Method for producing sugar and fine fiber

Abstract

To provide a novel method for producing a cellulose nanofiber from biomass, and to provide an enzyme composition used in the method.SOLUTION: Provided is a method for producing a cellulose nanofiber from biomass, comprising: reacting cellulosic biomass with an enzyme composition containing Trichoderma cellulase, the enzyme composition containing no endoglucanase belonging to Glycoside Hydrolase family 7 (GH7) consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing sugar and fine fibers. [Background technology]

[0002] Biomass is an organic resource derived from living organisms, excluding fossil fuels, and is one of the renewable energy sources. Among biomass, cellulosic biomass derived from plants and algae has attracted attention. Cellulosic biomass is composed primarily of cellulose, hemicellulose, and lignin, and can be decomposed into sugars using saccharifying enzymes such as cellulase and hemicellulase. Technologies for producing sugar from cellulosic biomass and technologies for producing useful resources such as petroleum substitutes and biofuels from the resulting sugars using chemical or microbiological methods are being developed worldwide.

[0003] Cellulosic biomass is also a source of nanocellulose. Nanocellulose is an ultrafine aggregate of cellulose molecules. Depending on its shape, it can be called cellulose nanofibers (CNFs) when they are micron-sized fibers, or cellulose nanocrystals (CNCs) when they are rod- or spindle-shaped crystals less than 150 nm long. Nanocellulose is five times stronger than steel but only one-fifth the weight, and has a low linear thermal expansion equivalent to that of quartz glass. It is attracting attention as a lightweight, high-strength, highly elastic, low-linear thermal expansion, and safe, natural renewable resource. CNFs, which are long fibers, are particularly useful because they can be used in food additives, biodegradable packaging materials, electronic materials, high-strength materials, automotive components, optical materials, medical and pharmaceutical applications.

[0004] Conventional methods for producing nanocellulose from cellulosic biomass include removing lignin and hemicellulose from plant fiber pulp or wood pulp, processing it in a refiner, and then kneading and defibrating it. However, this method produces a mixture of cellulose fibers with fiber diameters of approximately 10 nm to 5 μm, but it is not possible to obtain nanocellulose with a uniform fiber diameter. Non-Patent Document 1 describes a method for producing nanocellulose with a width of approximately 3 to 5 nm by introducing carboxyl groups onto the cellulose surface through TEMPO oxidation to significantly reduce the interactions between cellulose molecules, and then agitating the pulp slurry in a blender. However, this method requires the introduction of carboxyl groups onto the cellulose surface, making it impossible to obtain nanocellulose that has not been chemically modified. Furthermore, because the pulp is treated with a strong acid such as high-concentration sulfuric acid for TEMPO oxidation, the resulting nanocellulose is significantly degraded and difficult to form into a fibrous form.

[0005] Patent Document 1 describes a method for producing nanocellulose by stirring a plant-derived fiber aggregate in a liquid substance such as water using a blender or mixer at 1,000 to 50,000 rpm. This method produces nanocellulose that is not chemically modified and has been defibrated to the basic element state in the plant cell wall, with a fiber diameter of 10 to 50 nm. However, this method requires powerful mechanical processing, which increases production costs.

[0006] Patent Document 2 describes a method for co-producing biofuel and nanocellulose from lignocellulose using a composition containing one or more enzymes, and describes the use of cellulase as the enzyme, and that the cellulase can be endoglucanase, commercially available cellulase preparations, etc. However, this method requires the biomass to react with a high concentration of enzyme for a long period of time in order to co-produce sugar and nanocellulose, resulting in a decrease in nanocellulose yield and non-uniformity in the shape of the nanocellulose. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Laid-Open No. 2010-216021 [Patent Document 2] U.S. Patent No. 8,835,141 [Non-patent literature]

[0008] [Non-Patent Document 1] Biomacromolecules, 2006, 7(6):1687-1691 Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention provides a method for producing cellulose fine fibers from biomass. [Means for solving the problem]

[0010] The present inventors have discovered that an enzyme composition containing a group of saccharifying enzymes, which are produced from Trichoderma fungi and do not include endoglucanase 1 belonging to glycoside hydrolase family 7 (GH7), can produce fibrous nanocellulose, i.e., CNF, from cellulosic biomass, and therefore can produce CNF from cellulose that is not chemically modified.The present inventors have also discovered that the enzyme composition can be used for the simultaneous production of sugar and CNF. The present invention provides a novel method for producing CNF from biomass and an enzyme composition for use in the method. The present invention also provides a method for co-producing sugars and CNF using the enzyme composition.

[0011] Therefore, the present invention provides a method for producing cellulose nanofibers from biomass, comprising: The method comprises reacting cellulosic biomass with an enzyme composition containing cellulase from a fungus of the genus Trichoderma, The enzyme composition does not contain an endoglucanase belonging to glycosidase family 7 (GH7) consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1. A method is provided. The present invention also provides an enzyme composition for producing cellulose nanofibers from biomass, which contains a cellulase from a fungus of the genus Trichoderma and does not contain an endoglucanase belonging to carbohydrate hydrolase family 7 (GH7) consisting of an amino acid sequence that has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. [Effects of the Invention]

[0012] According to the present invention, it is possible to produce fibrous nanocellulose, i.e., CNF, from biomass, in which cellulose is not chemically modified. More preferably, the CNF produced by the present invention has a shape with a median fiber diameter of 15 nm or less and a median fiber length of 1 μm or less. Furthermore, according to the present invention, it is possible to simultaneously produce sugar and nanocellulose from cellulosic biomass. DETAILED DESCRIPTION OF THE INVENTION

[0013] Herein, identity between amino acid sequences and nucleotide sequences can be calculated by the Lipman-Pearson method (Science, 1985, 227:1435-41). Specifically, identity can be calculated by performing analysis using the search homology program in the genetic information processing software Genetyx-Win (Ver. 5.1.1; software development) with a unit size to compare (ktup) of 2.

[0014] As used herein, "at least 90% identity" in reference to amino acid sequences and nucleotide sequences refers to 90% or more, preferably 95% or more, more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, and even more preferably 99.5% or more identity. As used herein, "at least 90% identity" encompasses 100% identity.

[0015] As used herein, the expression "operably linked" between a gene and a control region (such as a promoter or secretion signal) means that the polynucleotide of the gene and the polynucleotide of the control region are positioned so that the protein encoded by the gene can be expressed under the control of the control region.

[0016] As used herein, unless otherwise specified, "biomass" refers to cellulosic biomass containing cellulose produced by plants or algae. Specific examples of biomass include at least one selected from the group consisting of various woods obtained from conifers such as larch and bald cedar, and broad-leaved trees such as oil palm (stem) and cypress; processed or crushed wood products such as wood chips; pulps such as wood pulp produced from wood, pulp produced from plant stems and leaves, and cotton linter pulp obtained from the fibers surrounding cotton seeds; papers such as newspapers, cardboard, magazines, and fine paper; plant stems, leaves, and fruit bunches such as bagasse (sugarcane pomace), palm empty fruit bunches (EFB), rice straw, and corn stalks or leaves; plant shells such as rice husks, palm husks, and coconut shells; and algae. From the viewpoints of availability and raw material cost, examples of biomass preferably used in the present invention include wood, plant stems, leaves, fruit bunches, processed products thereof, crushed products thereof, and pulp thereof. In the present invention, the above-mentioned biomass may be used alone or in combination of two or more kinds. The biomass may also be dried.

[0017] As used herein, "saccharification of biomass" refers to a reaction that produces monosaccharides such as glucose and xylose from cellulose, hemicellulose, and the like in biomass. As used herein, "saccharification residue" refers to the solid content remaining after saccharifying the cellulose and hemicellulose in biomass with a saccharification enzyme. The saccharification residue may contain components that were not decomposed into sugars in the saccharification reaction, such as nanocellulose.

[0018] As used herein, the term "saccharifying enzyme" refers to an enzyme involved in the saccharification of biomass, such as cellulase and hemicellulase.

[0019] Cellulases are enzymes that hydrolyze the glycosidic bonds of β-1,4-glucans (e.g., cellulose). Cellulases are mainly divided into two types: endo-cellulases (endoglucanases), which randomly cleave the cellulose chain to produce cellooligosaccharides, and exo-cellulases (cellobiohydrolases), which cleave the cellulose chain from its terminal end to produce cellobiose. The cellooligosaccharides and cellobiose produced from cellulose by these cellulases are further decomposed by β-glucosidase and ultimately broken down into glucose.

[0020] Hemicellulases are a group of enzymes involved in the degradation of hemicellulose. They include endoenzymes such as xylanases, endomannanases, and xyloglucan endoglucanases, which cleave the main chain of the heteropolysaccharide of hemicellulose, as well as various exoenzymes that act on various side chains of the heteropolysaccharide. For example, xylan is degraded at the main chain by xylanase, and then further degraded by β-xylosidase to produce xylose.

[0021] Carbohydrate hydrolases, including saccharifying enzymes, are classified into glycoside hydrolase families (GH families) based on their sequence similarity. Approximately 130 GH families exist, and several of these GH families contain cellulases. Information about the characteristics of GH families and the enzymes that belong to them is published in the Carbohydrate Active Enzyme Database (CAZy database [www.cazy.org / ]). Based on this information, those skilled in the art can identify the GH family to which a given known cellulase belongs. For example, cellulases produced by wild-type Trichoderma species include cellobiohydrolase 1 (CBH1; GH7, EC 3.2.1.176), cellobiohydrolase 2 (CBH2; GH6, EC 3.2.1.91), endoglucanase 1 (EG1; GH7, EC 3.2.1.4), and endoglucanase 2 (EG2; GH5, EC 3.2.1.4).

[0022] Nanocellulose (NC) is an ultrafine aggregate of cellulose molecules. Depending on their shape, NCs are sometimes called cellulose nanofibers (CNFs) if they are long fibrous, and cellulose nanocrystals (CNCs) if they are short crystalline. Generally, fibrous NCs with lengths of several micrometers or more and widths of 5-60 nm are called CNFs, while shorter NCs are called CNCs (Carbohydrate Polymers, 2014, 99:649-665). Long fibrous CNFs are useful for applications such as food additives, biodegradable packaging materials, electronic materials, high-strength materials, automotive components, optical materials, medical and pharmaceutical applications.

[0023] The present invention provides a method for producing nanocellulose from biomass. The method of the present invention can produce fibrous nanocellulose. Preferably, the method of the present invention is a method for producing cellulose nanofibers (CNF). The method of the present invention comprises reacting cellulosic biomass with an enzyme composition containing cellulase from a fungus of the genus Trichoderma. The enzyme composition containing cellulase from a fungus of the genus Trichoderma used in the method of the present invention may be referred to hereinafter in this specification as the "enzyme composition of the present invention."

[0024] The cellulase from a fungus of the genus Trichoderma contained in the enzyme composition of the present invention may be produced microbiologically or chemically synthesized. Preferably, the enzyme composition of the present invention contains one or more cellulases produced from a fungus of the genus Trichoderma. Examples of Trichoderma fungi from which the cellulases contained in the enzyme composition of the present invention are derived include Trichoderma reesei, Trichoderma viride, Trichoderma longibrachiatum, Trichoderma harzianum, and Trichoderma koningii. Preferred examples include Trichoderma reesei or mutant strains thereof, and more preferred examples include Trichoderma reesei strains PC-3-7 and QM9414, or mutant strains thereof.

[0025] Examples of the cellulase contained in the enzyme composition of the present invention include cellobiohydrolase, endoglucanase, exoglucanase, β-glucosidase, etc. More preferably, the enzyme composition of the present invention contains one or more types selected from the group consisting of cellobiohydrolases and endoglucanases derived from fungi of the genus Trichoderma. Even more preferably, the enzyme composition of the present invention contains one or more types of cellobiohydrolases derived from fungi of the genus Trichoderma and one or more types of endoglucanases derived from fungi of the genus Trichoderma.

[0026] On the other hand, the enzyme composition of the present invention does not contain endoglucanase 1 (EG1) from Trichoderma spp. Trichoderma EG1 is an endoglucanase belonging to GH family 7 (GH7) and is defined herein as an endoglucanase consisting of an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1. An example of Trichoderma spp. EG1 is Trichoderma reesei EG1 (GH7, EC 3.2.1.4) consisting of the amino acid sequence of SEQ ID NO: 1. Trichoderma EG1 belongs to a different GH family from other Trichoderma spp. endoglucanases, such as endoglucanase 2 (EG2) belonging to GH5, and also has a different amino acid sequence. Preferably, the enzyme composition of the present invention does not contain a Trichoderma spp. endoglucanase belonging to GH7. More preferably, the enzyme composition of the present invention does not contain an endoglucanase belonging to GH7.

[0027] Examples of Trichoderma cellobiohydrolases that can be contained in the enzyme composition of the present invention include the above-mentioned Trichoderma cellobiohydrolases, and preferred examples include Trichoderma reesei cellobiohydrolases. More preferred examples of Trichoderma cellobiohydrolases include cellobiohydrolases consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2 and cellobiohydrolases consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 3. The cellobiohydrolase of SEQ ID NO: 2 corresponds to cellobiohydrolase 1 (CBH1; GH7, EC 3.2.1.176) from Trichoderma reesei. The cellobiohydrolase of SEQ ID NO: 3 corresponds to cellobiohydrolase 2 (CBH2; GH6, EC 3.2.1.91) from Trichoderma reesei.

[0028] The endoglucanase from a fungus of the genus Trichoderma that can be contained in the enzyme composition of the present invention may be any endoglucanase from a fungus of the genus Trichoderma other than EG1, such as EG2. A more preferred example of the endoglucanase from a fungus of the genus Trichoderma that can be contained in the enzyme composition of the present invention is an endoglucanase consisting of an amino acid sequence that has at least 90% identity to the amino acid sequence of SEQ ID NO: 4. The endoglucanase of SEQ ID NO: 4 corresponds to endoglucanase 2 (EG2; GH5, EC 3.2.1.4) of Trichoderma reesei.

[0029] In a preferred embodiment, the enzyme composition of the present invention contains at least one cellulase selected from the group consisting of a cellobiohydrolase consisting of an amino acid sequence having at least 90% or more identity to the amino acid sequence of SEQ ID NO: 2, a cellobiohydrolase consisting of an amino acid sequence having at least 90% or more identity to the amino acid sequence of SEQ ID NO: 3, and an endoglucanase consisting of an amino acid sequence having at least 90% or more identity to the amino acid sequence of SEQ ID NO: 4. In a more preferred embodiment, the enzyme composition of the present invention contains a cellobiohydrolase consisting of an amino acid sequence having at least 90% or more identity to the amino acid sequence of SEQ ID NO: 2, a cellobiohydrolase consisting of an amino acid sequence having at least 90% or more identity to the amino acid sequence of SEQ ID NO: 3, and an endoglucanase consisting of an amino acid sequence having at least 90% or more identity to the amino acid sequence of SEQ ID NO: 4.

[0030] In the method of the present invention, the above-mentioned cellulase from Trichoderma may be used in combination with other saccharifying enzymes from Trichoderma. Therefore, the enzyme composition of the present invention can contain one or more other saccharifying enzymes from Trichoderma in addition to the above-mentioned cellulase from Trichoderma. Examples of such other saccharifying enzymes include hemicellulases such as xylanase, β-xylosidase, galactanase, and α-arabinofuranosidase. For example, hemicellulase from Trichoderma reesei is preferred.

[0031] In the method of the present invention, the above-mentioned cellulase from a fungus of the genus Trichoderma may be used in combination with a saccharifying enzyme from a fungus other than Trichoderma. Thus, the enzyme composition of the present invention can contain, together with the above-mentioned cellulase from a fungus of the genus Trichoderma, one or more saccharifying enzymes from a fungus other than Trichoderma, as long as it does not contain an endoglucanase belonging to GH7.Examples of such saccharifying enzymes include cellulases from Pyrococcus horikoshii or Bacillus; endoglucanases from Clostridium thermocellum, Bacillus, Thermobifida, or Cellulomonas; β-glucosidases from Aspergillus aculeatus, Aspergillus niger, or Penicillium emersonii; mutant β-glucosidases from Aspergillus aculeatus described in WO2019 / 167788; P-19729, Aspergillus niger, Humicola insolens, or Bacillus alcalophilus, Thermomyces, Aureobasidium, Streptomyces, Clostridium, Thermotoga, Thermoascus, Caldocellum, Xylanimonas, Thermobifida, or Thermomonospora; Bacillus pumilus, or Selenomonas ruminantium ruminantium) β-xylosidase; mutant xylanases described in JP 2013-243953 A, JP 2013-243954 A, JP 2015-167552 A, JP 2016-119877 A, JP 2017-012006 A, and JP 2017-035001 A; and mutants thereof.

[0032] In the method of the present invention, the above-mentioned cellulase of the genus Trichoderma may be used in combination with a commercially available cellulase preparation. Thus, the enzyme composition of the present invention can contain a commercially available cellulase preparation together with the above-mentioned cellulase of the genus Trichoderma, as long as it does not contain an endoglucanase belonging to GH7.

[0033] If necessary, the enzyme composition of the present invention may contain enzymes other than the saccharifying enzyme and other components, such as a buffer, a pH adjuster, a surfactant, a storage stabilizer, or a disinfectant.

[0034] In one embodiment, the enzyme composition of the present invention contains β-glucosidase. Due to the presence of β-glucosidase in the reaction solution, cellulose in the biomass is ultimately converted to glucose. In this case, the method of the present invention can simultaneously produce nanocellulose and sugar. In another embodiment, the enzyme composition of the present invention contains, in addition to cellulase, a hemicellulase such as xylanase or β-xylosidase. Due to the presence of hemicellulase in the reaction solution, xylose can be produced from hemicellulose in the biomass. In this case, too, the method of the present invention can simultaneously produce nanocellulose and sugar. In yet another embodiment, the enzyme composition of the present invention contains β-glucosidase and hemicellulase.

[0035] Preferred examples of β-glucosidases include β-glucosidases from the genus Trichoderma, such as Trichoderma reesei and Trichoderma viride, β-glucosidases from the genus Aspergillus, such as Aspergillus acritus and Aspergillus niger, mutant β-glucosidases derived from Aspergillus acritus described in WO2019 / 167788, and mutants thereof. Preferred examples of hemicellulases include hemicellulases from the genus Trichoderma, such as Trichoderma reesei or Trichoderma viride, hemicellulases from the genus Bacillus, mutant xylanases described in JP 2013-243954 A, JP 2015-167552 A, JP 2016-119877 A, JP 2017-012006 A, and JP 2017-035001 A, and mutants thereof.

[0036] The enzyme composition of the present invention can be prepared by producing one or more of the above-mentioned cellulases from the fungus of the genus Trichoderma microbiologically or by chemical synthesis, and mixing them as needed. If necessary, the enzyme composition of the present invention can be prepared by mixing the one or more Trichoderma cellulases with the above-mentioned other enzymes, other ingredients, etc.

[0037] In the microbiological production of Trichoderma enzymes, Trichoderma fungi naturally having cellulase-producing ability or recombinant microorganisms expressing the desired Trichoderma cellulase can be cultured, and the desired cellulase can be recovered from the culture. Cultivation of Trichoderma fungi or recombinant microorganisms can be carried out according to standard methods appropriate for the microbial species. Preferably, the culture is carried out in the presence of a cellulase inducer. Examples of cellulase inducers include, but are not limited to, any substance that induces cellulase production in the cultured microorganism, including one or more compounds selected from cellulose, sophorose, and cellooligosaccharides such as cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose. For example, commercially available microcrystalline cellulose (e.g., Avicel (registered trademark)) can be used.

[0038] Recombinant microorganisms expressing a desired Trichoderma cellulase can be produced using standard methods. For example, a gene encoding the desired cellulase is isolated from a Trichoderma fungus (e.g., Trichoderma reesei) or chemically synthesized and then incorporated into a vector. The resulting vector is amplified, if necessary, and then introduced into a host microorganism to produce a recombinant microorganism. The host microorganism may be the same species (e.g., Trichoderma) or a different species (e.g., Escherichia coli). Examples of the desired Trichoderma cellulase include at least one selected from the group consisting of cellobiohydrolases having an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 2, cellobiohydrolases having an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 3, and endoglucanases having an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 4. Genes encoding these cellulases are disclosed in publicly known databases (e.g., the NCBI database [www.ncbi.nlm.nih.gov]).

[0039] The vector into which the gene for the target cellulase is inserted can be selected appropriately depending on the type of host microorganism. Preferably, on the vector, the gene encoding the target cellulase is operably linked to a promoter that promotes expression of the gene. Preferably, the promoter functions as a promoter with high transcription activity (high activity promoter) in the host microorganism. Also preferably, on the vector, the gene encoding the target cellulase is operably linked to a secretion signal sequence, which allows the expressed target cellulase to be secreted outside the cell.

[0040] When the host microorganism is a Trichoderma fungus that naturally has the ability to produce cellulase, the expression of the target cellulase may be improved by replacing the promoter of the gene for the target cellulase on the genome with a highly active promoter instead of incorporating the gene for the target cellulase. In Trichoderma fungus that naturally has the ability to produce cellulase, the expressed cellulase can be secreted outside the cell.

[0041] Alternatively, Trichoderma cellulases may be expressed from the polynucleotides encoding them or their transcription products using a cell-free translation system. A "cell-free translation system" is an in vitro transcription / translation system or an in vitro translation system constructed by adding reagents such as amino acids necessary for protein translation to a suspension obtained by mechanically disrupting host (e.g., Trichoderma) cells.

[0042] The target cellulase produced in the culture or cell-free translation system can be recovered by using common techniques used in protein purification, such as centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., either alone or in appropriate combination. If the produced cellulase is secreted extracellularly, the product can be more easily recovered from the culture. For example, the culture supernatant can be recovered by centrifugation or the like, and, if necessary, further purified by the protein purification method described above to recover the target cellulase.

[0043] Alternatively, the enzyme composition of the present invention may contain a saccharifying enzyme group produced by a Trichoderma fungus. In this case, however, it is not preferable for the saccharifying enzyme group to contain an endoglucanase belonging to GH7 (hereinafter also referred to as GH7 endoglucanase), such as endoglucanase 1 (EG1). Therefore, it is preferable that the microorganism used to produce the Trichoderma fungus enzyme contained in the enzyme composition of the present invention is a microorganism that does not express GH7 endoglucanase. Preferably, the microorganism is a Trichoderma fungus that is deficient in GH7 endoglucanase.

[0044] Thus, in one embodiment, the enzyme composition of the present invention contains saccharifying enzymes produced by Trichoderma fungi lacking GH7 endoglucanase. In a more specific embodiment, the enzyme composition of the present invention contains a composition containing saccharifying enzymes recovered from a culture of Trichoderma fungi lacking GH7 endoglucanase. For example, according to the procedure described above, Trichoderma fungi lacking GH7 endoglucanase are cultured in the presence of a cellulase inducer, and then a fraction (e.g., culture supernatant) containing the expressed saccharifying enzymes is recovered from the culture. If necessary, the fraction can be further purified by the protein purification method described above to recover a composition containing saccharifying enzymes. The resulting composition may contain the desired saccharifying enzymes, such as Trichoderma cellulases and hemicellulases, but does not contain GH7 endoglucanase.

[0045] GH7 endoglucanase-deficient Trichoderma strains can be prepared by standard methods. Examples of Trichoderma strains to be deficient in GH7 endoglucanase include Trichoderma reesei, Trichoderma viride, Trichoderma longibrachiatum, Trichoderma harriseum, and Trichoderma koningii, with Trichoderma reesei being preferred, and Trichoderma reesei strains PC-3-7, QM9414, or mutants thereof being more preferred.

[0046] An example of a GH7 endoglucanase in Trichoderma is the aforementioned EG1. Genes encoding EG1 in Trichoderma are disclosed in publicly known databases (e.g., the NCBI database [www.ncbi.nlm.nih.gov]). For example, EG1 in Trichoderma reesei is encoded by a gene consisting of the nucleotide sequence of SEQ ID NO: 5. Those skilled in the art can identify the EG1 to be deleted in Trichoderma or the gene encoding it.

[0047] One way to eliminate GH7 endoglucanase in Trichoderma fungi is to delete or inactivate the gene encoding the GH7 endoglucanase in the genome of Trichoderma fungi. Examples of ways to delete or inactivate the gene include deleting part or all of the sequence of the gene, or deleting or mutating the promoter region of the gene to inactivate the promoter.

[0048] Other methods for deleting GH7 endoglucanase in Trichoderma include reducing the expression level of the enzyme or eliminating the function of the expressed enzyme. Examples of methods for reducing expression level include translational inhibition of the enzyme using antisense oligonucleotides, siRNA, etc. Examples of methods for eliminating enzyme function include inactivating the enzyme by substituting or inserting a different sequence into the gene sequence of the enzyme, or by introducing a mutation, or by using an inhibitor such as an aptamer or antibody.

[0049] Specific techniques for deleting, substituting, or inserting the above-mentioned gene sequences or for introducing mutations include, for example, mutagenesis using chemical mutagens such as ethyl methanesulfonate, N-methyl-N-nitrosoguanidine, and nitrous acid, or physical mutagens such as ultraviolet light, X-rays, gamma rays, and ion beams; site-directed mutagenesis; and the method described by Dieffenbach et al. (Cold Spring Harbar Laboratory Press, New York, pp. 581-621, 1995). Site-directed mutagenesis techniques include recombination, splicing overlap extension (SOE) PCR (Horton et al., Gene 77, 61-68, 1989), the ODA method (Hashimoto-Gotoh et al., Gene 152, 271-276, 1995), the Kunkel method (Kunkel, TA, Proc. Natl. Acad. Sci. USA, 1985, 82, 488), and genome editing using artificial DNA cleavage enzymes (artificial DNA nucleases or programmable nucleases). Alternatively, commercially available site-directed mutagenesis kits such as the Site-Directed Mutagenesis System Mutan-SuperExpress Km Kit (Takara Bio), the Transformer™ Site-Directed Mutagenesis Kit (Clonetech), and the KOD-Plus-Mutagenesis Kit (Toyobo) can also be used.

[0050] If necessary, in addition to deleting the GH7 endoglucanase, Trichoderma fungi can be modified to improve expression of a target cellulase. Examples of target cellulases include at least one selected from the group consisting of cellobiohydrolases having an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 2, cellobiohydrolases having an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 3, and endoglucanases having an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 4. Techniques for improving expression of a target cellulase in Trichoderma fungi are similar to the techniques used for producing recombinant microorganisms that express the target cellulase.

[0051] The enzyme composition of the present invention can be used for producing nanocellulose from biomass. Furthermore, when the enzyme composition of the present invention contains β-glucosidase or hemicellulase, it can be used for producing nanocellulose and sugar from biomass. The nanocellulose produced by the enzyme composition of the present invention can have a fibrous shape. Therefore, the enzyme composition of the present invention is preferably used for producing CNF, or for producing CNF and sugar.

[0052] The basic procedure for the reaction between biomass and the enzyme composition of the present invention in the method of the present invention can be carried out in accordance with the conventionally known procedure for saccharification of biomass.

[0053] For example, the biomass used in the method of the present invention can be one or a combination of two of the cellulosic biomasses listed above. From the viewpoint of improving nanocellulose productivity, the biomass is pretreated as necessary before reaction with the enzyme composition of the present invention. Examples of such pretreatments include one or more treatments selected from the group consisting of grinding, alkali treatment, and hydrothermal treatment. From the viewpoint of improving saccharification efficiency, alkali treatment is preferred as the pretreatment. From the viewpoint of further improving saccharification efficiency, alkali treatment and grinding are preferably performed together, and more preferably, alkali treatment and grinding are performed in parallel. The grinding treatment may be wet or dry grinding, but dry grinding is preferred. More preferably, solid alkali and biomass are subjected to grinding treatment together, and dry grinding is performed in parallel with the alkali treatment (alkali mixed grinding treatment).

[0054] In the reaction of biomass with the enzyme composition of the present invention, a reaction solution containing biomass and the enzyme composition of the present invention is prepared. For example, it is preferable to add the enzyme composition of the present invention to a suspension containing biomass. The reaction conditions are not particularly limited as long as they do not inactivate the enzyme contained in the enzyme composition of the present invention. Appropriate reaction conditions can be determined appropriately by those skilled in the art depending on the type, shape, and amount of biomass, as well as the type and amount of enzyme used. Examples of reaction conditions for the method of the present invention are described below.

[0055] From the viewpoint of improving nanocellulose productivity, the content (initial concentration) of biomass in the reaction solution is preferably 0.5 to 20% by mass, more preferably 3 to 15% by mass, and even more preferably 5 to 10% by mass. The content of the enzyme composition of the present invention in the reaction solution is appropriately determined depending on the type, shape, and amount of biomass, the composition of the enzyme composition, and the type of enzyme contained therein. For example, in the reaction solution, the total mass of Trichoderma cellulase contained in the enzyme composition of the present invention (initial amount in the reaction solution) is preferably 0.1 to 10% by mass, more preferably 0.1 to 5% by mass, and even more preferably 0.1 to 3% by mass, based on 100% dry mass of biomass. Alternatively, in the reaction solution, the total mass of saccharifying enzymes contained in the enzyme composition of the present invention (initial amount in the reaction solution) is preferably 0.1 to 15% by mass, more preferably 0.1 to 5% by mass, and even more preferably 0.1 to 3% by mass, based on 100% dry mass of biomass.

[0056] The pH conditions for the reaction are preferably pH 4 to 9, more preferably pH 4 to 8, and even more preferably pH 4 to 7, from the viewpoints of improving nanocellulose productivity and reducing production costs. The temperature conditions for the reaction are preferably 20 to 90°C, more preferably 25 to 85°C, even more preferably 30 to 80°C, even more preferably 40 to 75°C, even more preferably 45 to 65°C, and even more preferably 45 to 60°C, from the viewpoints of improving nanocellulose productivity and reducing production costs. The reaction time is preferably 1 to 14 days, more preferably 1 to 7 days, and even more preferably 1 to 5 days, from the viewpoints of improving nanocellulose productivity and reducing production costs. From the viewpoint of improving nanocellulose productivity, it is preferable to inactivate the enzyme after the reaction by heating or the like.

[0057] The solid fraction of the resulting reaction solution contains nanocellulose, while the liquid fraction of the reaction solution may contain sugar. Nanocellulose can be recovered from the solid fraction of the reaction solution. Preferably, nanocellulose and sugar are recovered separately from the reaction solution by solid-liquid separation of the reaction solution. For example, the reaction solution is subjected to solid-liquid separation by filtration, centrifugation, or the like, to separate it into a liquid fraction containing sugar and a solid fraction (saccharification residue) containing nanocellulose. Nanocellulose can be recovered by defibrating the solid fraction, if necessary, using mechanical treatment such as ultrasonication. On the other hand, the liquid fraction containing sugar can be used as a sugar solution as is, or can be recovered as a highly concentrated sugar solution by concentrating it using a membrane or heat. Furthermore, mechanical treatment of the solid fraction can be omitted by adding a dispersant or the like before solid-liquid separation of the reaction solution.

[0058] The nanocellulose produced by the method of the present invention includes cellulose nanofibers (CNF). Preferably, the CNF has a uniform shape with a median fiber diameter of 15 nm or less and a median fiber length of 1 μm or less. Preferably, the nanocellulose produced by the method of the present invention is CNF with a median fiber diameter of 15 nm or less and a median fiber length of 1 μm or less. The shape of the nanocellulose can be measured by electron microscopy, particle size analysis using a particle analyzer, or the like. In a preferred embodiment, the nanocellulose produced by the method of the present invention is obtained by defibrating saccharification residue, staining with uranyl acetate, and observing it under a transmission electron microscope. When the nanocellulose is observed, the median fiber diameter of 100 nanocellulose molecules observed is 15 nm or less. Furthermore, when the particle size distribution is measured using a laser diffraction particle size analyzer (e.g., Particle Size Analyzer SALD-2100; Shimadzu Corporation), with the relative particle amount on the vertical axis and the particle size on the horizontal axis, the median particle size in the resulting distribution is 1 μm or less.

[0059] In the method of the present invention, sugar can be produced from biomass along with nanocellulose. However, as the saccharification rate of biomass (sugar yield) increases, the productivity (yield) of nanocellulose decreases. Furthermore, excessive saccharification of biomass can result in the degradation of nanocellulose into smaller molecules. To increase nanocellulose productivity and obtain nanocellulose with a moderate degree of fibrous structure, it is preferable to adjust the saccharification rate of biomass in the method of the present invention to approximately 90% or less. For example, in the procedure of the method of the present invention described above, the saccharification rate of biomass can be adjusted to an appropriate range by adjusting the reaction conditions: biomass content 5-10% by mass, total mass of saccharifying enzyme 0.1-3% by mass relative to 100% dry mass of biomass, pH 4.5-5.5, temperature 45-60°C, and reaction time 1-5 days.

[0060] The saccharification rate of biomass in the method of the present invention is determined by the following procedure: the concentrations of glucose and xylose in the reaction liquid or its liquid fraction after the saccharification reaction are measured, and this is regarded as the amount of holocellulose (cellulose + xylan) decomposed from the biomass. The ratio of the amount of decomposed holocellulose to the amount of holocellulose (cellulose + xylan) contained in the biomass is calculated, and the saccharification rate of the biomass can be determined. More specifically, the saccharification rate of biomass can be calculated using the following formula. Biomass saccharification rate (%) ={([G]+[X])×0.9 / ([B]×[HinB] / 100)}×100 [G] = glucose concentration in the reaction solution after the reaction (mg / mL) [X] = xylose concentration in the reaction solution after the reaction (mg / mL) [B] = initial biomass concentration in the reaction solution (mg / mL) [HinB] = Amount of holocellulose in biomass (%)

[0061] The yield of nanocellulose in the method of the present invention is determined by the following procedure: freeze-dry the residue after the saccharification reaction (the solid content of the reaction solution) and measure its mass. The ratio of the measured residue mass to the mass of the substrate before the saccharification reaction (the initial amount of biomass in the reaction solution) is calculated, and the yield of nanocellulose can be determined.

[0062] As exemplary embodiments of the present invention, the following substances, manufacturing methods, uses, methods, etc. are further disclosed in this specification, but the present invention is not limited to these embodiments.

[0063] [1] A method for producing cellulose nanofibers from biomass, The method comprises reacting cellulosic biomass with an enzyme composition containing cellulase from a fungus of the genus Trichoderma, The enzyme composition does not contain an endoglucanase belonging to glycosidase family 7 (GH7) consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1. method. [2] Preferably, the enzyme composition comprises at least one cellulase selected from the group consisting of cellobiohydrolases having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2, cellobiohydrolases having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 3, and endoglucanases having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 4. [3] Preferably, the enzyme composition comprises a cellobiohydrolase having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2, a cellobiohydrolase having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 3, and an endoglucanase having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 4. [4] The method according to any one of [1] to [3], wherein the enzyme composition preferably does not contain an endoglucanase belonging to GH7. [5] The method according to any one of [1] to [4], wherein the enzyme composition preferably contains one or more other saccharifying enzymes, as long as the enzyme composition does not contain an endoglucanase belonging to GH7. [6] Preferably, the enzyme composition contains a group of saccharifying enzymes produced by a Trichoderma fungus lacking an endoglucanase belonging to GH7. [7] The method according to any one of claims [1] to [6], wherein the enzyme composition preferably contains a composition containing a group of saccharifying enzymes recovered from a culture of a Trichoderma fungus lacking an endoglucanase belonging to GH7. [8] The method according to any one of [1] to [7], wherein the Trichoderma fungus is preferably Trichoderma reesei or a mutant strain thereof. [9] Preferably, the enzyme composition contains at least one enzyme selected from the group consisting of β-glucosidase and hemicellulase, and sugar is produced from the biomass together with cellulose nanofibers, Preferably, the sugar is at least one selected from the group consisting of glucose and xylose. The method described in any one of [1] to [8].

[10] Preferably, the method described in [9] further comprises recovering cellulose nanofibers and sugars separately from the reaction product obtained by the reaction of the cellulosic biomass with the enzyme composition by solid-liquid separation.

[11] The method according to any one of [1] to

[10] , wherein the cellulose nanofibers preferably have a median fiber diameter of 15 nm or less and a median fiber length of 1 μm or less.

[12] The method according to any one of [1] to

[11] , wherein the saccharification rate of the cellulosic biomass is preferably 90% or less in the reaction between the cellulosic biomass and the enzyme composition.

[13] Preferably, in the reaction between the cellulosic biomass and the enzyme composition, the initial concentration of the biomass in the reaction solution is 5 to 10 mass%, the total mass of the saccharifying enzyme is 0.1 to 3 mass% relative to 100% dry mass of the biomass, the pH of the reaction solution is 4.5 to 5.5, the temperature is 45 to 60°C, and the reaction time is 1 to 5 days.

[0064]

[14] An enzyme composition for producing cellulose nanofibers from biomass, which contains a saccharifying enzyme produced from a fungus of the genus Trichoderma and does not contain an endoglucanase belonging to the carbohydrate hydrolase family 7 (GH7) consisting of an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1.

[15] The enzyme composition described in

[14] , preferably comprising at least one cellulase selected from the group consisting of cellobiohydrolases having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2, cellobiohydrolases having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 3, and endoglucanases having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 4.

[16] The enzyme composition according to

[15] , preferably comprising a cellobiohydrolase having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2, a cellobiohydrolase having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 3, and an endoglucanase having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 4.

[17] The enzyme composition according to any one of

[14] to

[16] , preferably not containing an endoglucanase belonging to GH7.

[18] The enzyme composition according to any one of

[14] to

[17] , which preferably contains one or more other saccharifying enzymes, as long as it does not contain an endoglucanase belonging to GH7. 〔19〕The enzyme composition according to any one of 〔14〕~〔18〕, containing a group of saccharifying enzymes produced from a Trichoderma fungus lacking an endoglucanase belonging to GH7. 〔20〕The enzyme composition according to any one of 〔14〕~〔19〕, containing a composition containing a group of saccharifying enzymes recovered from a culture of a Trichoderma fungus lacking an endoglucanase belonging to GH7. 〔21〕The enzyme composition according to any one of 〔14〕~〔20〕, wherein preferably, the Trichoderma fungus is Trichoderma reesei or a mutant thereof. 〔22〕Preferably, it is an enzyme composition for the production of sugars and cellulose nanofibers from biomass, containing at least one selected from the group consisting of β-glucosidase and hemicellulase, Preferably, the sugar is at least one selected from the group consisting of glucose and xylose, The enzyme composition according to any one of 〔14〕~〔21〕.

Examples

[0065] Hereinafter, the present invention will be described in more detail with reference to examples.

[0066] Example 1: Preparation of a saccharifying enzyme solution (1) Construction of a plasmid for gene disruption <Plasmids for egl2, egl4, egl5, egl6, egl7, xyn1, xyn2, and xyn4 disruption> From the genomic DNA of Trichoderma reesei, the genes of endoglucanases 2, 4, 5, 6, 7 (egl2, egl4, egl5, egl6, egl7 respectively), and the genes of xylanases 1, 2, 4 (xyn1, xyn2, xyn4 respectively) were cloned. The DNA containing each gene and its upstream and downstream regions (for egl2, SEQ ID NO: 6; for egl4, SEQ ID NO: 7; for egl5, SEQ ID NO: 8; for egl6, SEQ ID NO: 9; for egl7, SEQ ID NO: 10; for xyn1, SEQ ID NO: 11; for xyn2, SEQ ID NO: 12; and for xyn4, SEQ ID NO: 13) were amplified by PCR using the primers for amplifying gene fragments shown in Table 1 respectively, and cloned into the HincII cleavage site of pUC118 (Takara Bio), thereby cloning each gene. Using the cloned plasmid as a template, PCR was performed using the inverse PCR primers for each plasmid shown in Table 2. Then, using the obtained fragment and acetamidase amdS (SEQ ID NO: 14) derived from Aspergillus nidurans as a template, PCR was performed using the In-Fusion primers shown in Table 3. The obtained DNA fragment was processed according to the protocol of the In-Fusion HD Cloning Kit (Takara Bio), thereby constructing plasmids for each gene disruption.

[0067]

Table 1

[0068]

Table 2

[0069]

Table 3

[0070] <Plasmid for cbh1 disruption> The cbh1 upstream region was obtained by PCR using the primers cbh1-SphI Fw and cbh1-SphI Rv shown in Table 4, with the plasmid pBcbh1up (SEQ ID NO: 16) containing a DNA fragment containing a portion of the cellobiohydrolase 1 gene (cbh1) and its upstream region inserted into the BamHI digestion site of pBluescript II KS+ (Stratagene) as a template. The resulting fragment was cloned into pT7blueT (Takara Bio) to obtain pT7cbh1. The approximately 0.4 kbp DNA fragment obtained by treating pT7cbh1 with the restriction enzymes BglII and SalI was inserted between the BglII and SalI sites of pBcbh1up to obtain the plasmid pBcbh1P. The plasmid pT7gus-1 containing the E. coli-derived β-glucuronidase gus (SEQ ID NO: 15) was digested with the restriction enzyme NcoI, blunted at the ends, and then digested with the restriction enzyme SalI to obtain a DNA fragment of approximately 1.8 kbp. pBcbh1P was treated with the restriction enzyme AscI, the ends were blunted, and then treated with the restriction enzyme SalI. The resulting approximately 1.8 kbp DNA fragment was ligated to this to obtain the plasmid pBcbh1P-gus. Plasmid pBcbh1down (SEQ ID NO: 17) was constructed by inserting a portion of cbh1 and its downstream region into the EcoRI cleavage site of pBluescript II KS+. Using this as a template, the downstream region of cbh1 was obtained by PCR using the primers cbh1-SpeI Fw and cbh1-SpeI Rv shown in Table 4. The resulting fragment was cloned into plasmid pT7blueT to obtain pT7cbh1down. pT7cbh1down was treated with restriction enzymes AscI and EcoRI, and the resulting DNA fragment was inserted between the AscI and EcoRI sites of pBcbh1down to obtain plasmid pT7cbh1T. amdS (SEQ ID NO: 14) was treated with restriction enzymes SpeI and XbaI, and the resulting fragment was inserted into the EcoRV cleavage site of pT7cbh1T to obtain the cbh1-disrupting plasmid pT7cbh1T-amdS.

[0071] [Table 4]

[0072] <Plasmid for cbh2 disruption> Using plasmid pBcbh2up (SEQ ID NO: 18), which was obtained by introducing a part of the gene (cbh2) of cellobiohydrolase 2 and its upstream region into the EcoRV cleavage site of pBluescript II KS+, as a template, the upstream region of cbh2 was obtained by PCR using the primers cbh2-NcoI Fw and cbh2-NcoI Rv shown in Table 5. The obtained fragment was cloned into plasmid pT7blueT to obtain pT7cbh2up. The fragment obtained by treating pTcbh2up with the restriction enzyme HincII was introduced into the HincII cleavage site of pT7cbh2up to obtain pTcbh2UPfull. After treating pTcbh2UPfull with BamHI, it was blunted and further cleaved with NcoI to obtain a plasmid fragment. Plasmid pT7gus-1 having β-glucuronidase gus (SEQ ID NO: 15) derived from E. coli was treated with HindIII, blunted, and further treated with NcoI. The obtained gus gene fragment was introduced into the above plasmid fragment to obtain plasmid pT7cbh2UP-gus. A plasmid pBcbh2down (SEQ ID NO: 19) was constructed by introducing a part of cbh2 and its downstream region into the HindIII cleavage site of pBluescript II KS+. Using this as a template, a downstream region of cbh2 was obtained by PCR with the primers cbh2-SpeI Fw and cbh2-SpeI Rv shown in Table 5. The resulting fragment was cloned into plasmid pT7blueT to obtain pT7cbh2down. pTcbh2down was treated with restriction enzymes ClaI and HindIII, and the resulting fragment was introduced between the ClaI site and the HindIII site of pT7cbh2down to obtain pT7cbh2downfull. After treating pT7cbh2downfull with ClaI, it was blunt-ended to obtain a plasmid fragment. amdS (SEQ ID NO: 14) was treated with restriction enzymes SpeI and XbaI, and the resulting DNA fragment of approximately 3.1 kbp was blunt-ended. This was introduced into the above plasmid fragment to obtain plasmid pT7cbh2down-amdS. pT7cbh2UP-gus was treated with restriction enzyme XbaI, then blunt-ended and treated with SpeI. A fragment obtained by treating pT7cbh2down-amdS with SpeI and ScaI was introduced into this to obtain a plasmid pT7cbh2-gus for cbh2 disruption.

[0073] [Table 5]

[0074] <Plasmid for egl1 disruption> A fragment was obtained by PCR using the primers Egl1-SspI Fw and Egl1-SspI Rv shown in Table 6, with the plasmid pEgl1E (SEQ ID NO: 20), which had been inserted into the EcoRV cleavage site of pBluescript II KS+, as a template. This fragment was cloned into the plasmid pT7blueT to obtain pT7Egl1up. The fragment excised from pT7Egl1up by digestion with the restriction enzymes NcoI and XbaI was inserted between the NcoI and XbaI sites of pEgl1E to obtain pPEgl1-2. The plasmid pEgl1SU (SEQ ID NO: 21), which had been inserted into the SalI cleavage site of pBluescript II KS+ with the upstream region of egl1, was digested with the restriction enzymes EcoRV and KpnI, and the resulting fragment was inserted between the EcoRV and KpnI sites of pPEgl1-2 to obtain plasmid pPEgl1-3. pPEgl1-3 was digested with SspI, blunted, and then digested with KpnI to obtain the egl1 upstream region. The obtained egl1 upstream region was introduced into the plasmid pT7gus-1, which had been digested with NcoI, blunted, and KpnI-treated and contained the E. coli-derived β-glucuronidase gus (SEQ ID NO: 15), to obtain the plasmid pPEgl1gus. Plasmid pEgl1KD (SEQ ID NO: 22) was constructed by inserting a portion of egl1 and its downstream region into the KpnI cleavage site of pBluescript II KS+. Using this as a template, the egl1 downstream region was extracted by PCR using the primers Egl1-SpeI Fw and Egl1-SpeI Rv shown in Table 6. The resulting fragment was cloned into pT7blueT to obtain pTEgl1-1. amdS (SEQ ID NO: 14) was digested with the restriction enzymes SpeI and XbaI, resulting in a blunt-ended fragment. This fragment was then inserted into the EcoRV cleavage site of pTEgl1-1 to obtain plasmid pTegl1-2. pTegl1-2 was digested with the restriction enzyme SpeI to obtain a fragment containing the egl1 downstream region and amdS. The resulting fragment was then inserted into the SpeI cleavage site of pPEgl1gus to construct the egl1 disruption plasmid pEgl1GUS.

[0075] [Table 6]

[0076] <Plasmid for egl3 disruption> Using plasmid pL1 (SEQ ID NO: 23) in which the gene of endoglucanase 3 (egl3) and its upstream region were introduced into the BamHI cleavage site of pBluescript II KS+ as a template, a DNA fragment of the egl3 upstream region was obtained by PCR using the primers Egl3-NcoI Fw and Egl3-NcoI Rv shown in Table 7. The obtained fragment was cloned into pT7blueT to obtain pT7pegl3. pL1 was treated with restriction enzymes BamHI and XbaI to disrupt the restriction enzyme NcoI recognition sequence present in the obtained fragment, and then introduced between the BamHI site and the XbaI site of pT7pegl3 to obtain pLPegl3. A part of egl3 and its downstream region were introduced between the PstI site and the SalI site of pBluescript II KS+ to obtain plasmid pL301 (SEQ ID NO: 24). In pL301, the recognition sequences of restriction enzymes BamHI, XbaI, and NcoI present in the egl3 downstream region, and the recognition sequence of restriction enzyme ApaI present in the plasmid were disrupted to obtain plasmid pL301dBXNA. Using pL301dBXNA as a template, PCR was performed using the primers Egl3-SpeI Fw and Egl3-SpeI Rv shown in Table 7. The obtained fragment was treated with SpeI and ApaI and introduced between the SpeI site and the ApaI site of pL301dBXNA to obtain plasmid pLTegl3. Fragments obtained by treating pLPegl3 with BamHI and NcoI, and fragments obtained by treating pLTegl3 with SpeI and SalI were obtained. These fragments were respectively introduced between the BamHI site and the NcoI site, and between the SpeI site and the SalI site of plasmid pT7gus-1 having β-glucuronidase gus (SEQ ID NO: 15) derived from E. coli to obtain pGEFL. pGEFL was treated with restriction enzyme XhoI to make it blunt-ended to obtain a plasmid fragment. A fragment obtained by treating amdS (SEQ ID NO: 14) with restriction enzymes SpeI and XbaI was made blunt-ended. This was introduced into the above plasmid fragment to obtain the plasmid pGEFLS for egl3 disruption.

[0077]

Table 7

[0078] <Plasmid for xyn3 disruption> For the plasmid pBxyn3S’ (SEQ ID NO: 25) in which the gene (xyn3) of xylanase 3 and its upstream region n3 and downstream region were introduced into the SalI cleavage site of pBluescript II SK+, the restriction enzyme EcoRI recognition sequence existing in the downstream region of xyn3 was disrupted to obtain the plasmid pBxyn3SE’. After treating pBxyn3SE’ with the restriction enzyme NarI and then blunting it, a plasmid fragment was obtained. The fragment obtained by treating amdS (SEQ ID NO: 14) with the restriction enzymes SpeI and XbaI was blunted. This was introduced into the above plasmid fragment to obtain the plasmid pBxyn3amdS. Using pBxyn3S’ as a template, a fragment of the upstream region of xyn3 was obtained by PCR using the primers xyn3-EcoRI(U) and xyn3-NcoI shown in Table 8. The obtained fragment was cloned into pT7blueT (Takara Bio) to obtain pTxyn3P. Using pBxyn3S’ as a template, a fragment of the downstream region of xyn3 was obtained by PCR using the primers xyn3-SpeI and xyn3-EcoRI(D) shown in Table 8. The obtained fragment was cloned into pT7blueT to obtain pTxyn3T. pTxyn3P was treated with BamHI and NcoI, and the obtained fragment was introduced between the BamHI site and the NcoI site of the plasmid pT7gus-1 having β-glucuronidase gus (SEQ ID NO: 15) derived from E. coli to obtain the plasmid pTgus-2. pTxyn3T was treated with SpeI and EcoRI, and the obtained fragment was introduced between the SpeI site and the EcoRI site of pTgus-2 to obtain the plasmid pTgus-3. pTgus-3 was treated with EcoRI and NsiI, and the obtained fragment was introduced between the EcoRI site and the NsiI site of pBxyn3amdS to obtain the plasmid for xyn3 disruption pBxyn3ag-D0.

[0079]

Table 8

[0080] (2) Preparation of transformants Trichoderma reesei strain PC-3-7 was transformed with the gene disruption plasmid constructed in (1) above. The plasmid was introduced into the cells using the protoplast PEG method. The plasmid was linearized with a restriction enzyme before use. Transformants were selected on selective medium containing acetamide as the sole nitrogen source (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, 0.06% acetamide, and 0.1% Trace element 1; all percentages are w / v%). Trace element 1 consisted of the following: 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 stably retaining the target gene were selected by colony PCR.

[0081] (3) Cultivation of transformants Spores of the strain selected in (2) were added to Avicel medium (1% Avicel (Sigma-Aldrich), 0.14% (NH4)2SO4, 0.2% KH2PO4, 0.03% CaCl2·2H2O, 0.03% MgSO4·7H2O, 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 with shaking at 28°C for 5 days. The composition of Trace element 2 is as follows: 6 mg H3BO3, 26 mg (NH4)6Mo7O 24 ·4H2O, 100mg FeCl3·6H2O, 40mg CuSO4·5H2O, 8mg MnCl2·4H2O, and 200mg ZnCl2, make up to 100mL with distilled water.

[0082] (4) Preparation of saccharification enzyme solution The culture obtained in (3) above was centrifuged and then filtered to obtain a culture supernatant. The obtained culture supernatant was used as a saccharification enzyme solution in the following examples. The protein concentration of the saccharification enzyme solution was quantified by the Bradford method. Using the Quick Start Protein Assay (BioRad) 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.

[0083] Reference Example 1: Preparation of biomass Rice straw pulp (manufactured by Toho Tokushu Pulp Co., Ltd.) was used as the biomass. The pulp formed into a sheet was sheared into approximately 5 cm x 2 cm pieces using scissors. The sheared pulp was pulverized into powder using a hammer crusher (NH-34S; Sansho Industry Co., Ltd.) equipped with a mesh screen (mesh diameter 3 mm). Composition analysis of the rice straw pulp was performed by Tokai Techno Co., Ltd. The composition of the rice straw pulp used in this example was as follows: Glucose 78.8(wt)% Xylose 18.7(wt)% (Cellulose 70.9(wt)%) (Xylan 16.5(wt)%) Acid-insoluble lignin 0.8(wt)% Ash content 1.7(wt)%

[0084] Reference Example 2: Measurement of saccharification rate The saccharification rate (%) of biomass was calculated as follows: The glucose and xylose concentrations (mg / mL) in the supernatant after the saccharification reaction were measured using an enzyme electrode (BF-7; Oji Scientific Instruments Co., Ltd.), and the amount of decomposed holocellulose (cellulose + xylan) was calculated based on this. The ratio (%) of decomposed holocellulose to the holocellulose contained in the biomass was calculated as the biomass saccharification rate (%) according to the following formula: Biomass saccharification rate (%) = ([glucose concentration in the supernatant (mg / mL)] + [xylose concentration in the supernatant (mg / mL)]) × 0.9 / ([initial biomass concentration in the reaction solution (mg / mL)] × [amount of holocellulose in the biomass (%)] / 100) × 100 The amount of holocellulose in the biomass was calculated using the following value for rice straw pulp: [Amount of holocellulose in the biomass (87.4%) = Amount of cellulose (70.9%) + Amount of hemicellulose (xylan) (16.5%)]

[0085] Reference Example 3: Shape analysis of nanocellulose The morphology of the produced nanocellulose was analyzed as follows. (1) Pretreatment The saccharification residue in the reaction solution was subjected to bead crushing or ultrasonic treatment to eliminate aggregation of the nanocellulose contained therein. For bead crushing, a FastPrep 24 Instrument (MP-Biomedicals) bead crusher was used, and Toraceram® (Toray Industries, Inc.) φ1.0 mm crushing beads were used, at 6.5 m / sec for 1 minute. For ultrasonic treatment, an ultrasonic cleaning device W-113 (Honda Electronics Co., Ltd.) was used, at 100 W, 28 kHz, and 30 minutes. The saccharification residue after pretreatment was used to measure the nanocellulose fiber length and fiber diameter as described below.

[0086] (2) Measurement of nanocellulose fiber diameter The saccharification residue after pretreatment (1) was stained with uranyl acetate and imaged using a transmission electron microscope (H7600, Hitachi, Ltd., accelerating voltage 80 kV). The resulting images were converted into electronic files, and the cellulose fiber diameter was measured using Image J (public domain, ver. 2.0.0-rc-69 / 1.52p). The fiber diameters of approximately 100 cellulose fibers per sample were determined, and the median was calculated.

[0087] (3) Measurement of nanocellulose fiber length The particle size distribution of the saccharification residue after pretreatment (1) was measured using a laser diffraction particle size distribution analyzer (SALD-2100; Shimadzu Corporation), and a histogram was created with the relative particle amount on the vertical axis and particle size on the horizontal axis. The median particle size of the sample was taken as the median fiber length of the nanocellulose.

[0088] Example 2: Production of sugar and nanocellulose from biomass using saccharification enzymes from enzyme gene-deficient strains A test for the simultaneous production of sugar and nanocellulose was conducted using the saccharification enzymes prepared in Example 1. The saccharification enzyme solutions obtained from the cultures of the 13 strains lacking saccharification-related enzyme genes prepared in Example 1 and the control saccharification enzyme solution obtained from PC-3-7 (parent strain) were used. In a 20 mL plastic tube, 2 mL of a solution containing biomass (rice straw pulp from Reference Example 1, 50 mg / mL (dry weight)) as a substrate, saccharification enzyme solution (3 mg protein per 1 g dry weight biomass), and Aspergillus acritus-derived β-glucosidase (final concentration 10 μg / mL), and a final concentration of 50 mM sodium acetate buffer (pH 5.0) was reacted for 7 days with rotary shaking at 50 °C and 150 rpm. The reaction solution was then heat-treated at 98 °C for 5 minutes to inactivate the enzyme. The biomass saccharification rate in the reaction solution was determined using the procedure described in Reference Example 2. In addition, the saccharification residue during the reaction was subjected to ultrasonic treatment using the procedure of Reference Example 3, and the fiber diameter and fiber length of the nanocellulose contained therein were measured.

[0089] The saccharification rate and the measurement results of nanocellulose fiber diameter and fiber length are shown in Table 9. With the saccharification enzyme (control) obtained from the PC-3-7 strain (parent strain), the biomass saccharification rate was 91.2%, and the nanocellulose fiber diameter and fiber length were 29.7 nm and 0.4 μm, respectively. This indicates that the nanocellulose yield was low due to the relatively high sugar yield (see Example 5), and that it was difficult to produce fine fibrous nanocellulose. On the other hand, with the saccharification enzyme obtained from the PCΔegl1 strain, an EG1-deficient strain in which the egl1 gene of the PC-3-7 strain was deleted, the biomass saccharification rate was reduced to 76.9%, thereby improving the nanocellulose yield. The nanocellulose produced was finely fibrous with a fiber diameter of 19.3 nm and a fiber length of 1.1 μm. This indicates that, of the 14 saccharification enzymes examined, this enzyme was the most efficient at producing fine fibrous nanocellulose (cellulose nanofibers).

[0090] [Table 9]

[0091] Example 3: Production of sugar and nanocellulose from biomass using saccharification enzymes from enzyme gene-deficient strains A test for simultaneous production of sugar and nanocellulose was conducted using the same procedure as in Example 2, using the saccharification enzyme solution (6 mg protein per 1 g dry weight of biomass) obtained from the PCΔegl1 and PCΔegl2 strains prepared in Example 1 as the saccharification enzyme. The biomass saccharification rate in the reaction solution after 1, 3, and 7 days of reaction was determined using the procedure in Reference Example 2. Furthermore, using the procedure in Reference Example 3, the saccharification residue was subjected to a bead crushing treatment, and the fiber diameter of the fine fibrous nanocellulose was measured.

[0092] The measurement results for the saccharification rate and nanocellulose fiber diameter are shown in Table 10. With the saccharification enzyme obtained from the PCΔegl1 strain, the biomass saccharification rate after 7 days of reaction was 83.3%, and the nanocellulose fiber diameter was 10.0 nm, indicating that the desired nanocellulose was obtained in relatively high yield. On the other hand, with the saccharification enzyme obtained from the PCΔegl2 strain, the biomass saccharification rate after 7 days of reaction was 95.8%, and the nanocellulose fiber diameter was 37.7 nm, indicating that the nanocellulose size was relatively large and the yield was low. Furthermore, with the saccharification enzyme obtained from the PCΔegl2 strain, the nanocellulose size was relatively large under all conditions, and fine fibers were not obtained.

[0093] [Table 10]

[0094] Example 4: Effect of saccharification enzyme amount on the production of sugar and nanocellulose from biomass A test for simultaneous production of sugar and nanocellulose was carried out using the saccharification enzyme solutions obtained from the PCΔegl1 and PCΔegl2 strains prepared in Example 1 as the saccharification enzyme, with the exception that the enzyme amount was changed to 3 mg protein, 4.5 mg protein, or 6 mg protein per gram of dry weight biomass and the reaction period was changed to 3 days, following the same procedure as in Example 2. The biomass saccharification rate in the reaction solution was determined using the procedure in Reference Example 2. Furthermore, the saccharification residue was subjected to a bead crushing treatment using the procedure in Reference Example 3, and the nanocellulose fiber diameter and fiber length were measured.

[0095] The saccharification rate and the measurement results of nanocellulose fiber diameter and fiber length are shown in Table 11. When the saccharification enzyme obtained from the PCΔegl1 strain was used, a saccharification rate of 78.1% was achieved and fine CNF with a cellulose fiber diameter of 12.3 nm and a fiber length of 0.7 μm was obtained when the enzyme amount was 6 mg per gram of biomass. On the other hand, when the enzyme amount was 3 mg, larger CNF with a fiber diameter of 22.1 nm and a fiber length of 2.2 μm was obtained. From these results, it was found that adjusting the enzyme concentration during processing makes it possible to prepare nanocellulose with longer or shorter fiber diameters and lengths. On the other hand, when the saccharification enzyme obtained from the PCΔegl2 strain was used, the shape of the nanocellulose produced did not depend on the enzyme concentration.

[0096] [Table 11]

[0097] Example 5: Correlation between biomass saccharification rate and saccharification residue in the production of sugar and nanocellulose from biomass The correlation between the estimated residue amount from the biomass saccharification rate and the actual measured residue amount in the reaction solution was verified. A sugar and nanocellulose co-production test was conducted using the same procedure as in Example 2, except that the reaction system was 20 mL. The saccharification enzyme solution (1, 3, 4, 6, or 8 mg of protein per 1 g of biomass dry weight) obtained from the PCΔegl1 strain prepared in Example 1 and PC-3-7 (parent strain) was used, and the reaction period was 3 days. The reaction solution was centrifuged (12,000 × g, 4°C, 15 minutes) to separate the supernatant and residue. A portion of the supernatant was collected, and the biomass saccharification rate was determined using the procedure in Reference Example 2. The residue was freeze-dried and weighed. The results are shown in Table 12.

[0098] [Table 12]

[0099] The residue amount and biomass saccharification rate shown in Table 12 were plotted to obtain an approximate straight line. The approximate straight line obtained was as follows: y = -10.44x + 1047, R 2 = 0.87 (x: biomass saccharification rate, y: residue volume, R 2 : Coefficient of determination) The amount of residue showed a high negative correlation with the saccharification rate. These results confirmed that the lower the saccharification rate of nanocellulose biomass, the higher the amount of nanocellulose produced.

[0100] Example 6: Crystallization Index (CrI) of nanocellulose in saccharification residue The crystallization index (CrI) of nanocellulose was determined using the Segal method (Textile Res. J. 1959, 29:786-794). The crystallization of nanocellulose is thought to affect the strength and elastic modulus of materials containing it. In the Segal method, the X-ray diffraction profile of amorphous cellulose using CuKα radiation (wavelength: 0.1542 nm) exhibits a broad peak with a maximum at 2θ = 18°. The intensity at this angle is used as a representative value for the amorphous component, and the peak intensity appearing around 2θ = 22.8° is used as a representative value for the crystalline component. The crystallization index is calculated from the ratio of these intensities using the following formula: CrI = (X-ray intensity at 2θ = 22.8° - X-ray intensity at 2θ = 18°) / X-ray intensity at 2θ = 22.8° × 100

[0101] A reaction solution was prepared using the saccharifying enzyme solution derived from the PCΔegl1 strain prepared in Example 1, following the same procedure as in Example 2 (Δegl1-treated group). An enzyme-untreated group was also prepared as a control. The reaction solution was centrifuged (12,000 × g, 4°C, 15 minutes) to recover the supernatant and residue. The biomass saccharification rate and fiber length of the enzyme-treated reaction solution were determined according to the procedures in Reference Examples 2 and 3. The saccharification rate was 89.7%, and the fiber length was 0.8 μm. The remaining residue was freeze-dried and then ground in a mortar. The resulting pulverized material was used as a sample for X-ray diffraction. The sample was measured using a powder X-ray diffractometer (Miniflex II; Rigaku Corporation), and the crystallization index was calculated from the obtained X-ray diffraction profile.

[0102] The results are shown in Table 13. The CrI of the saccharification residue was 62.8% in the untreated group and 72.3% in the Δegl1-treated group. The saccharification residue obtained by reacting biomass with the saccharification enzyme derived from the PCΔegl1 strain had a higher CrI than the residue without enzyme treatment, demonstrating its usefulness as a raw material for high-strength, high-elasticity materials.

[0103] [Table 13]

[0104] Example 7: Enzyme composition of saccharification enzyme solution The saccharifying enzymes contained in the saccharifying enzyme solutions obtained from the cultures of the PC-3-7, PCΔegl1, and PCΔegl2 strains were comprehensively quantified by two-dimensional electrophoresis. Auto 2D (SHARP) was used for two-dimensional electrophoresis. Two-dimensional electrophoresis consisted of isoelectric focusing over a pH range of 4 to 7, followed by SDS-PAGE on a 10% acrylamide gel. After electrophoresis, the gel was stained with SYPRO Ruby (Invitrogen) and subjected to fluorescence detection using a ChemiDoc (Bio-Rad). Gel images were captured using a GelDoc EZ imager (Bio-Rad), and the spot intensities were quantified. The enzyme component ratios calculated from the spot intensities are shown in Table 14. It was confirmed that the enzyme produced by the PCΔegl1 strain did not contain EG1, while the enzyme produced by the PCΔegl2 strain did not contain EG2.

[0105] [Table 14]

[0106] While the embodiments of the present invention have been described above, it should be understood that they are not intended to limit the invention to the particular embodiments described. Various other changes and modifications within the scope of the present invention will be apparent to those skilled in the art. The publications and patent applications cited herein are incorporated by reference as if fully set forth herein.

Claims

1. A method for producing cellulose nanofibers from biomass, comprising: The method comprises reacting cellulosic biomass with an enzyme composition containing cellulase from a fungus of the genus Trichoderma, The cellulase of the Trichoderma fungus includes a cellobiohydrolase having an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 2, a cellobiohydrolase having an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 3, and an endoglucanase having an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 4, The enzyme composition does not contain an endoglucanase belonging to glycosidase family 7 (GH7) consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

1. method.

2. The method of claim 1 , wherein the enzyme composition does not contain an endoglucanase belonging to GH7.

3. 3. The method according to claim 1, wherein the enzyme composition contains a group of saccharifying enzymes produced by a fungus of the genus Trichoderma that lacks an endoglucanase belonging to GH7.

4. The method according to any one of claims 1 to 3, wherein the enzyme composition contains a composition comprising a group of saccharifying enzymes recovered from a culture of a Trichoderma fungus lacking an endoglucanase belonging to GH7.

5. The method according to any one of claims 1 to 4, wherein the Trichoderma fungus is Trichoderma reesei or a mutant thereof that expresses the Trichoderma fungus cellulase.

6. The method according to any one of claims 1 to 5, wherein the enzyme composition comprises at least one selected from the group consisting of β-glucosidase and hemicellulase, and sugar is produced from the cellulosic biomass together with cellulose nanofibers.

7. The method according to claim 6, further comprising recovering cellulose nanofibers and sugars separately from the reaction product obtained by the reaction of the cellulosic biomass with the enzyme composition by solid-liquid separation.

8. An enzyme composition for producing cellulose nanofibers from cellulosic biomass, comprising a saccharifying enzyme produced from a fungus of the genus Trichoderma, and the saccharifying enzymes include a cellobiohydrolase consisting of an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 2, a cellobiohydrolase consisting of an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 3, and an endoglucanase consisting of an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 4, The enzyme composition does not contain an endoglucanase belonging to glycosidase family 7 (GH7) consisting of an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

1. Enzyme composition.

9. The enzyme composition according to claim 8, which contains at least one enzyme selected from the group consisting of β-glucosidase and hemicellulase, and is an enzyme composition for producing sugars and cellulose nanofibers from cellulosic biomass.

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