composition. When the enzymatic treatment with amylase is carried out separately from the cellulase composition of the present invention, the temperature of the enzymatic treatment reaction is preferably in the range of 25 to 60°C, more preferably in the range of 30 to 55°C, the enzymatic treatment time is preferably in the range of 2 to 200 hours, and the pH during the enzymatic treatment reaction is preferably in the range of pH 3.0 to 7.0, more preferably in the range of pH 3.5 to 6.0.

[0088] Amylase can also be used as an enzyme to promote the hydration treatment of biomass containing cellulose and starch. As amylase in this case, it is suitable to have α-amylase activity. As the timing of allowing amylase to act to promote the hydration treatment of biomass containing cellulose and starch, it can be simultaneous with the hydration treatment or after the hydration treatment, but it is preferably to act simultaneously with the hydration treatment.

[0089] Regarding the conditions for enzymatic treatment with amylase for promoting the hydration treatment of biomass containing cellulose and starch, when carried out simultaneously with the hydration treatment, it is sufficient to follow the hydration treatment conditions. When the enzymatic treatment with amylase is carried out separately from the hydration treatment, the temperature of the enzymatic treatment reaction is preferably in the range of 25 to 110°C, the enzymatic treatment time is preferably 30 minutes or more, and the pH during the enzymatic treatment reaction is preferably in the range of pH 3.0 to 7.0.

[0090] The amylase used in the present invention can utilize commercially available amylase preparations, and either a microbial culture solution containing amylase or a purified amylase can be used. Among commercially available amylase preparations, as examples of α-amylase preparations, α-Amylase, heat-stable (Sigma-Aldrich Japan), α-Amylase from Bacillus sp. (Sigma-Aldrich Japan), Clistase T10S (Amano Enzyme), FUNGAL ALPHAAMYLASE (Megazyme), etc. can be cited. In addition, as examples of glucoamylase preparations, amyloglucosidase from Aspergillus niger (Sigma-Aldrich Japan), Gluczyme AF6 (Amano Enzyme), amyloglucosidase (Megazyme), Spirizyme (Novozymes), etc. can be cited.

[0091] The viscosity of the sugar solution obtained from biomass containing cellulose and starch by using a cellulase composition prepared from a cellulase composition derived from filamentous fungi of the genus Talaromyces and a cellulase composition derived from filamentous fungi of the genus Trichoderma is lower than that of the sugar solution obtained by using only the cellulase composition derived from filamentous fungi of the genus Talaromyces or the cellulase composition derived from filamentous fungi of the genus Trichoderma. The viscosity of the sugar solution herein refers to the viscosity measured by a digital rotational viscometer. The sugar solution after the hydrolysis reaction is taken into a specified container, the rotor is immersed in the culture solution and rotated, and the torque, which is the viscous resistance acting on the rotor at this time, is measured under room temperature conditions, whereby the viscosity of the sugar solution can be measured. The unit of viscosity is centipoise (cP), and 1 poise is defined as the viscosity of a stress that generates a force of 1 dyne per 1 cm in the direction of the velocity in a plane perpendicular to the direction of the velocity gradient in a fluid when the velocity gradient is 1 cm / second per 1 cm 2 The viscosity of the sugar solution obtained in the present invention is preferably 100 cP or less, more preferably 80 cP or less, still more preferably 60 cP or less, and particularly preferably 50 cP or less.

[0092] The enzyme-treated product obtained by the above method can be separated by solid-liquid separation into a liquid part (sugar solution) and a solid part containing hydrolysis residues. As the method of solid-liquid separation, filtration treatments such as pressure filtration, vacuum filtration, and pressure filtration are preferred. In the case of performing pressure filtration, for example, a sand layer filter or a bag filter can be used. In addition, in the case of performing vacuum filtration, devices such as a suction filter and a pre-coated filter (cartridge filter) can be used. In the case of performing pressure filtration, devices such as a candle filter, a leaf filter, a filter press, and a bag filter can be used. In the filtration treatment, vacuum filtration or pressure filtration is preferred, and pressure filtration is more preferred. As the device for performing pressure filtration, a leaf filter or a filter press is preferred, and a filter press is more preferred.

[0093] As the filter cloth used for filtration treatment as solid-liquid separation, commercially available products can be used. For example, if it is a double-layer fabric, examples include TR246D3K (Nakao Filter), T2731C (Shikishima Canvas), TR132AK (Nakao Filter), PPG930B1K (Nakao Filter); if it is a twill fabric, examples include P851 (Shikishima Canvas), P1140 (Shikishima Canvas), P866 (Shikishima Canvas), PP9F (Nakao Filter), TR9F (Nakao Filter), P866C (Shikishima Canvas), P851 (Shikishima Canvas), P1140FC (Shikishima Canvas), P850 (Shikishima Canvas); if it is a satin fabric, an example is P1267C (Shikishima Canvas); if it is a plain fabric, examples include PP1172106 (Platinum), PP412HK (Nakao Filter); if it is a weft-backed twill weave, an example is P91C (Nakao Filter).

[0094] In addition, when filtering the sugar solution, filter aids represented by diatomaceous earth can also be used, and examples include the Radiolite series (Showa Chemical Industry Co., Ltd.) and the like.

[0095] As described above, by subjecting the biomass containing cellulose to enzymatic treatment with the cellulase composition of the present invention, the turbidity of the resulting liquid portion can be reduced. The so-called turbidity is defined in JIS K0101 "Industrial Water Test Methods" as "The so-called turbidity indicates the degree of turbidity of water, and is classified into visual turbidity, transmitted light turbidity, scattered light turbidity, and integrating sphere turbidity for representation. When measured by comparison with a kaolin standard solution, it is expressed in units of "degree (kaolin)", and when measured by comparison with a formazin standard solution, it is expressed in units of "degree (formazin)". In the present invention, the turbidity "NTU" measured by the scattered light measurement method using a formazin standard solution with excellent reproducibility and stability compared to the kaolin standard solution is used for evaluation. The so-called NTU refers to "Nephelometric Tubidity Unit (scattered turbidity unit)". Considering the subsequent processes, the lower the turbidity of the liquid portion is, the more preferable it is. Specifically, it is preferably 100 NTU or less, more preferably 75 NTU or less, and further preferably 50 NTU or less. If the turbidity of the sugar solution exceeds 100 NTU, the filterability may decrease during the membrane separation process and other processes after solid-liquid separation.

[0096] Although the liquid part (sugar solution) from which glucoamylase has been removed can be obtained by the above solid-liquid separation, the protein components contained in glucoamylase do not need to be completely removed, and protein components derived from glucoamylase can be included in the sugar solution. Specifically, proteins derived from filamentous fungi of the genus Talaromyces and proteins derived from filamentous fungi of the genus Trichoderma.

[0097] By using the sugar solution derived from biomass containing cellulose and starch obtained by the method of the present invention as a fermentation raw material, chemicals can be produced through microbial fermentation. The microorganisms used in the production of chemicals are not particularly limited as long as they have the ability to produce the target chemicals. Examples include yeasts represented by baker's yeast, bacteria such as Escherichia coli and coryneform bacteria, filamentous fungi, and actinomycetes. The microorganisms can be those isolated from natural environments. In addition, they can also be microorganisms whose properties have been changed by mutation or genetic recombination techniques.

[0098] The chemicals obtained through fermentation are not particularly limited as long as they are substances produced by microbial or cell culture. As specific examples, substances such as alcohols, organic acids, amino acids, and nucleic acids that are produced in large quantities in the fermentation industry can be cited. For example, as alcohols, ethanol, butanol, 2,3-butanediol, 1,4-butanediol, glycerol, etc. can be cited; as organic acids, formic acid, acetic acid, lactic acid, succinic acid, malic acid, etc. can be cited; as amino acids, lysine, glutamic acid, etc. can be cited; as nucleic acids, inosinic acid, guanylic acid, inosine, guanosine, etc. can be cited. In addition, it can also be applied to the production of substances such as proteins like enzymes and antibiotics.

[0099] In addition, the chemicals obtained through the above microbial fermentation can be used as raw materials for the final target substances. As specific examples, alcohols obtained through fermentation can be used as raw materials to produce aviation fuels according to known techniques. Aviation fuels using chemicals from non-petroleum sources as raw materials are also called sustainable alternative aviation fuels (SAF: Sustainable Aviation Fuel). As the ASTM D7566 standard, Annex 1 to 7 have been approved. By using the chemicals obtained through the above microbial fermentation as raw materials, any SAF in Annex 1 to 7 of the ASTM D7566 standard can be produced.

[0100] Examples

[0101] The following examples specifically illustrate the present invention.

[0102] <Reference Example 1> Protein Concentration Measurement Conditions

[0103] Protein concentration measurement reagent used: "Quick Start Bradford" protein assay (Bio-Rad)

[0104] Measurement conditions

[0105] Protein concentration measurement reagent: 250 μL

[0106] Measurement sample: 5 μL (the sample is appropriately diluted at appropriate stages in the range of 2 - 256 times)

[0107] Standard: 0.03 - 0.5 g / L BSA

[0108] Color reaction: The protein concentration measurement reagent and the measurement sample were mixed on a 96-well microplate, and shaken at 30 °C and 9,000 rpm for 5 minutes using a maximizer (BioShaker Μ·BR-022UP).

[0109] Quantification of protein: Using a microplate reader (BioTek Synergy HTX), the absorbance of the color-developed solution at 595 nm was measured, and the protein concentration of each stage of the diluted solution was calculated from the values of the standard curve based on the standard. The protein concentration of the measurement sample was calculated by averaging the values obtained by multiplying the protein concentration of each stage of the diluted solution included in the range of the standard curve by their respective dilution factors.

[0110] <Reference Example 2> Measurement of enzyme activity

[0111] (Measurement of galactanase activity)

[0112] Substrate: 90 μL of 100 mM sodium acetate (pH 5.0) containing 5.5 g / L galactan derived from potatoes

[0113] Enzyme dilution: 10 μL (diluted so that the protein concentration becomes 0.25 g / L)

[0114] Standard: 0.25 - 4 g / L galactose

[0115] Color reagent: DNS reagent (a substance obtained by dissolving 5 g / L 3,5-dinitrosalicylic acid (DNS) and 300 g / L potassium sodium tartrate in 0.4 M sodium hydroxide)

[0116] Enzyme reaction: The enzyme dilution was mixed with the substrate cooled to 4 °C on a 96-well PCR plate, and incubated at 50 °C for 10 minutes using a thermal cycler (BioRad T100 Thermal Cycler). Then, the reaction solution was rapidly cooled to 4 °C, and 10 μL of 1 M sodium hydroxide was added to stop the enzyme reaction.

[0117] Color reaction: 40 μL of the enzyme reaction solution and 80 μL of DNS reagent were mixed on a new 96-well PCR plate and heated at 95 °C for 5 minutes using a thermal cycler (BioRad T100 Thermal Cycler). The color reaction solution was cooled to room temperature, mixed with 120 μL of water, and then 180 μL of it was transferred to a new 96-well microplate.

[0118] Quantification of activity: The absorbance of the color reaction solution at 540 nm was measured using a microplate reader (BioTek Synergy HTX), and the amount of reducing sugar released in the reaction solution was calculated from the values of the standard curve based on the standards. One unit of activity was defined as the amount of enzyme that released 1 μmol of reducing sugar per minute, and the pectinase activity was calculated from the amount of reducing sugar contained in the enzyme dilution. The activity per 1 mg of protein was set as the value obtained by dividing the pectinase activity contained in the enzyme dilution by the protein concentration contained in the enzyme dilution.

[0119] (Determination of pectinase activity)

[0120] Substrate: 90 μL of 100 mM sodium acetate (pH 5.0) containing 5.5 g / L of pectin derived from citrus fruits

[0121] Enzyme dilution: 10 μL (diluted to a protein concentration of 0.25 g / L)

[0122] Standards: 0.25 - 4 g / L of galacturonic acid

[0123] Color reagent: DNS reagent

[0124] Enzyme reaction: The enzyme dilution was mixed with the substrate cooled to 4 °C on a 96-well PCR plate and incubated at 50 °C for 10 minutes using a thermal cycler. Then, the reaction solution was quickly cooled to 4 °C, and 10 μL of 1 M sodium hydroxide was added to stop the enzyme reaction.

[0125] Color reaction: 40 μL of the enzyme reaction solution and 80 μL of DNS reagent were mixed on a new 96-well PCR plate and heated at 95 °C for 5 minutes using a thermal cycler. The color reaction solution was cooled to room temperature, mixed with 120 μL of water, and then 180 μL of it was transferred to a new 96-well microplate.

[0126] Quantification of activity: The absorbance of the color reaction solution at 540 nm was measured using a microplate reader, and the amount of reducing sugar released in the reaction solution was calculated from the values of the standard curve based on the standards. One unit of activity was defined as the amount of enzyme that released 1 μmol of reducing sugar per minute, and the pectinase activity was calculated from the amount of reducing sugar contained in the enzyme dilution. The activity per 1 mg of protein was set as the value obtained by dividing the pectinase activity contained in the enzyme dilution by the protein concentration contained in the enzyme dilution.

[0127] (Determination of Avicelase Activity)

[0128] Substrate: 180 μL of 100 mM sodium acetate (pH 5.0) containing 5.5 g / L "Avicel" PH-101

[0129] Enzyme dilution: 20 μL (diluted to a protein concentration of 0.25 g / L)

[0130] Standard: 0.25~4g / L glucose

[0131] Color development reagent: DNS reagent

[0132] Enzyme reaction: The enzyme dilution was mixed with the substrate cooled to 4°C in a 96-well plate and shaken at 50°C and 9,000 rpm for 120 minutes using a Macchiaizer. The reaction solution was then rapidly cooled to 4°C and 20 μL of 1M sodium hydroxide was added to stop the enzyme reaction.

[0133] Color development reaction: 40 μL of the supernatant of the enzyme reaction solution and 80 μL of the DNS reagent were mixed in a new 96-well PCR plate and heated at 95°C for 5 minutes using a thermal cycler. The color development solution was cooled to room temperature, mixed with 120 μL of water, and 180 μL of it was transferred to a new 96-well microplate.

[0134] Quantification of activity: The absorbance of the colorimetric solution at 540 nm was measured by a microplate reader, and the amount of reducing sugars released in the reaction solution was calculated from the value of the standard curve based on the standard product. Activity 1U is defined as the amount of enzyme that releases 1 μmol of reducing sugar per minute, and the Avicelase activity is calculated from the amount of reducing sugars contained in the enzyme dilution. The activity per 1 mg of protein is the value obtained by dividing the Avicelase activity contained in the enzyme dilution by the protein concentration contained in the enzyme dilution.

[0135] (Determination of CMCase Activity)

[0136] Substrate: 90 μL of 100 mM sodium acetate (pH 5.0) containing 5.5 g / L sodium carboxymethyl cellulose

[0137] Enzyme diluent: 10 μL (diluted to a protein concentration of 0.25 g / L)

[0138] Standard: 0.25~4g / L glucose

[0139] Color development reagent: DNS reagent

[0140] Enzyme reaction: The enzyme dilution was mixed with the substrate cooled to 4°C in a 96-well PCR plate and incubated at 50°C for 10 minutes using a thermal cycler. Then, the reaction solution was rapidly cooled to 4°C, and 10 μL of 1 M sodium hydroxide was added to stop the enzyme reaction.

[0141] Color reaction: 40 μL of the enzyme reaction solution and 80 μL of DNS reagent were mixed in a new 96-well PCR plate and heated at 95°C for 5 minutes using a thermal cycler. The color reaction solution was cooled to room temperature, and after mixing with 120 μL of water, 180 μL of it was transferred to a new 96-well microplate.

[0142] Quantification of activity: The absorbance of the color reaction solution at 540 nm was measured by a microplate reader, and the amount of reducing sugar released in the reaction solution was calculated from the values of the standard curve based on the standards. One unit of activity was defined as the amount of enzyme that released 1 μmol of reducing sugar per minute, and the CMCase activity was calculated from the amount of reducing sugar contained in the enzyme dilution. The activity per 1 mg of protein was set as the value obtained by dividing the CMCase activity contained in the enzyme dilution by the protein concentration contained in the enzyme dilution.

[0143] (Measurement conditions for xylanase activity)

[0144] Substrate: 90 μL of 100 mM sodium acetate (pH 5.0) containing 5.5 g / L of beech-derived xylan

[0145] Enzyme dilution: 10 μL (diluted to a protein concentration of 0.25 g / L)

[0146] Standards: 0.25 - 4 g / L xylose

[0147] Color reagent: DNS reagent

[0148] Enzyme reaction: The enzyme dilution was mixed with the substrate cooled to 4°C in a 96-well PCR plate and incubated at 50°C for 10 minutes using a thermal cycler. Then, the reaction solution was rapidly cooled to 4°C, and 10 μL of 1 M sodium hydroxide was added to stop the enzyme reaction.

[0149] Color reaction: 40 μL of the enzyme reaction solution and 80 μL of DNS reagent were mixed in a new 96-well PCR plate and heated at 95°C for 5 minutes using a thermal cycler. The color reaction solution was cooled to room temperature, and after mixing with 120 μL of water, 180 μL of it was transferred to a new 96-well microplate.

[0150] Quantification of activity: Using a microplate reader, the absorbance of the color-developing solution at 540 nm was measured, and the amount of reducing sugar liberated in the reaction solution was calculated from the values of the standard curve based on the standards. One unit (U) of activity was defined as the amount of enzyme that liberated 1 μmol of reducing sugar per minute, and the xylanase activity was calculated from the amount of reducing sugar contained in the enzyme dilution solution. The activity per 1 mg of protein was determined by dividing the xylanase activity contained in the enzyme dilution solution by the protein concentration contained in the enzyme dilution solution.

[0151] <Reference Example 3> Hydrolysis Treatment of Cassava Meal

[0152] To 8.5 kg of cassava meal (produced in Thailand, moisture content 83%) obtained from EBP Ethanol Co., Ltd., 3.5 L of RO water and 0.2 U of heat-stable amylase (Sigma-Aldrich Japan) were added and mixed, and then 4N sodium hydroxide (Nacalai Tesque) was added to adjust the pH to 5.0, followed by autoclave treatment (90 °C, 2 hours) to hydrate the cassava meal. 100 g of the hydrated cassava meal was dispensed into a 500 mL baffled flask, and 3.0 U of amyloglucosidase derived from Aspergillus niger (Sigma-Aldrich Japan), which is a glucoamylase, and the cellulase composition prepared in Example 1 or Example 2 described below were added thereto, and hydrolysis treatment (50 °C, 24 hours) was carried out using a bench-top constant temperature shaker BR-40LF (Taitec) under the condition of 210 rpm.

[0153] <Reference Example 4> Determination of Glucose Concentration

[0154] The sugar solution after the saccharification reaction was boiled at 100 °C for 10 minutes to stop the reaction. Further, the boiled sugar solution was centrifuged at 20,000 × g at 4 °C for 10 minutes to obtain a supernatant. The supernatant was filtered through a 0.45 μm filter, and the resulting substance was quantitatively analyzed under the following conditions.

[0155] Glucose was quantitatively analyzed using an ACQUITY UPLC system (Waters) under the following conditions. Quantitative analysis was carried out based on the standard curve prepared from the glucose standard.

[0156] Column: AQUITY UPLC BEH Amide 1.7 μm 2.1 × 100 mm Column

[0157] Separation method: HILIC

[0158] Mobile phase: Mobile phase A was set as 80% acetonitrile and 0.2% aqueous TEA solution, and mobile phase B was set as 30% acetonitrile and 0.2% aqueous TEA solution, according to the following gradient. The gradient was set as a linear gradient to reach the mixing ratio corresponding to the following time.

[0159] Starting conditions: (A 99.90%, B 0.10%). After 2 minutes from the start: (A 96.70%, B 3.30%). After 3.5 minutes from the start: (A 95.00%, B 5.00%). After 3.55 minutes from the start: (A 99.90%, B 0.10%). After 6 minutes from the start: (A 99.90%, B 0.10%).

[0160] Detection method: ELSD (Evaporative Light Scattering Detector)

[0161] Flow rate: 0.3 mL / min

[0162] Temperature: 55 °C

[0163] <Reference Example 5> Viscosity measurement of sugar solution

[0164] Regarding the viscosity obtained in Example 1, the sugar solution was measured using a digital rotational viscometer Viscometer DV2T-LV (Brookfield) and an appropriate rotor (Brookfield) according to each viscosity. Note that for the rotor, if the viscosity was low (less than 500 cP), LV-02 was used; if the viscosity was medium (500 cP to 2,000 cP), LV-03 was used; if the viscosity was high (2,000 cP or more), LV-04 was used. First, 60 mL of the sugar solution was added to a 50 mL centrifuge tube (Corning), the rotor was immersed, and the measurement was carried out under the specified conditions (liquid temperature 40 ± 2 °C, 60 rpm). Note that for the measurement time, 9 points were measured every 20 seconds, and the average value thereof was set as the viscosity.

[0165] Regarding the viscosity of the sugar solution obtained in Example 3, the sugar solution was measured using a digital rotational viscometer VISCO-895 (ATAGO) and rotor A1 (ATAGO). First, 15 mL of the sugar solution was added to a beaker S (ATAGO), the rotor was immersed, and the measurement was carried out under the specified conditions (liquid temperature 40 ± 2 °C, 100 rpm). Note that for the viscosity, 1 point was measured for each rotation of the rotor, and the moving average of the final 5 points in a measurement time of 1 minute was set as the viscosity.

[0166] <Reference Example 6> Suction filter test

[0167] Using a polysulfone holder (Advantec) as a plastic holder for vacuum filtration, a commercially available filter cloth (TR9F (Nakao Filter), T2731C (Shima Canvas), P866C (Shima Canvas), PP412HK (Nakao Filter), or P91C (Nakao Filter)) cut to an appropriate size was sandwiched between the support net and the funnel, and 20 mL of the sugar solution prepared in Reference Example 3 was supplied from the upper part of the funnel onto the filter cloth. Further, suction filtration was performed using a diaphragm-type dry vacuum pump DAP-15 (Albac Vacuum Technology Co., Ltd.), and the resulting filtrate was used to measure the turbidity using a portable turbidimeter 2100P (HACH).

[0168] <Reference Example 7> Preparation of a culture solution derived from a filamentous fungus of the genus Trichoderma

[0169] The culture solution derived from a filamentous fungus of the genus Trichoderma, which is a raw material for the cellulase composition prepared in Example 1, was prepared by the following method.

[0170] (Pre-culture)

[0171] The spores of Trichoderma reesei PC-3-7 strain (ATCC#66589) were diluted with physiological saline to a concentration of 1.0×10 7 / mL, and 1 mL of this diluted spore solution was inoculated into 100 mL of the pre-culture medium having the composition shown in Table 1 placed in a 500 mL baffled flask, and cultured using an orbital shaker at 28°C and 120 rpm for 72 hours.

[0172] [Table 1]

[0173]

[0174] ﹡ The 5×mandels solution has the following composition

[0175]

[0176] ﹡﹡ The 10×ammonium tartrate solution contains 92 g / L ammonium tartrate

[0177] ﹡﹡﹡ The trace element solution has the following composition

[0178]

[0179] (Main culture)

[0180] Using pulp (“Arbocel” B800 (Rettenmaier)), a cellulose enzyme inducer, a formal culture was carried out. 10 mL of the pre-culture solution was inoculated into 100 mL of the formal culture medium with the composition shown in Table 2, and deep culture was performed. The culture device used was a micro fermenter Bio-Jr.8 (Biott). Under the culture conditions of 28 °C, 900 rpm, and an aeration rate of 100 mL / min, while controlling the pH to 5, the culture was carried out for 96 hours.

[0181] [Table 2]

[0182]

[0183] ﹡ Same as Table 1.

[0184] ﹡﹡﹡ Same as Table 1.

[0185] (Sampling of the culture solution)

[0186] 1 mL of the culture solution was sampled 96 hours after the start of the culture. The culture solution was centrifuged at 15,000×g and 4 °C for 10 minutes to obtain the supernatant. The supernatant was filtered through a 0.45 μm filter, and the resulting filtrate was set as the culture solution derived from Trichoderma filamentous fungi.

[0187] (Measurement of protein concentration)

[0188] The protein concentration of the resulting culture solution derived from Trichoderma filamentous fungi was measured by the method of Reference Example 1. As a result, the protein concentration was 8.2 g / L.

[0189] <Reference Example 8> Preparation of the culture solution of Talaromyces filamentous fungi on corn bran

[0190] The culture solution of Talaromyces filamentous fungi on corn bran, which is the raw material of the cellulase composition prepared in Example 2, was prepared by the following method.

[0191] (Pre-culture)

[0192] Talaromyces cellulolyticus C1 strain (FERM P-18508) was inoculated into 100 mL of the pre-culture medium with the composition shown in Table 3 in a 500 mL baffled flask, and cultured at 28 °C and 120 rpm for 72 hours using an orbital shaker.

[0193] [Table 3]

[0194]

[0195] ﹡﹡﹡﹡The aqueous urea solution has the following composition. It is added after filter sterilization and autoclaving of other components.

[0196] 200 g / L urea

[0197] ﹡﹡﹡﹡﹡ The trace element solution (2) has the following composition. It is added after filter sterilization and autoclaving of other components.

[0198] 10 g / L ZnSO 4 ·7H 2 O

[0199] 10 g / L MnSO 4 ·6H 2 O

[0200] 8.7 g / L CuSO 4 ·5H 2 O

[0201] (Formal culture)

[0202] Formal culture was carried out using the pulverized corn seed coat as a cellulase inducer. 10 mL of the pre-culture solution was inoculated into 100 mL of the formal culture medium of the composition shown in Table 2 prepared by adding the pulverized corn seed coat having a relative particle amount peak at a particle diameter of 114.5 μm in the volume-based particle size distribution obtained by the measurement method using laser diffraction / scattering method as described in International Publication No. 2021 / 235419, and deep culture was carried out. A micro fermenter Bio-Jr.8 (Biott) was used as the culture device, and the culture was carried out for 192 hours while controlling the pH to 4 under the culture conditions of 30 °C, 1000 rpm, and an aeration rate of 100 mL / min.

[0203] (Collection of the culture solution)

[0204] The formal culture solution was centrifuged and filtered in the same manner as in Reference Example 7, and the resulting filtrate was designated as the culture solution of Talaromyces filamentous fungus on corn seed coat.

[0205] <Reference Example 9> Preparation of the culture solution of Talaromyces filamentous fungus on lactose

[0206] The lactose culture solution of the filamentous fungus of the genus Talaromyces used as a raw material for the cellulase composition prepared in Example 2 was obtained by culturing Talaromyces cellulolyticus C1 strain (FERM P-18508) under the same conditions as in Reference Example 8, except that lactose (Mullins) was used as a cellulase inducer. The obtained culture solution was centrifuged and filtered in the same manner as in Reference Example 7, and the resulting filtrate was designated as the lactose culture solution of the filamentous fungus of the genus Talaromyces.

[0207] <Reference Example 10> Preparation of the culture solution of the filamentous fungus of the genus Trichoderma

[0208] The culture solution of the filamentous fungus of the genus Trichoderma used as a raw material for the cellulase composition prepared in Example 2 was prepared by the following method.

[0209] (Pre-culture)

[0210] Spores of Trichoderma reesei PC-3-7 strain (ATCC#66589) were inoculated into 250 mL of the pre-culture medium placed in a 1000 mL baffled flask in the same manner as in Reference Example 7, and cultured using an orbital shaker at 28 °C and 120 rpm for 72 hours.

[0211] (Main culture)

[0212] 10 mL of the pre-culture solution was added to 100 mL of the main culture medium having the composition shown in Table 2, which was prepared by adding the ground corn seed coat having a relative particle amount peak at a particle diameter of 114.5 μm in the volume-based particle size distribution obtained by the measurement method using laser diffraction / scattering method as described in International Publication No. 2021 / 235419, and deep culture was carried out. As the culture apparatus, a micro fermenter Bio-Jr.8 (Biott) was used, and the culture was carried out under the culture conditions of 30 °C, 1000 rpm, and an aeration rate of 100 mL / min while controlling the pH to 4 for 240 hours.

[0213] In addition, from 10 hours after the start of the main culture to 240 hours, 250 mL of the liquid sugar medium having the composition shown in Table 4 containing glucose and lactose as a cellulase inducer was added every day.

[0214] [Table 4]

[0215]

[0216] (Collection of the culture solution)

[0217] The formal culture solution was centrifuged and filtered in the same manner as in Reference Example 7, and the resulting filtrate was designated as the Trichoderma filamentous fungus culture solution.

[0218] <Example 1> Production of sugar solution from cassava meal 1

[0219] The Acremonium cellulase (Meiji Seika Pharma), which is a cellulase derived from Acremonium filamentous fungus belonging to the genus Talaromyces, and the cellulase derived from Trichoderma filamentous fungus obtained in Reference Example 7 were mixed separately as shown in A to G in Table 5 to prepare a cellulase composition. At this time, the total amount of protein contained in the cellulase composition added to 1 g of biomass containing cellulose was 0.2 mg. Using the cellulase compositions prepared under each of the conditions A to G, the cassava meal was hydrolyzed under the conditions of Reference Example 3 to obtain a sugar solution.

[0220] [Table 5]

[0221]

[0222] The results of measuring the glucose concentration contained in the obtained sugar solution according to Reference Example 4 are shown in Table 6. In the case of using the cellulase composition under any of the conditions, the glucose concentration contained in the obtained sugar solution was at the same level.

[0223] The results of measuring the viscosity of the obtained sugar solution according to Reference Example 5 are shown in Table 6. Compared with the cellulase composition derived only from Acremonium filamentous fungus (Condition A) and the cellulase composition derived only from Trichoderma filamentous fungus (Condition B), by using the cellulase composition composed of the cellulase composition derived from Acremonium filamentous fungus and the cellulase composition derived from Trichoderma filamentous fungus (Conditions C to G), the viscosity of the sugar solution was reduced.

[0224] The results of measuring the turbidity of the obtained sugar solution according to Reference Example 6 are shown in Table 6. For the Trichoderma filamentous fungus culture solution only (Condition B), the turbidity of the sugar solution was significantly high. On the other hand, for the Acremonium filamentous fungus culture solution (Condition A) and the cellulase composition obtained by mixing the Acremonium filamentous fungus culture solution and the Trichoderma filamentous fungus culture solution (Conditions C to G), the turbidity of the sugar solution was significantly reduced compared to Condition B.

[0225] [Table 6]

[0226]

[0227] <Example 2> Preparation of cellulase composition using Acremonium filamentous fungus culture solution and Trichoderma filamentous fungus culture solution

[0228] A culture solution of corn seed husks of filamentous fungi of the genus Talaromyces was prepared according to Reference Example 8, a culture solution of lactose of filamentous fungi of the genus Talaromyces was prepared according to Reference Example 9, and a culture solution of filamentous fungi of the genus Trichoderma was prepared according to Reference Example 10. The protein concentration of each cellulase was measured by the method described in Reference Example 1, and the cellulase composition was prepared by mixing them in a quantitative ratio of each condition J to M in Table 9.

[0229] In addition, as a comparison object, condition H was set to be only Acremonium cellulase (Meiji Seika Pharma), which is a commercially available cellulase preparation derived from a filamentous fungus of the genus Talaromyces, and condition I was set to be only a culture solution of a filamentous fungus of the genus Trichoderma. The results of measuring the enzyme activity of the cellulase composition prepared under each condition of H to M according to Reference Example 2 are recorded in Table 7.

[0230] [Table 7]

[0231]

[0232] <Example 3> Production of Sugar Liquid from Cassava Meal 2

[0233] The cassava meal was hydrolyzed under the conditions of Reference Example 3 using the cellulase composition prepared under the conditions H to M described in Example 2 to obtain a sugar solution. At this time, the total amount of protein contained in the cellulase composition added per 1 g of cellulose-containing biomass was 0.4 mg.

[0234] The results of measuring the glucose concentration contained in the obtained sugar solution according to Reference Example 4 are shown in Table 8. When the cellulase composition under any conditions was used, the glucose concentration contained in the obtained sugar solution was about the same.

[0235] The results of measuring the viscosity of the obtained sugar solution according to Reference Example 5 are recorded in Table 8. For the culture solution of Trichoderma filamentous fungi alone (condition I), the viscosity of the sugar solution was significantly high, while for the culture solution of Talaromyces filamentous fungi (condition H) and the cellulase composition obtained by mixing the culture solution of Talaromyces filamentous fungi and the culture solution of Trichoderma filamentous fungi (conditions J to M), the viscosity of the sugar solution was significantly reduced compared with condition I.

[0236] The results of measuring the turbidity of the obtained sugar solution according to Reference Example 6 are shown in Table 8. For the culture solution of Trichoderma filamentous fungi only (Condition I), filtration was impossible. For the culture solution composition derived from Talaromyces filamentous fungi only (Condition H), the turbidity of the sugar solution was significantly high. For the cellulase composition prepared by mixing the culture solution of Talaromyces filamentous fungi and the culture solution of Trichoderma filamentous fungi (Conditions J to M), the turbidity of the sugar solution was greatly reduced. In addition, compared with the viscosity of the sugar solution in the cellulase composition prepared by mixing the Talaromyces filamentous fungi corn cob culture solution or the Talaromyces filamentous fungi lactose culture solution with the cellulase composition derived from Trichoderma filamentous fungi (Conditions J and K), the turbidity of the sugar solution in the cellulase composition prepared by mixing the two types of Talaromyces filamentous fungi corn cob culture solution and Talaromyces filamentous fungi lactose culture solution with the culture solution of Trichoderma filamentous fungi (Conditions L and M) was further reduced.

[0237] [Table 8]

[0238] Table 8

[0239]

Claims

1. A sugar solution, which is obtained by a step of subjecting biomass containing cellulose and starch to enzymatic treatment with a saccharifying enzyme, and the saccharifying enzyme contains a cellulase composition derived from filamentous fungi of the genus Talaromyces.

2. A sugar solution, which is obtained by the following steps, the steps including: a step of subjecting biomass containing cellulose and starch to hydration treatment; and a step of subjecting the hydrated product of the step to enzymatic treatment with a saccharifying enzyme, and the saccharifying enzyme contains a cellulase composition derived from filamentous fungi of the genus Talaromyces.

3. The sugar solution according to claim 1 or 2, wherein the saccharifying enzyme is a saccharifying enzyme containing a cellulase composition derived from filamentous fungi of the genus Talaromyces and a cellulase composition derived from filamentous fungi of the genus Trichoderma.

4. The sugar solution according to claim 1 or 2, wherein the saccharifying enzyme contains a cellulase composition derived from filamentous fungi of the genus Talaromyces and a cellulase composition derived from filamentous fungi of the genus Trichoderma, and has the enzyme activities of (a) to (c). (a) The enzyme activity for decomposing galactan is 0.5 to 20 U per 1 mg of protein. (b) The enzyme activity for decomposing pectin is 2.5 to 40 U per 1 mg of protein. (c) The enzyme activity for decomposing crystalline cellulose is 0.5 to 1.5 U per 1 mg of protein.

5. The sugar solution according to claim 1 or 2, wherein the step of obtaining the sugar solution includes a step of performing solid-liquid separation on the saccharifying enzyme-treated product.

6. The sugar solution according to claim 1 or 2, wherein the step of obtaining the sugar solution includes a step of performing pressure filtration on the saccharifying enzyme-treated product.

7. The sugar solution according to claim 1 or 2, wherein the biomass containing cellulose and starch is cassava residue.

8. A cellulase composition, which contains a cellulase composition derived from filamentous fungi of the genus Talaromyces and a cellulase composition derived from filamentous fungi of the genus Trichoderma, and has the enzyme activities of (a) to (c). (a) The enzyme activity for decomposing galactan is 0.5 to 20 U per 1 mg of protein. (b) The enzyme activity for decomposing pectin is 2.5 to 40 U per 1 mg of protein. (c) The enzyme activity for decomposing crystalline cellulose is 0.5 to 1.5 U per 1 mg of protein.

9. The cellulase composition according to claim 8, wherein the enzyme activity for decomposing carboxymethyl cellulose is 5 to 15 U per 1 mg of protein.

10. The cellulase composition according to claim 8, wherein the enzyme activity for decomposing xylan is 10 to 45 U per 1 mg of protein.

11. The cellulase composition according to claim 8, wherein the filamentous fungi of the genus Talaromyces is Talaromyces cellulolyticus.

12. The cellulase composition according to claim 8, wherein the filamentous fungi of the genus Trichoderma is Trichoderma reesei.

13. A method for manufacturing a cellulase composition, which includes the following steps (i) to (iii). (i) A step of culturing filamentous fungi of the genus Talaromyces in a medium containing a cellulase inducer mainly composed of crushed corn seed coats. (ii) A step of culturing filamentous fungi of the genus Talaromyces in a medium containing a cellulase inducer mainly composed of lactose. (iii) A step of mixing the culture solution obtained in (i) and / or (ii) with a cellulase composition derived from a filamentous fungus of the genus Trichoderma to prepare a cellulase composition having the enzyme activities of (a) to (c). (a) The enzyme activity for decomposing galactan is 0.5 to 20 U per 1 mg of protein. (b) The enzyme activity for decomposing pectin is 2.5 to 40 U per 1 mg of protein. (c) The enzyme activity for decomposing crystalline cellulose is 0.5 to 1.5 U per 1 mg of protein.

14. The method for producing a cellulase composition according to claim 13, which comprises a step of culturing a filamentous fungus of the genus Trichoderma as a step of obtaining the cellulase composition derived from a filamentous fungus of the genus Trichoderma.

15. A method for producing a sugar solution, which comprises a step of subjecting a biomass containing cellulose and starch to enzymatic treatment with a cellulase composition, the cellulase composition containing a cellulase composition derived from a filamentous fungus of the genus Talaromyces and a cellulase composition derived from a filamentous fungus of the genus Trichoderma and having the enzyme activities of (a) to (c). (a) The enzyme activity for decomposing galactan is 0.5 to 20 U per 1 mg of protein. (b) The enzyme activity for decomposing pectin is 2.5 to 40 U per 1 mg of protein. (c) The enzyme activity for decomposing crystalline cellulose is 0.5 to 1.5 U per 1 mg of protein.

16. A sugar solution derived from a biomass containing cellulose and starch, which contains a protein derived from a filamentous fungus of the genus Talaromyces and a protein derived from a filamentous fungus of the genus Trichoderma.

17. A method for producing a chemical, which uses the sugar solution according to claim 1 or 2, the sugar solution obtained by the method for producing a sugar solution according to claim 15, or the sugar solution derived from a biomass containing cellulose and starch according to claim 16 as a fermentation raw material.

18. A method for producing an aviation fuel, which comprises a step of producing a chemical by the method according to claim 17 and a step of producing an aviation fuel using the chemical obtained in the step as a raw material.

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