Inducer for protein production

Abstract

Plant fibers derived from blended fibers containing plant fibers and fibers other than plant fibers have excellent protein induction ability in protein production by filamentous fungus culture and thus can be used suitably as an inducer for enhancing the productivity of protein production by filamentous fungus culture.

Description

Inducers for protein production

[0001] The present invention relates to an inducer for protein production by filamentous fungal culture and a method for producing a protein by filamentous fungal culture using the inducer.

[0002] Filamentous fungi, such as those of the genus Trichoderma and Talaromyces, are known to have high productivity in protein production, and since genetic recombination techniques for filamentous fungi have been established and they can be modified to produce any protein by genetic recombination, they are attracting attention as platform microorganisms for protein production (Non-Patent Document 1). Therefore, currently, active research is being conducted into improving the productivity of protein production by culturing filamentous fungi.

[0003] Representative examples of approaches to increasing the productivity of protein production by filamentous fungal culture include genetic modification of the genome of filamentous fungi involved in protein production and optimization of filamentous fungal culture conditions for protein production. In addition to these approaches, for example, filamentous fungi of the genus Trichoderma have the technical feature that the productivity of cellulase, which is classified as a saccharifying enzyme, is improved when cultured with inducers such as glucose, lactose, cellulose, and xylan (Non-Patent Document 2). Based on this technical feature, the development of inducers that increase the productivity of protein production by filamentous fungal culture is also underway. Furthermore, in the development of inducers, acquisition cost is also an important evaluation criterion in development from the perspective of reducing protein production costs.

[0004] Specific examples of inducers for protein production by filamentous fungal culture, which have been discovered through development aimed at improving productivity and reducing costs in protein production, include lignocelluloses such as rice straw (Patent Document 1), corn hulls (Patent Document 2), and cotton fibers (Non-Patent Document 3).

[0005] International Publication No. WO 2013 / 190064 International Publication No. WO 2021 / 235419

[0006] Nevalainen, Helena, and Robyn Peterson. “Heterologous expression of proteins in Trichoderma.”Biotechnology and biology of Trichoderma. Elsevier, 2014.89-102. Pirayre, Aurelie, et al. “Glucose-lactose mixture feeds in industry-like conditions: a gene regulatory network analysis on the hyperproducing Trichoderma reesei strain Rut-C30.”BMC genomics 21 (2020): 1-17. Allen, A. L. , and C. D. Roche. “Effects of strain and fermentation conditions on production of cellulase by Trichoderma reesei.” (1989): 650-656.

[0007] The present invention addresses the problem of providing a novel inducer for increasing the productivity of protein production by filamentous fungal culture.

[0008] The present inventors came up with the idea of ​​using textile products used in clothing, etc. as raw materials for inducers because of their ease of availability, and after extensive research, found that plant fibers derived from blended fibers containing plant fibers and fibers other than plant fibers are unexpectedly superior inducers to plant fibers derived from plant fibers that are not blended fibers, and that they are particularly superior inducers for producing cellulase by culturing filamentous fungi of the genus Trichoderma or Talaromyces, thereby completing the present invention.

[0009] That is, the present invention comprises the following (1) to (26). (1) An inducer for protein production by filamentous fungal culture, comprising, as an active ingredient, plant fiber derived from a blended fiber containing plant fiber and a fiber other than plant fiber. (2) The inducer according to (1), wherein the plant fiber is seed hair fiber, bast fiber, leaf vein fiber, or fruit fiber. (3) The inducer according to (2), wherein the seed hair fiber is cotton fiber. (4) The inducer according to (1), wherein the fiber other than plant fiber is a chemical fiber. (5) The inducer according to (4), wherein the chemical fiber is a chemical fiber containing at least polyester fiber. (6) The inducer according to any one of (1) to (5), comprising, as an active ingredient, plant fiber obtained by treating the blended fiber with an acid, a cellulase, or a subcritical hydrolysis. (7) The inducer according to (6), wherein the acid is an inorganic acid or an organic acid. (8) The inducer according to (7), wherein the inorganic acid is phosphoric acid. (9) The inducer according to (7), wherein the organic acid is a divalent or higher carboxylic acid. (10) The inducer according to (9), wherein the divalent or higher carboxylic acid is citric acid or oxalic acid. (11) A culture medium comprising the inducer according to any one of (1) to (10). (12) A method for producing a protein, comprising culturing a filamentous fungus capable of producing a protein in the culture medium according to (11). (13) The method for producing a protein according to (12), wherein the protein is an enzyme. (14) The method for producing a protein according to (13), wherein the enzyme is cellulase. (15) The method for producing a protein according to any one of (12) to (14), wherein the filamentous fungus is a filamentous fungus of the genus Trichoderma or a filamentous fungus of the genus Talaromyces. (16) A protein composition comprising a protein and a fiber-derived component other than plant fiber contained in a blended fiber comprising plant fiber and a fiber other than plant fiber. (17) The protein composition according to (16), wherein the fiber-derived component other than plant fiber is a fiber fragment other than plant fiber. (18) The protein composition according to (16) or (17), wherein the fiber other than plant fiber is a chemical fiber. (19) The protein composition according to (18), wherein the chemical fiber is a polyester fiber. (20) The protein composition according to any one of (16) to (19), wherein the protein is an enzyme.(21) The protein composition according to (20), wherein the enzyme is cellulase. (22) A method for producing a sugar solution, comprising a step of hydrolyzing cellulose with cellulase obtained by the method according to (14) or the protein composition according to (21). (23) A method for producing a sugar solution according to (22), wherein the cellulose is cellulose contained in a blended fiber containing plant fibers and fibers other than plant fibers. (24) A method for recovering fibers other than plant fibers from a blended fiber containing plant fibers and fibers other than plant fibers, comprising a step of hydrolyzing a blended fiber containing plant fibers and fibers other than plant fibers with cellulase obtained by the method according to (14) or the protein composition according to (21), and a step of removing the hydrolyzed plant fibers from the treated product obtained in the hydrolysis treatment step. (25) A method for producing a chemical product, comprising a step of culturing a microorganism capable of producing a chemical product using the sugar solution obtained by the method according to (22) or (23) as a fermentation feedstock. (26) A method for producing a protein, comprising a step of culturing a microorganism capable of producing a protein using the sugar solution obtained by the method according to (22) or (23) as a fermentation raw material.

[0010] The inducer of the present invention has excellent protein induction ability in protein production by filamentous fungal culture, and can therefore be used as an inducer for increasing the productivity of protein production. In particular, in cellulase production by culturing filamentous fungi of the genus Trichoderma or Talaromyces, the inducer can be used as an inducer for producing cellulase with excellent enzymatic activity.

[0011] Micrograph of a Trichoderma culture stained with Congo red

[0012] The present invention is characterized by the use of plant fibers derived from a blended fiber containing plant fibers and fibers other than plant fibers as an active ingredient of an inducer for protein production by filamentous fungal culture. The inducer here refers to an additive that promotes protein production by a filamentous fungus when the filamentous fungus is cultured to produce the protein.

[0013] Blended fibers are a general term for fibers spun by mixing two or more different types of staple fibers. Specific examples of blended fibers include blended fibers made by mixing different types of natural fibers, blended fibers made by mixing natural fibers and chemical fibers, and blended fibers made by mixing different types of chemical fibers. However, the blended fibers used in the present invention are blended fibers made by mixing natural plant fibers with fibers other than plant fibers.

[0014] The plant fibers contained in the blended fibers used in the present invention are fibers whose main component is cellulose, which is the skeletal component of plant cell walls, and examples thereof include seed hair fibers, bast fibers, leaf vein fibers, and fruit fibers derived from plant tissues, as well as cellulose fibers obtained by melt-spinning cellulose, with seed hair fibers being preferred. Specific examples of seed hair fibers include cotton fiber, kapok fiber, and birch fiber, with cotton fiber being preferred.

[0015] The fibers other than plant fibers contained in the blended fibers used in the present invention are not particularly limited and may be natural fibers other than plant fibers, specifically animal fibers or mineral fibers, or may be chemical fibers, but are preferably chemical fibers. Specific examples of chemical fibers include one or more types selected from the group consisting of polyester fibers, polyurethane fibers, polyamide fibers, and polyacrylonitrile fibers (acrylic fibers). Chemical fibers containing at least polyester fibers are preferred, polyester fibers and / or polyurethane fibers are more preferred, and polyester fibers are even more preferred. Specific examples of polyester fibers include polyethylene terephthalate fibers (PET fibers), polybutylene terephthalate fibers (PBT fibers), polytrimethylene terephthalate fibers (PTT fibers, also known as 3GT fibers), and polylactic acid fibers (PLA fibers), but PET fibers are preferred.

[0016] One of the most preferred combinations of fibers constituting the blended fiber used in the present invention is a blended fiber of cotton fiber, which is a vegetable fiber, and PET fiber, and the blended fiber of cotton fiber and PET fiber is sometimes commonly called a T / C blended fiber.

[0017] The plant fiber derived from the blended fiber, which is an active ingredient of the inducer of the present invention, is the plant fiber separated from the blended fiber. The treatment method for separating the blended fiber into the plant fiber and the other fiber is not particularly limited, and methods known to those skilled in the art include acid treatment, subcritical hydrolysis treatment, enzyme treatment using cellulase or the like, microwave treatment, and glycolysis treatment. In the present invention, the acid treatment in which the blended fiber is immersed in an acidic aqueous solution, cellulase treatment, and subcritical hydrolysis treatment are preferably applied.

[0018] The acid used in the acid treatment is not particularly limited and may be an inorganic acid or an organic acid. Among inorganic acids, preferred are phosphoric acid, hydrochloric acid, sulfuric acid, nitric acid, boric acid, and hydrofluoric acid, more preferably phosphoric acid. Among organic acids, preferred are divalent or higher carboxylic acids, more preferably citric acid, oxalic acid, formic acid, acetic acid, lactic acid, glycolic acid, malonic acid, succinic acid, malic acid, and tartaric acid, and even more preferably citric acid and oxalic acid. The acid treatment is preferably carried out while heating, and the temperature conditions during the acid treatment are preferably 50 to 130°C, more preferably 60 to 120°C, and even more preferably 70 to 120°C. The pH conditions are preferably pH 5.0 or lower, more preferably pH 4.0 or lower, and even more preferably pH 3.0 or lower.

[0019] The cellulase used in the cellulase treatment is not particularly limited and is characterized by including cellulases and hemicellulases having cellulose and hemicellulose hydrolysis activity. Specific examples of cellulases include endoglucanases (EC 3.2.1.4), which hydrolyze cellulose from the inside, cellobiohydrolases (EC 3.2.1.91), which hydrolyze cellulose and cellooligosaccharides from the terminals to release cellobiose, and β-glucosidases (EC 3.2.1.21), which hydrolyze cellulose and cellooligosaccharides from the terminals to release glucose. Specific examples of hemicellulases that can be included in the cellulases include endoxylanases (EC 3.2.1.8), which hydrolyze xylan from the inside, and β-xylosidases (EC 3.2.1.37), which hydrolyze xylooligosaccharides from the terminals. Furthermore, the cellulases are characterized by being cellulases derived from filamentous fungi. The cellulase derived from filamentous fungi may be a commercially available cellulase preparation, or the culture solution of a filamentous fungus of the genus Talaromyces or a filamentous fungus of the genus Trichoderma may be used directly without removing the fungal cells from the culture solution, or a crude cellulase obtained by simply removing the fungal cells from the culture solution of a filamentous fungus of the genus Talaromyces or a filamentous fungus of the genus Trichoderma through solid-liquid separation may be used, or the culture solution of a filamentous fungus of the genus Talaromyces and a filamentous fungus of the genus Trichoderma may be subjected to solid-liquid separation, and the filtrate may be mixed with purified cellulase that has been treated with a membrane or column.Examples of commercially available cellulase preparations include cellulase preparations derived from Talaromyces fungi, such as Acremonium Cellulase (Meiji Seika Pharma) derived from Talaromyces cellulolyticus and Filtrase NL (DSM) derived from Talaromyces emersonii, and cellulase preparations derived from Trichoderma fungi, such as Meicelase (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), and Trichoderma Examples of such enzymes include sucrase X (Mitsubishi Chemical), sucrase C (Mitsubishi Chemical), and citrase CL (DSM) derived from Trichoderma reesei, cellulosin TP25 (HBI), Optimase CX (Danisco Japan), Multifect GC (Danisco Japan), Multifect B (Danisco Japan), GODO-TCF (Goudou Shusei), GODO-TCL (Goudou Shusei), Besselex (Goudou Shusei), and Bakezyme X-CELL (DSM).

[0020] The culture method for Talaromyces and Trichoderma filamentous fungi is not particularly limited as long as it can produce a culture solution having cellulase activity. For example, culture can be performed by liquid culture using centrifuge tubes, flasks, jar fermenters, tanks, etc., or solid culture using plates, etc. Filamentous fungi must be cultured under aerobic conditions. Among these culture methods, submerged culture in a jar fermenter or tank, in which culture is performed while aerating and stirring, is particularly preferred. The aeration rate is preferably 0.1 to 2.0 vvm, more preferably 0.3 to 1.5 vvm, and particularly preferably 0.5 to 1.0 vvm. The culture temperature is preferably 25 to 35°C, more preferably 25 to 31°C. The pH condition during culture is preferably 3.0 to 7.0, more preferably 4.0 to 6.0. The culture is performed under conditions sufficient for protein production until a recoverable amount of protein is accumulated. Usually, it is about 24 to 288 hours, preferably 36 to 240 hours.

[0021] By subjecting the acid- or cellulase-treated blended fiber to a physical action in water, powdered plant fiber can be liberated from the blended fiber, and the plant fiber can be suitably used as the inducer of the present invention. Agitation using a stirrer, turbine, or the like is preferred as a method for applying a physical action in water. By filtering the treated product after physical action in water using a sieve, the chemical fiber can be separated, and the powdered plant fiber can be recovered as a filtrate. The filtrate containing the separated plant fiber powder can be used as an inducer directly, or the filtrate can be dried to recover the plant fiber powder and then used as the active ingredient of the inducer.

[0022] The subcritical hydrolysis treatment is a treatment in which the blended fibers are brought into contact with subcritical water to decompose fibers other than plant fibers contained in the blended fibers and separate the plant fibers contained in the blended fibers. In the present invention, the subcritical hydrolysis treatment using an alkaline component described in WO 2019 / 140245 is suitable. Specifically, blended fibers containing plant fibers and fibers other than plant fibers are treated in a subcritical water reactor using an alkaline component at a temperature of 105 to 190°C, with the lower limit of the temperature being preferably 138°C or higher, more preferably 140°C or higher, at a pressure of 0.2 to 10 MPa, with the lower limit of the pressure being preferably 0.3 MPa or higher, more preferably 0.4 MPa or higher, and the upper limit of the pressure being preferably 8.0 MPa or lower, more preferably 6.0 MPa or lower, for a time of 0 to 120 minutes, with the lower limit of the time being preferably 5 minutes or higher, more preferably 10 minutes or higher, and the upper limit of the time being preferably 180 minutes or lower, more preferably 120 minutes or shorter, thereby eluting the fibers other than the plant fibers contained in the blended fibers, and plant fibers suitable for use as the inducer of the present invention can be recovered.

[0023] Examples of alkaline components used in subcritical hydrolysis include alkali metals such as lithium, sodium, and potassium, alkaline earth metals such as magnesium and calcium, hydroxides such as ammonium, carbonates, bicarbonates, ammonia, and organic amines such as trimethylamine. Of these, alkali metal salts are preferred.

[0024] Examples of alkali metal salts include alkali metal hydroxides such as lithium hydroxide, sodium hydroxide, potassium hydroxide, and cesium hydroxide; alkali metal carbonates such as lithium carbonate, sodium carbonate, potassium carbonate, cesium carbonate, lithium hydrogencarbonate, sodium hydrogencarbonate, potassium hydrogencarbonate, and cesium hydrogencarbonate; alkali metal phosphates such as lithium phosphate, sodium phosphate, potassium phosphate, cesium phosphate, dilithium hydrogenphosphate, dipotassium hydrogenphosphate, dicesium hydrogenphosphate, lithium monohydrogenphosphate, sodium monohydrogenphosphate, potassium monohydrogenphosphate, and cesium monohydrogenphosphate; and alkali metal borates such as lithium borate, sodium borate, potassium borate, and cesium borate. Among these, it is preferable that the alkali metal salt be at least one selected from alkali metal hydroxides and alkali metal carbonates. Specifically, lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium hydrogencarbonate, sodium hydrogencarbonate, potassium hydrogencarbonate, lithium carbonate, sodium carbonate, and potassium carbonate are more preferable.

[0025] Depending on the treatment method for the blended fibers, it is preferable to defibrate the blended fibers in advance. Specifically, when the treatment method is acid treatment or cellulase treatment, it is preferable to defibrate the blended fibers in advance. On the other hand, when the treatment method is subcritical hydrolysis treatment, it is not necessary to defibrate the blended fibers in advance, and therefore it is preferable not to defibrate them from the viewpoint of simplifying the process. There are no particular limitations on the defibration method for the blended fibers, and the fibers do not need to be completely defibrated; they can be defibrated by cutting, a pin mill, a hammer mill, a jet mill, a ball mill, or the like, or a combination of these methods can be used.

[0026] The plant fiber may contain, as a secondary component, a fiber-derived component other than the plant fiber contained in the blended fiber. The fiber-derived component other than the plant fiber contained in the blended fiber is a fragment or decomposition product of the fiber other than the plant fiber contained in the blended fiber. Specific examples of the decomposition product of the fiber other than the plant fiber contained in the blended fiber include polymers, oligomers, and monomers constituting the fiber other than the plant fiber contained in the blended fiber.

[0027] The inducer of the present invention, which contains the plant fiber as an active ingredient, can be added to a medium for culturing a filamentous fungus capable of producing a protein, thereby allowing it to function as an inducer for producing a protein.

[0028] The medium for culturing a filamentous fungus capable of producing a protein is not particularly limited as long as it has a medium composition that allows the filamentous fungus to be cultured to produce a protein, and any medium composition known to those skilled in the art can be used.

[0029] The amount of the inducer of the present invention to be added to the medium is not particularly limited as long as it is an amount that allows the inducer to function, but it is sufficient to add the inducer so that the final concentration of the plant fiber, which is an active ingredient of the inducer of the present invention, is preferably 1 to 50% by weight, more preferably 5 to 30% by weight, and even more preferably 5 to 25% by weight, based on dry weight.

[0030] The medium may contain glucose and / or lactose as a carbon source to serve as nutrients for the filamentous fungi to be cultured, and preferably contains both glucose and lactose. The amounts of glucose and / or lactose added to the medium may be such that the final concentrations of each are about 1 g / L.

[0031] Glucose and / or lactose may also be used as inducers for protein production, and in this case, they are preferably added to the medium during the culture. When glucose and / or lactose are added during the culture, preferably 10 g or more of glucose and / or 1 g or more of lactose, more preferably 25 g or more of glucose and / or 2.5 g or more of lactose, and even more preferably 50 g or more of glucose and / or 5 g or more of lactose are added per 1 L of culture solution per 24 hours of culture. The glucose and / or lactose may be added in liquid or solid form to the medium, but it is preferable to use them dissolved in water or the like.

[0032] When a sugar mixture of glucose and lactose is added during the culture, the ratio of glucose to lactose is preferably equal or the amount of glucose is greater than the amount of lactose. The timing for starting the addition of liquid sugar is preferably within 144 hours from the start of culture, more preferably within 72 hours, and particularly preferably within 48 hours. The sugar may be added once, multiple times, or continuously.

[0033] Cellulose, xylan, or the like may also be used as an inducer for inducing protein production. In this case, biomass containing cellulose or xylan may be added as an inducer. Specific examples of biomass containing cellulose or xylan include plants such as seed plants, ferns, mosses, algae, and aquatic plants, as well as waste building materials. Seed plants are classified into gymnosperms and angiosperms, and both are preferably used. Angiosperms are further classified into monocotyledons and dicotyledons. Specific examples of monocotyledons include bagasse, switchgrass, napier grass, erianthus, corn stover, corn cob, corn hull, rice straw, and wheat straw. Specific examples of dicotyledons include beet pulp, eucalyptus, oak, and birch.

[0034] When biomass containing cellulose or xylan is used as an inducer for inducing protein production, it may be pretreated. The pretreatment method is not particularly limited, and known techniques such as acid treatment, sulfuric acid treatment, dilute sulfuric acid treatment, alkali treatment, hydrothermal treatment, subcritical hydrolysis treatment, pulverization treatment, and steaming treatment can be used. Pulp may also be used as such pretreated biomass containing cellulose or xylan.

[0035] In addition, the medium may contain, for example, polypeptone, meat juice, CSL, soybean meal, etc. as a nitrogen source that serves as nutrition for the filamentous fungi being cultured.

[0036] The filamentous fungi to which the inducer of the present invention is applied are not particularly limited as long as they are filamentous fungi capable of producing proteins, but are preferably filamentous fungi of the genus Tricoderma or Talaromyces, which have high protein-producing ability.

[0037] The filamentous fungi of the genus Trichoderma are also called the genus Hypocrea, but are described herein as the genus Trichoderma. The filamentous fungi of the genus Trichoderma are preferably Trichoderma reesei, Trichoderma viride, Trichoderma atroviride, or Trichoderma longibrachiatum, and more preferably Trichoderma reesei.

[0038] Specific examples of Trichoderma reesei include Trichoderma parareesei (ATCC MYA-4777), which is an ancestor of Trichoderma reesei, and Trichoderma Examples of known mutant strains derived from B. reesei include the 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)), and MCG80 strain (Biotechnol. Bioeng., 12, 451-459 (1982)), as well as derivative strains thereof. The QM6a, QM9414, and QM9123 strains are available from the NITE Biological Resource Center (NBRC), and the RutC-30 strain is available from the American Type Culture Collection (ATCC).

[0039] The filamentous fungi of the genus Talaromyces are also called filamentous fungi of the genus Acremonium, but will be referred to as filamentous fungi of the genus Talaromyces in this specification. The filamentous fungi of the genus Talaromyces are preferably Talaromyces marneffei, Talaromyces proteolyticus, Talaromyces stipitatus, Talaromyces rugulosus, Talaromyces pinophilus, Talaromyces amestolkiae, Talaromyces atroloseus, Talaromyces verruculosus, Talaromyces islandicus, Talaromyces wortmannii, Talaromyces funiculosus, Talaromyces purpureogenus, Talaromyces variabilis, Talaromyces stollii, Talaromyces sp., Talaromyces cellulolyticus, Talaromyces emersonii or Talaromyces australis, more preferably Talaromyces cellulolyticus.

[0040] Specific examples of Talaromyces cellulolyticus include known mutant strains derived from Talaromyces cellulolyticus, such as Y-94 strain (FERM BP-5826), TN strain (FERM BP-11452), C1 strain (FERM P-18508), and CF-2612 strain (FERM BP-10848), as well as derivative strains thereof.

[0041] The filamentous fungi to which the inducer of the present invention is applied are not limited to wild-type strains; mutant strains that have been improved to enhance protein production ability can also be preferably used. For example, mutant strains that have been subjected to mutation treatment using drugs, ultraviolet irradiation, heavy ion beam irradiation, or the like, and that have reduced viscosity of the culture solution during culture or improved protein productivity can be used. Furthermore, genetically modified strains that have been cultured using genetic engineering techniques and have reduced viscosity of the culture solution or improved protein productivity can also be used. Furthermore, mutant strains obtained by combining the above-mentioned mutation treatment using drugs, ultraviolet irradiation, heavy ion beam irradiation, or the like with genetic engineering techniques can also be used.

[0042] The culture method for the filamentous fungus to which the inducer of the present invention is applicable is not particularly limited. For example, culture can be carried out by liquid culture using centrifuge tubes, flasks, jar fermenters, tanks, etc., or solid culture using plates, etc. When culture is required under aerobic conditions, among these culture methods, submerged culture in a jar fermenter or tank, in which culture is carried out while aerating and stirring, is particularly preferred. The aeration rate is preferably 0.1 to 2.0 vvm, more preferably 0.3 to 1.5 vvm, and even more preferably 0.5 to 1.0 vvm. The culture temperature is preferably 25 to 35°C, more preferably 25 to 31°C. The pH condition during culture is preferably 3.0 to 7.0, more preferably 4.0 to 6.0. The culture time is sufficient as long as culture is carried out under conditions sufficient for protein production until a recoverable amount of protein accumulates, and is typically about 24 to 288 hours, preferably 36 to 240 hours.

[0043] The protein produced using the inducer of the present invention is not particularly limited, but is suitable for producing proteins that are secreted outside the bacterial cell, and is preferably an enzyme, more preferably a saccharifying enzyme such as cellulase, amylase, invertase, chitinase, or pectinase, and even more preferably cellulase.

[0044] The cellulases produced by the present invention include various hydrolases, including enzymes with decomposition activity against xylan, cellulose, and hemicellulose. Specific examples include cellobiohydrolase (EC 3.2.1.91), which produces cellobiose by hydrolyzing cellulose chains; endoglucanase (EC 3.2.1.4), which hydrolyzes cellulose chains starting from the central portion; β-glucosidase (EC 3.2.1.21), which hydrolyzes cellooligosaccharides and cellobiose; xylanase (EC 3.2.1.8), which is characterized by acting on hemicellulose and particularly on xylan; and β-xylosidase (EC 3.2.1.37), which hydrolyzes xylooligosaccharides. According to the protein production method of the present invention, the protein concentration and activity of the above-mentioned cellulases are improved, with the effect being particularly pronounced in β-glucosidase activity.

[0045] Protein concentration is measured as follows. The culture medium obtained by culturing a Trichoderma filamentous fungus using the method of the present invention is centrifuged at 15,000 × g for 10 minutes, and the supernatant is used as a protein solution. 5 μL of the diluted cellulase solution is added to 250 μL of Cellulase Quick Start Bradford Protein Assay (Bio-Rad), and the solution is left to stand at room temperature for 15 minutes. The absorbance at 595 nm is measured after the addition. Using bovine serum albumin solution as a standard solution, the protein concentration contained in the saccharifying enzyme solution is calculated based on a calibration curve. When measuring the protein concentration of cellulase, the protein solution can be treated as a cellulase solution.

[0046] β-Glucosidase activity is measured as follows. First, 10 μL of the enzyme dilution is added to 90 μL of 50 mM acetate buffer containing 1 mM 4-nitrophenyl-β-glucopyranoside (Sigma-Aldrich Japan), and the mixture is allowed to react at 30°C for 10 minutes. Next, 10 μL of 2 M sodium carbonate is added and mixed well to stop the reaction, and the increase in absorbance at 405 nm is measured. Finally, the activity is calculated as 1 U, which is the activity that liberates 1 μmol of 4-nitrophenol per minute.

[0047] When producing a protein by culturing a filamentous fungus using the inducer of the present invention, the culture solution containing the filamentous fungal cells may be used as is as a protein composition, or, if necessary, may be subjected to a treatment that prevents the growth of the filamentous fungal cells, specifically, a treatment such as heat treatment, chemical treatment, acid / alkali treatment, or UV treatment. Alternatively, the culture solution containing the filamentous fungal cells may be subjected to a treatment such as solid-liquid separation to remove the filamentous fungal cells, and the resulting culture supernatant may be used as the protein composition. Alternatively, a purified product obtained by removing unnecessary substances other than the protein from the culture solution containing the filamentous fungal cells may be used as the protein composition.

[0048] The protein composition produced using the inducer of the present invention may contain fiber-derived components other than plant fibers contained in the blended fiber, which is the raw material for the inducer of the present invention (hereinafter referred to as "non-plant fiber-derived components"). Non-plant fiber-derived components are fragments or decomposition products of fibers other than plant fibers, and the protein composition may frequently contain non-plant fiber fragments. Whether the protein composition contains fiber fragments other than plant fibers can be confirmed visually using a microscope, and optical microscopes such as digital microscopes and stereomicroscopes, or scanning electron microscopes (SEMs) can be used as the microscope. Furthermore, whether the protein composition contains decomposition products of fibers other than plant fibers can be confirmed by detecting the decomposition products using various chromatographic methods. When a protein is produced using a method other than the protein production method using the inducer of the present invention, it is unlikely that the resulting protein composition will contain fiber-derived components other than plant fibers. Therefore, if a protein composition contains fiber-derived components other than plant fibers, the protein composition can be determined to have been produced using the inducer of the present invention.

[0049] When the target of production in the protein production method using the inducer of the present invention is cellulase, a sugar solution can be produced by hydrolyzing cellulose using the cellulase. The cellulose used as the raw material for producing the sugar solution can be cellulose contained in cellulose-containing biomass described as an inducer other than the inducer of the present invention, or cellulose contained in a blended fiber containing plant fiber and a fiber other than plant fiber. When producing a sugar solution using cellulose contained in a blended fiber containing plant fiber and a fiber other than plant fiber as the raw material, as shown in the examples described below, a blended fiber containing plant fiber and a fiber other than plant fiber is preferably used as the raw material, because hydrolysis efficiency is improved by using a blended fiber having the same composition as the blended fiber used as the raw material for the inducer of the present invention.

[0050] The conditions for the saccharification reaction are not particularly limited, but the temperature of the saccharification reaction is preferably in the range of 25 to 60°C, more preferably 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 3.0 to 7.0, more preferably 4.0 to 6.0. In the case of cellulase derived from filamentous fungi, the optimum pH for the reaction is pH 5.0. Furthermore, because pH changes occur during the hydrolysis process, it is preferable to add a buffer solution to the reaction solution or to carry out the reaction while maintaining a constant pH using an acid or alkali.

[0051] When cellulase and the like are separated and recovered from the saccharified solution, the saccharified solution can be filtered using an ultrafiltration membrane or the like, and the cellulase and the like can be recovered in the non-permeated side. If necessary, solids can be removed from the saccharified solution as a pre-filtration step. The recovered enzymes can be reused in the saccharification reaction.

[0052] Furthermore, when a blended fiber containing plant fibers and fibers other than plant fibers is treated with cellulase, the plant fibers are completely or partially hydrolyzed, while the fibers other than plant fibers remain unhydrolyzed and retain their fibrous shape. Therefore, by removing the hydrolyzed plant fibers from the treated product obtained by hydrolyzing a blended fiber containing plant fibers and fibers other than plant fibers with cellulase obtained using the inducer of the present invention, it is possible to recover fibers other than plant fibers from the blended fiber containing plant fibers and fibers other than plant fibers. Furthermore, as described above, the cellulase obtained using the inducer of the present invention has excellent hydrolysis efficiency for blended fibers having the same composition as the blended fiber used as the raw material for the inducer of the present invention, and is therefore preferably used when recovering fibers other than plant fibers from blended fibers having the same composition as the blended fiber used as the raw material for the inducer of the present invention.

[0053] When plant fibers are completely hydrolyzed, a hydrolysate containing sugar as the main component is obtained, and in this case, the non-plant fibers can be recovered by subjecting the cellulase-treated product of a blended fiber containing plant fibers and fibers other than plant fibers to a solid-liquid separation process known to those skilled in the art.When plant fibers are partially hydrolyzed, a hydrolysate containing crystalline cellulose as the main component is obtained, and in this case, the non-plant fibers can be recovered by filtering the cellulase-treated product of a blended fiber containing plant fibers and fibers other than plant fibers using a sieve or the like.

[0054] When recovering non-plant fibers from blended fibers containing plant fibers and non-plant fibers by cellulase treatment, it is preferable to perform the above-mentioned defibration treatment before the cellulase treatment. There are no particular restrictions on the cellulase treatment conditions, and the above-mentioned hydrolysis conditions for saccharifying cellulose are preferably applied.

[0055] The recovered fibers other than plant fibers can be reused according to chemical recycling methods or material recycling methods known to those skilled in the art, and in particular, if the fibers other than plant fibers are chemical fibers, they can be reused as raw materials for textile products or resin products.

[0056] Chemical products and proteins can be produced by microbial fermentation using the sugar solution obtained by the above-mentioned method as a fermentation feedstock. There are no particular limitations on the microorganisms used in producing chemical products and proteins, as long as they are capable of producing the desired chemical product or protein, and examples include yeasts such as baker's yeast, bacteria such as Escherichia coli and coryneform bacteria, filamentous fungi, and actinomycetes. Microorganisms may be isolated from the natural environment, or may have properties modified by mutation or genetic engineering.

[0057] The chemical products obtainable by microbial fermentation are not particularly limited, and specific examples include substances mass-produced in the fermentation industry, such as alcohols, organic acids, amino acids, nucleic acids, etc. For example, alcohols include ethanol, butanol, 1,3-propanediol, 1,3-butanediol, 2,3-butanediol, 1,4-butanediol, glycerol, etc.; organic acids include formic acid, acetic acid, lactic acid, succinic acid, malic acid, etc.; amino acids include lysine and glutamic acid; and nucleic acids include inosinic acid, guanylic acid, inosine, guanosine, etc.

[0058] The chemical products obtained by microbial fermentation may be used as raw materials for the final target product. A specific example is the production of aviation fuel using alcohol obtained by microbial fermentation as a raw material according to known techniques. Aviation fuels made from non-petroleum-derived chemical products are also called sustainable aviation fuels (SAFs), and are approved as Annexes 1 to 7 of the ASTM D7566 standard. By using the chemical products obtained by microbial fermentation as raw materials, SAFs conforming to any of Annexes 1 to 7 of the ASTM D7566 standard can be produced.

[0059] There are no particular limitations on the proteins obtained by the microbial fermentation, and there are no particular limitations on their uses. Specific examples include enzymes, peptides, protein materials, etc. that can be used for industrial purposes, medical purposes, or food and feed purposes.

[0060] The present invention will be specifically described below with reference to examples.

[0061] <Reference Example 1> Protein concentration measurement conditions Protein concentration measurement reagent used: Quick Start Bradford Protein Assay (Bio-Rad) Measurement conditions Measurement temperature: room temperature Protein concentration measurement reagent: 250 μL Filamentous fungus culture solution: 5 μL Reaction time: 5 minutes Absorbance: 595 nm Standard: BSA.

[0062] <Reference Example 2> Conditions for measuring β-glucosidase activity Substrate: 4-nitrophenyl-β-glucopyranoside (Sigma-Aldrich Japan) Reaction solution: 90 μL of 50 mM acetate buffer containing 1 mM 4-nitrophenyl-β-glucopyranoside Enzyme diluent: 10 μL Reaction temperature: 30°C Reaction time: 10 minutes Reaction stopper: 10 μL of 2 M sodium carbonate Absorbance: 405 nm

[0063] Reference Example 3: Preparation of Plant Fiber 1. Acid Treatment Plant fiber powder was prepared from 100% cotton fiber fabric, T / C blended fiber fabric, and absorbent cotton. The 100% cotton fiber fabric and T / C blended fiber fabric (TY_HT-8050W; Toyoshima) were each cut into approximately 1 cm square pieces with scissors and then defibrated in a blender. The defibrated material and absorbent cotton were each immersed in a 20% phosphoric acid solution, heated at 90°C for 4 hours, and then stirred with a stirrer to obtain an acid-treated material. The acid-treated material was transferred to a sieve and washed several times with water. The washings that passed through the sieve were collected and poured into a Buchner funnel lined with filter paper (FILTER PAPER 185 mm; ADVANTEC). The mixture was subjected to suction filtration using a suction pump (DAP-15; ULVAC), and the powdered plant fiber remaining on the filter paper was collected.

[0064] 2. Cellulase Treatment Plant fiber powder was prepared from T / C blended fiber fabric. The fabric was pre-cut into 1 cm squares with scissors and defibrated in a blender. Cellulase derived from the main culture of a filamentous fungus belonging to the genus Trichoderma cultured with inducer 3 in Example 1 (described below) was added to 1 kg of the defibrated material so that the protein content was 4 g. The mixture was stirred at 50°C to obtain a cellulase-treated product. Powdered plant fiber was recovered from the cellulase-treated product in the same manner as in the recovery of plant fiber powder from the acid-treated product described above.

[0065] 3. Subcritical Hydrolysis Treatment 6.0 g of T / C blended fiber fabric, 60 g of deionized water, and 2.1 g of sodium hydroxide (Fujifilm Wako Pure Chemical Industries) were added to a SUS316L autoclave equipped with a stirrer. The atmosphere inside the reaction vessel was replaced with nitrogen, and the mixture was stirred at 200 rpm at 180°C for 30 minutes under a nitrogen pressure of 1.0 MPa in a sealed container. The pressure inside the system during the reaction was 0.4 MPa. After the reaction, the mixture was cooled to 80°C, and the aqueous solution in which the plant fibers were dispersed was filtered to recover the plant fibers.

[0066] Reference Example 4 Saccharification Reaction 0.1 g of plant fiber powder derived from the T / C blended fiber fabric prepared in Reference Example 3 was used as the saccharification target, and a cellulase solution derived from the main culture broth of filamentous fungi of the genus Trichoderma obtained in Example 1 described below was added to the saccharification target so that the protein amount was 2 mg per 1 g of the target, and the resulting mixture was subjected to hydrolysis (50°C, 24 hours) using a 50 mL Thermoblock Rotator SN-06BN ​​(Nichishin Rika).

[0067] <Reference Example 5> Measurement of glucose concentration After the saccharification reaction, the sugar solution was centrifuged at 15,000 × g and 4°C for 10 minutes, and the resulting supernatant was filtered through a 0.45 µm filter. To the supernatant, 1 N NaOH solution was added to a volume of 10% to terminate the reaction, and quantitative analysis was carried out under the following conditions.

[0068] Glucose was detected using a high-performance liquid chromatograph (Prominence; Shimadzu Corporation) under the following conditions, and quantitative analysis was performed based on a calibration curve prepared with a glucose standard. Guard column: Shodex HILICpak VG-50G 4A 4.6 mm × 10 mm Column: Shodex HILICpak VG-50G 4E 4.6 mm × 250 mm Separation method: HILIC Mobile phase: MilliQ water:acetonitrile = 25:75 (v / v) Detector: RI Flow rate: 0.6 mL / min Column oven temperature: 40°C Sample dilution: 50% acetonitrile.

[0069] Example 1 Cultivation of Trichoderma genus filamentous fungi (pre-culture) 1.0 x 10 spores of Trichoderma reesei strain PC-3-7 (ATCC #66589) were cultured.７ The spores were diluted with saline to a concentration of 1 / mL, and 1 mL of the diluted spore solution was inoculated into 100 mL of pre-culture medium (Table 1) in a 500 mL baffled flask, followed by cultivation for 72 hours at 28°C and 120 rpm in a shaking incubator.

[0070]

[0071] (Main Culture) 10 mL of the preculture solution was inoculated into 100 mL of the main culture medium (Table 2) in a 500 mL baffled flask, and cultured in a shaking incubator at 28° C. and 120 rpm for 168 hours.

[0072] The various inducers added to the culture medium to induce protein production were the following inducers 1 to 8. (Inducer 1) Plant fiber powder derived from T / C blended fiber prepared by acid treatment in Reference Example 3 (Inducer 2) Lactose (Fujifilm Wako Pure Chemical Industries) (Inducer 3) Industrial pulp (Arbocel B800; Rettenmeyer Japan) (Inducer 4) Absorbent cotton (Inducer 5) Plant fiber powder derived from absorbent cotton prepared by acid treatment in Reference Example 3 (Inducer 6) 100% cotton fiber fabric (cut into approximately 1 cm squares) (Inducer 7) Plant fiber powder derived from 100% cotton fiber fabric prepared by acid treatment in Reference Example 3 (Inducer 8) Corn hull pulverized product prepared according to the method described in WO 2021 / 235419 (having a relative particle amount peak at a particle diameter of 75 μm in the volume-based particle size distribution obtained by the measurement method using the laser diffraction scattering method).

[0073]

[0074] (Collection of culture medium) 168 hours after the start of culture, 1 mL of the culture medium was collected. The culture medium was centrifuged at 15,000 × g and 4°C for 10 minutes, and the resulting supernatant was filtered through a filter with a pore size of 0.45 µm. The resulting filtrate was used as a cellulase solution in the following experiments.

[0075] (Measurement of protein concentration) The protein concentrations of various cellulase solutions were measured according to Reference Example 1. As a result, the cellulase solutions obtained by culture using inducers 1 and 3 had the highest protein concentration of 2.4 g / L. Furthermore, although inducers 1 and 4 to 7 all contain cellulose derived from cotton fibers as the main component, inducer 1 derived from T / C blended fibers had significantly superior ability to induce protein production. Detailed results are shown in Table 3.

[0076] (Measurement of β-glucosidase activity) According to Reference Example 2, the β-glucosidase activity in each culture medium was measured 168 hours after the start of culture. The activity was calculated by measuring the increase in absorbance at 405 nm, and the activity liberating 1 μmol of substrate per minute was defined as 1 U. As a result, the culture medium obtained by culture using inducer 1 had the highest activity at 2.5 U / mL. Detailed results are shown in Table 3.

[0077]

[0078] <Example 2> Saccharification reaction Using the various cellulase solutions obtained in Example 1, saccharification reactions were performed according to the method in Reference Example 4, and the amount of glucose contained in the saccharification reaction solution was quantified according to the method in Reference Example 5. As a result, the highest free glucose concentration was observed when the saccharification reaction was performed using the cellulase solution obtained by culture using inducer 1. The results are shown in Table 4.

[0079]

[0080] Example 3 Fiber-Derived Components Other Than Plant Fiber Contained in Protein Composition To 3 mL of the culture medium obtained by culturing using Inducer 1 in Example 1, 0.1 mL of a Congo Red solution (Fujifilm Wako Pure Chemical Industries, Ltd.) diluted with water to a final concentration of 0.2% was added, and the mixture was allowed to stand at room temperature and then examined under an optical microscope. As shown in Figure 1, the presence of numerous plant fibers derived from T / C blended fibers was confirmed, which were specifically stained with Congo Red. However, the presence of chemical fibers derived from T / C blended fibers, which were fiber fragments that were not specifically stained with Congo Red, was also confirmed at the same time.

[0081] Example 4 Cultivation of Trichoderma filamentous fungi using acid-treated, cellulase-treated, and subcritical hydrolysis-treated T / C blended fibers (pre-culture) A pre-culture solution was prepared in the same manner as in Example 1.

[0082] (Main Culture) The preculture solution was inoculated into the same main culture medium (Table 2) as in Example 1, and cultured under the same conditions for 192 hours.

[0083] The various inducers added to the culture medium to induce protein production were the following inducers 1, 3, 9, and 10: (Inducer 1) Plant fiber powder derived from T / C blended fiber prepared by acid treatment in Reference Example 3; (Inducer 3) Industrial pulp (Arbocel B800; Rettenmeyer Japan); (Inducer 9) Plant fiber powder derived from T / C blended fiber prepared by cellulase treatment in Reference Example 3; (Inducer 10) Plant fiber derived from T / C blended fiber prepared by subcritical hydrolysis in Reference Example 3.

[0084] (Collection of culture broth) Using the same procedure as in Example 1, various cellulase solutions were prepared from the culture broth collected 192 hours after the start of culture, and used in the following experiments.

[0085] (Measurement of protein concentration) The protein concentrations of various cellulase solutions were measured using the same procedure as in Example 1. The results are shown in Table 5, and the protein induction ability of inducers 1, 9, and 10 derived from T / C blended fiber was comparable to that of inducer 3 derived from industrial pulp.

[0086] (Measurement of β-glucosidase activity) The β-glucosidase activity in each culture solution was measured 192 hours after the start of culture using the same procedure as in Example 1. The results are shown in Table 5. The activity of the culture solution obtained in the culture using inducer 9 derived from T / C blended fiber was the highest at 2.4 U / mL. The activity of the culture solutions obtained in the culture using inducers 1 and 10 derived from T / C blended fiber was also about twice as high as the activity of the culture solution obtained in the culture using inducer 3 derived from industrial pulp.

[0087]

[0088] 1. Congo red stained area (plant fiber) 2. Fiber fragments not stained with Congo red (chemical fiber) 3. Aggregation of plant fiber and chemical fiber

Claims

1. An inducer for protein production by filamentous fungal culture, containing as an active ingredient plant fiber derived from a blend of plant fiber and fibers other than plant fiber.

2. The inducer according to claim 1, wherein the plant fiber is seed hair fiber, bast fiber, leaf vein fiber or fruit fiber.

3. The inducer of claim 2, wherein the seed hair fibers are cotton fibers.

4. The inducer according to claim 1, wherein the fiber other than plant fiber is a chemical fiber.

5. The inducer according to claim 4, wherein the chemical fiber is a chemical fiber containing at least polyester fiber.

6. The inducer according to claim 1, which contains as an active ingredient a plant fiber obtained by treating the blended fiber with an acid, cellulase, or subcritical hydrolysis.

7. The inducer of claim 6, wherein the acid is an inorganic acid or an organic acid.

8. The inducer of claim 7, wherein the inorganic acid is phosphoric acid.

9. The inducer according to claim 7, wherein the organic acid is a divalent or higher carboxylic acid.

10. The inducer according to claim 9, wherein the divalent or higher carboxylic acid is citric acid or oxalic acid.

11. A medium comprising the inducer according to any one of claims 1 to 10.

12. A method for producing a protein, comprising the step of culturing a filamentous fungus capable of producing a protein in the medium according to claim 11.

13. The method for producing a protein according to claim 12, wherein the protein is an enzyme.

14. The method for producing a protein according to claim 13, wherein the enzyme is cellulase.

15. The method for producing a protein according to claim 12, wherein the filamentous fungus is a filamentous fungus of the genus Trichoderma or Talaromyces.

16. A protein composition comprising a protein and a fiber-derived component other than plant fiber contained in a blended fiber containing plant fiber and fiber other than plant fiber.

17. The protein composition according to claim 16, wherein the fiber-derived component other than plant fiber is a fiber fragment other than plant fiber.

18. The protein composition according to claim 16, wherein the fiber other than plant fiber is a chemical fiber.

19. The protein composition of claim 18, wherein the chemical fiber is a polyester fiber.

20. The protein composition of claim 16, wherein the protein is an enzyme.

21. The protein composition of claim 20, wherein the enzyme is a cellulase.

22. A method for producing a sugar solution, comprising a step of hydrolyzing cellulose with the cellulase obtained by the method according to claim 14 or the protein composition according to claim 21.

23. The method for producing a sugar solution according to claim 22, wherein the cellulose is cellulose contained in a blended fiber containing plant fiber and a fiber other than plant fiber.

24. A method for recovering non-plant fibers from a blended fiber containing plant fibers and fibers other than plant fibers, comprising the steps of hydrolyzing a blended fiber containing plant fibers and fibers other than plant fibers with the cellulase obtained by the method described in claim 14 or the protein composition described in claim 21, and removing the hydrolyzed plant fibers from the treated product obtained in the hydrolysis treatment step.

25. A method for producing a chemical product, comprising the step of culturing a microorganism capable of producing the chemical product using the sugar solution obtained by the method according to claim 22 as a fermentation raw material.

26. A method for producing a protein, comprising the step of culturing a microorganism capable of producing a protein using the sugar solution obtained by the method according to claim 22 as a fermentation raw material.

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