Filter aid, filtering processing method, and cellulase production method

Abstract

According to the present invention, in filtering processing in a cellulase production method for removing fungus bodies and suspended matter in a culture medium containing cellulase from the culture medium, cellulase in which high enzyme activity is maintained can be obtained by using, as a filter aid, a mixture of an inorganic filter aid and a filamentous fungus culture medium.

Description

Filter aid, filtration treatment method, and method for producing cellulase

[0001] The present invention relates to a filter aid containing a mixture of an inorganic filter aid and a filamentous fungal culture solution as an active ingredient, a filtration treatment method using the filter aid, and a method for producing cellulase using the filter aid.

[0002] Trichoderma fungi are known to have a high protein-producing capacity, and studies have been conducted to use them to produce proteins. Trichoderma fungi use cellulose, lactose, cellobiose, and other inducers to produce cellulase, a type of saccharifying enzyme.

[0003] Cellulase is used as a saccharifying enzyme to hydrolyze cellulose-containing biomass to produce sugar. The first step in obtaining cellulase from a cellulase-containing Trichoderma culture is to remove Trichoderma fungal cells and turbidity from the culture. Removing these substances makes it possible to avoid membrane clogging in downstream membrane filtration processes (MF, UF). One method for removing turbidity is to use a filter aid, such as a suction filter (e.g., a precoat filter) or a pressure filter (e.g., a filter press). Known methods include using diatomaceous earth or perlite, particularly diatomaceous earth, as a filter aid (Non-Patent Document 1). In this case, filter aids primarily composed of biodegradable fibers such as cellulose or chitin may also be used (Patent Documents 1 and 2).

[0004] There are two types of filter aid usage methods: body feed, in which the filter aid is added directly to the liquid to be filtered, and precoat, in which a layer of filter aid is formed on the surface of the filter medium in advance. In solid-liquid separation using filter aids, filtration is usually carried out using both methods (Patent Document 3).

[0005] WO2013 / 018678 JP 8-224077 JP 2019-176837

[0006] Tomantschger Kurt et al. , MATHEMATICAL MODEL FOR THE PARTICLE SIZE DISTRIBUTION OF A KIESELGUHR FILTER GRANULATION, METALURGIA INTERNATIONAL, 17, 10, 192-197, 2012

[0007] The present inventors, in the course of investigating filtration as a solid-liquid separation method for removing microbial cells and turbid matters from a cellulase-containing culture solution, discovered a phenomenon in which the enzymatic activity of cellulase recovered after filtration is significantly reduced compared to before filtration. The reduction in enzymatic activity of cellulase leads to a reduction in the ability to decompose cellulose-containing biomass, which in turn causes a reduction in sugar yield when cellulose-containing biomass is hydrolyzed with cellulase to produce sugar.

[0008] Therefore, an object of the present invention is to establish a method for maintaining high enzymatic activity even after filtration of a microbial culture solution containing cellulase.

[0009] As a result of extensive research to solve the above problems, the present inventors have discovered that a mixture of an inorganic filter aid such as diatomaceous earth and a culture solution of a filamentous fungus such as a filamentous fungus of the genus Trichoderma or a filamentous fungus of the genus Talaromyces is useful as a filter aid, and that by performing a filtration process using this filter aid in the solid-liquid separation step described above, cellulase that maintains high enzymatic activity can be recovered, thereby completing the present invention.

[0010] That is, the present invention comprises the following [1] to

[16] . [1] A filter aid that is a mixture of an inorganic filter aid and a filamentous fungal culture solution. [2] The filter aid described in [1], wherein the inorganic filter aid is diatomaceous earth. [3] The filter aid described in [1] or [2], wherein the filamentous fungus is a filamentous fungus of the genus Trichoderma or Talaromyces. [4] The filter aid described in any of [1] to [3], wherein the culture solution is cultured until the dry cell weight (g-cell / L) of the filamentous fungus is at least 1.0 g / L. [5] The filter aid described in any of [1] to [4], wherein the culture solution contains filamentous fungal cells after culture. [6] A filtration treatment method comprising a step of filtration treatment together with the filter aid described in any of [1] to [5]. [7] The filtration treatment method described in [6], wherein the filtration treatment step is a filtration treatment step of adding the filter aid described in any of [1] to [5] to the liquid to be filtered. [8] The filtration method according to [6] or [7], wherein the filtration step is a filtration step in which the liquid to be filtered passes through a filter medium precoated with the filter aid according to any one of [1] to [5] from the precoat side. [9] A method for producing cellulase, comprising: a step (1) of culturing a microorganism capable of producing cellulase; and a step (2) of filtering the culture solution containing the microbial cells obtained in the step (1) together with the filter aid according to any one of [1] to [5] prepared independently of the step (1), and recovering the cellulase from which the microbial cells have been filtered out.

[10] The method for producing cellulase according to [9], wherein the microorganism in the step (1) is a microorganism capable of producing a cellulase having β-xylosidase activity, and the step (2) is a step of recovering the cellulase having β-xylosidase activity.

[11] The filtration treatment in the step (2) is a filtration treatment in which the filter aid according to any one of claims 1 to 5 prepared independently of the step (1) is added to the culture solution containing the microbial cells after culture obtained in the step (1). [9] or

[10] . The method for producing cellulase.

[12] The method for producing cellulase according to any one of [9] to

[11] , wherein the filtration treatment in step (2) is a filtration treatment in which the culture solution containing the microbial cells after culture obtained in step (1) is passed through the precoat side of a filter medium precoated with the filter aid according to any one of [1] to [5] prepared independently of step (1).

[13] The method for producing cellulase according to any one of [9] to

[12] , wherein the filtration treatment in step (2) is performed using a filter press.

[14] The method for producing cellulase according to any one of [9] to

[13] , wherein the microorganism capable of producing cellulase cultured in step (1) is a filamentous fungus of the genus Trichoderma or a filamentous fungus of the genus Talaromyces.

[15] The method for producing cellulase according to

[14] , comprising a step of mixing a preculture solution of the filamentous fungus cultured in step (1) with an inorganic filter aid to prepare the filter aid according to any one of [1] to [5].

[16] A method for producing sugar, comprising the steps of producing cellulase by the method according to any one of [9] to

[15] , and hydrolyzing cellulose-containing biomass with the cellulase obtained in the step.

[0011] According to the present invention, it is possible to recover cellulase with its enzymatic activity maintained at a high level even by filtering a culture solution containing cellulase.

[0012] The protein concentration (g / L) and β-xylosidase specific activity (U / mg protein) of the filtrate recovered by filtration in Comparative Example 2 and Example 3 are shown relative to the protein concentration and β-xylosidase activity of the unfiltered crude enzyme solution derived from Trichoderma fungi (Reference Example 2) taken as 100%.

[0013] The present invention will be described in detail below.

[0014] <Filter Aid and Filtration Treatment Method> A filter aid refers to a substance that has one or more of the following functions: reducing filtration resistance, reducing clogging of filter media, and improving the clarity of the filtrate. Generally, filter aids are broadly divided into organic filter aids and inorganic filter aids. However, organic filter aids (e.g., biodegradable fiber polymers such as cellulose and chitin) may be decomposed by the material to be filtered (e.g., cellulase). Therefore, the present invention is characterized by the use of an inorganic filter aid. The most representative type of inorganic filter aid is diatomaceous earth, which is produced as a fossil of diatoms, a type of phytoplankton. In addition to diatomaceous earth, perlite derived from lava erupted by volcanic activity is also cited as an example of an inorganic filter aid, but perlite is often used as an auxiliary to diatomaceous earth filter aids. Therefore, the inorganic filter aid used in the present invention is preferably mainly composed of diatomaceous earth and / or perlite as its active ingredient, more preferably diatomaceous earth as the main component, and even more preferably diatomaceous earth itself is used as the inorganic filter aid. The term "major component" means that it accounts for more than 50% by weight of the constituent components.

[0015] Diatomaceous earth differs from other algae in that diatoms are made of amorphous hydrated silica (SiO ２ ・nH ２ It is covered with a porous, hard cell wall consisting of silica. This is called a siliceous shell and has countless pores with a diameter of about 0.1 to 1 μm. Compared to the pore size (0.2 to 20 nm) of activated carbon and zeolite, these pores are macroscopic and do not have the adsorption performance of activated carbon or zeolite (J. Soc. Powder Technol., Japan, 39, pp. 114-121, 2002). The mined raw material is refined through operations such as drying, classification, and calcination. Filter aids made from diatomaceous earth are broadly divided into calcined products and flux-calcined products, but either can be used as the diatomaceous earth used as the main component of inorganic filter aids.

[0016] Commercially available diatomaceous earth can be used. For example, calcined products include Radiolite (registered trademark) series manufactured by Showa Chemical Industry Co., Ltd., such as "Radiolite #100," "Radiolite #200," "Radiolite #300," and "Radiolite #500," and Celite (trademark) series manufactured by Imerys, such as Standard Supercell, "Celite 350," "Celite 505," "Celite 512," and "Celite 577." Of these, "Celite 512" is preferred. Examples of flux-calcined products include Radiolite (registered trademark) series products such as Radiolite #700, Radiolite #800, Radiolite #900, and Radiolite #3000, and Imerys' Celite (trademark) series products such as Hyflo Supercell Celite 281, Celite 499, and Celite 503, with Radiolite #700 being preferred.

[0017] The filter aid of the present invention is characterized by being a mixture of the above-mentioned inorganic filter aid and filamentous fungal culture solution. In addition, the mixture that is the filter aid of the present invention is characterized by being a mixture prepared independently of the filtration treatment process. For example, when an inorganic filter aid is added during the filtration treatment of a filamentous fungal culture solution, a mixture of an inorganic filter aid and a filamentous fungal culture solution is prepared as the liquid to be filtered, but such a mixture does not fall under the filter aid of the present invention.

[0018] The filamentous fungi in the filamentous fungal culture solution used in the filter aid of the present invention are not particularly limited, and include fungi of the genus Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, and the like. m), Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora thora), Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Pleurotus, Rhizopus Rhizopus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, and Trichoderma filamentous fungi.Specific examples of these filamentous fungi include Acremonium, Aspergillus, Chrysosporium, Fusarium, Humicola, Myceliophthora, Neurospora, Penicillium, Pyromyces, Talaromyces, Thermoascus, Thielavia, and Trichoderma, with preferred being filamentous fungi of the genus Trichoderma or Talaromyces.

[0019] The filamentous fungus of the genus Trichoderma used for preparing the filamentous fungal culture solution may be a wild-type strain or a mutant strain of the filamentous fungus of the genus Trichoderma that has been improved to have enhanced protein production ability. Specific examples of the filamentous fungus of the genus Trichoderma include Trichoderma reesei, Trichoderma viride, Trichoderma atroviride, and Trichoderma longibrachiatum, with Trichoderma reesei being preferred. 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. Furthermore, mutant strains originating from the QM9414 strain and having the mutations described in WO2019 / 188980, WO2019 / 230860, WO2020 / 027010, WO2020 / 045472, WO2020 / 045473, WO2020 / 075787, and WO2020 / 075788 can also be used. The QM6a strain, QM9414 strain, and QM9123 strain are available from the NBRC (NITE Biological Resource Center), and the PC-3-7 strain and RutC-30 strain are available from the ATCC (American Type Culture Collection).

[0020] The filamentous fungus of the genus Talaromyces used to prepare the filamentous fungal culture solution may be a wild-type strain or a mutant strain of the filamentous fungus of the genus Talaromyces that has been improved to have enhanced protein production ability. Specific examples of Talaromyces filamentous fungi are not particularly limited as long as they have the above-described characteristics, but Talaromyces marneffei, Talaromyces stipitatus, Talaromyces amestolkiae, Talaromyces atroloseus, Talaromyces verruculosus, Talaromyces cellulolyticus, Talaromyces pinophilus, or Talaromyces emersonii are preferred, with Talaromyces cellulolyticus being more preferred. Specific examples of Talaromyces cellulolyticus include known mutant strains derived from Talaromyces cellulolyticus, such as the Y-94 strain (FERM BP-5826), the TN strain (FERM BP-11452), the C1 strain (FERM P-18508), and the CF-2612 strain (FERM BP-10848), as well as derivative strains thereof.

[0021] The culture method for filamentous fungi is not particularly limited, and can be, for example, liquid culture using centrifuge tubes, flasks, jar fermenters, tanks, etc., or solid culture using plates, etc. Trichoderma filamentous fungi are preferably cultured under aerobic conditions. Among these culture methods, submerged culture in flasks, particularly jar fermenters, or tanks with aeration and agitation is preferred. The aeration rate is preferably approximately 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 for culture is preferably 3.0 to 7.0, more preferably 4.0 to 6.0. The culture period is under conditions that allow fungal growth or protein production until a recoverable amount of fungal cells accumulates. The culture period is typically approximately 24 to 288 hours, more preferably 36 to 240 hours.

[0022] The filamentous fungal culture solution is preferably a culture solution in which the filamentous fungus has been cultured until the dry cell weight reaches 1 g / L or more, more preferably 3 g / L or more, even more preferably 5 g / L or more, and particularly preferably 10 g / L or more. The upper limit of the dry cell weight of the filamentous fungus after culturing is not particularly limited as long as it does not impair the effects of the present invention, but is preferably 50 g / L, more preferably 40 g / L. The dry cell weight can be determined by placing 2 mL of the filamentous fungal culture solution in a 2 mL Eppendorf tube, centrifuging it at 20,000 × g for 10 minutes at 4°C, discarding the supernatant, washing with distilled water, and then centrifuging it again at 20,000 × g for 10 minutes at 4°C. The recovered cell pellet is then dried in a dry heat sterilizer at 105°C for 2 hours, and the weight (g-cell / 2 mL) is measured using an electronic balance and converted to dry cell weight (g-cell / L).

[0023] The filamentous fungal culture medium may be a culture medium from which filamentous fungal cells have been removed, or a culture medium containing filamentous fungal cells, but a culture medium containing filamentous fungal cells is preferably used.

[0024] The mixing ratio of the inorganic filter aid and the filamentous fungal culture solution is preferably such that the concentration of the inorganic filter aid contained in the filamentous fungal culture solution is 1 to 15% (w / v), more preferably 3 to 12% (w / v), and even more preferably 5 to 10% (w / v). If the mixing ratio exceeds 15% (w / v), the fluidity of the liquid to be filtered may decrease, resulting in a deterioration in filterability.

[0025] The pH set when mixing the inorganic filter aid and the filamentous fungal culture solution is preferably pH 2.0 to 7.0, more preferably pH 3.0 to 6.0. If a filter aid mixed at a pH of 8.0 or higher is used, the desired effect cannot be obtained, which is not preferable.

[0026] The time for mixing and incubating the inorganic filter aid and the filamentous fungal culture solution is not particularly limited, but is preferably 1 hour or more, more preferably 6 hours or more, and even more preferably 12 hours or more.

[0027] Filtration methods that assume the use of the filter aid of the present invention include heavy pressure filtration, vacuum filtration, and pressure filtration. When heavy pressure filtration is performed, for example, a sand bed filter or a bag filter can be used. When vacuum filtration is performed, devices such as a Nutsche filter or a precoat filter (drum filter) can be used. When pressure filtration is performed, devices such as a candle filter, a leaf filter, a filter press, and a bag filter can be used. As a filtration method to which the filter aid of the present invention can be applied, vacuum filtration or pressure filtration is preferred, and pressure filtration is more preferred. Furthermore, as an apparatus for performing pressure filtration, a leaf filter or a filter press is preferred, and a filter press is more preferred. The filter press may be vertical or horizontal.

[0028] Further, as a filtration method using the filter aid of the present invention, a method can be mentioned in which the filter aid of the present invention is added to the liquid to be filtered (body feed) and then filtered according to the above-mentioned filtration method. In this method, the cake formed by filtering the liquid to be filtered containing the filter aid contains a mixture of turbid matter and filter aid, resulting in a high porosity and low filtration resistance. In this method, the filter aid of the present invention is preferably 1 to 15% (w / v) relative to the amount of the liquid to be filtered, as the amount of inorganic filter aid contained in the filter aid, more preferably 3 to 12% (w / v), and even more preferably 5 to 10% (w / v). Note that if the amount of inorganic filter aid exceeds 15% (w / v), the fluidity of the liquid to be filtered may decrease, resulting in a deterioration in filterability.

[0029] Furthermore, a filtration treatment method using the filter aid of the present invention may involve first applying (precoating) the filter aid of the present invention to the surface of a filter cloth, and then filtering the liquid to be filtered through the coated surface. The precoated filter aid of the present invention preferably contains filamentous fungi. For example, by forming a layer (precoat layer) of filter aid with a thickness of 2 to 6 mm on the filter cloth, turbid matter that clogs the filter is captured, and the operation of peeling the cake from the filter material after the filtration operation is also facilitated. The type of filter cloth to be precoated is not particularly specified as long as it is a filter cloth that can be used in the above-mentioned filtration treatment method. For example, it may be plain twill weave, twill twill weave, or reverse twill twill weave. Filter cloths can be purchased from Nakao Filter Kogyo Co., Ltd., Ataka Daiki Co., Ltd., etc.

[0030] The filter aid of the present invention can be used in any filtration process, including, but not limited to, filtering microbial cells from a microbial culture. A specific example is filtering microbial cells from a microbial culture capable of producing cellulase to recover the cellulase. In particular, in a filtration process for recovering cellulase having β-xylosidase activity by filtering microbial cells from a culture of a microorganism capable of producing cellulase having β-xylosidase activity, the filter aid of the present invention can be used in the filtration process to recover the cellulase without impairing the β-xylosidase activity.

[0031] <Method for Producing Cellulase> Cellulases include various hydrolases, including enzymes with decomposition activity against xylan, cellulose, hemicellulose, etc. 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 β-xylosidase (EC 3.2.1.37), which hydrolyzes xylooligosaccharides. Furthermore, cellulases secreted by filamentous fungi of the genus Talaromyces, which will be described later, include pectinase and galactanase, which are enzymes that decompose polysaccharides such as pectin and galactan. For example, the commercially available cellulase preparation derived from filamentous fungi of the genus Talaromyces, "Acremonium Cellulase" (Meiji Seika Pharma), has high pectinase activity and galactanase activity in addition to cellulase activity.

[0032] The filter aid of the present invention is characterized in that it produces cellulase by culturing a microorganism capable of producing cellulase, accumulating cellulase in the culture solution, and performing solid-liquid separation by filtration using the filter aid of the present invention.

[0033] Microorganisms capable of producing cellulase include bacteria, yeasts, and filamentous fungi, among which filamentous fungi are preferred. Examples of filamentous fungi include Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, and the like. s), Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocalima Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Pleurotus, Rhizopus Examples of fungi that may be useful include fungi of the genera Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, and Trichoderma.Among these filamentous fungi, preferred are filamentous fungi selected from the group consisting of the genera Acremonium, Aspergillus, Chrysosporium, Fusarium, Humicola, Myceliophthora, Neurospora, Penicillium, Pyromyces, Talaromyces, Thermoascus, Thielavia, and Trichoderma. Furthermore, from the viewpoints of cellulase productivity and the biomass saccharification performance of the resulting cellulase, filamentous fungi of the genus Trichoderma and filamentous fungi of the genus Talaromyces are more preferred. Specific examples of filamentous fungi of the genus Trichoderma and filamentous fungi of the genus Talaromyces include the filamentous fungi of the genus Trichoderma and Talaromyces used in the preparation of the filter aid of the present invention.

[0034] The filter aid of the present invention is also suitable for use in solid-liquid separation for recovering cellulases having these enzyme activities from the culture liquid of microorganisms capable of producing cellulases having β-xylosidase activity, preferably microorganisms capable of producing cellulases having pectinase activity and / or galactanase activity.

[0035] Microorganisms capable of producing cellulase having β-xylosidase activity include bacteria, yeasts, and filamentous fungi, among which filamentous fungi are preferred. Examples of filamentous fungi include Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, and the like. s), Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocalima Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Pleurotus, Rhizopus Examples of fungi that may be useful include fungi of the genera Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, and Trichoderma.Among these filamentous fungi, filamentous fungi selected from the group consisting of the genera Acremonium, Aspergillus, Chrysosporium, Fusarium, Humicola, Myceliophthora, Neurospora, Penicillium, Pyromyces, Talaromyces, Thermoascus, Thielavia, and Trichoderma are preferred, and more preferred than filamentous fungi of the genus Trichoderma. Specific examples of the filamentous fungi of the genus Trichoderma and the filamentous fungi of the genus Talaromyces include the filamentous fungi of the genus Trichoderma and the filamentous fungi of the genus Talaromyces described above for use in preparing the filter aid of the present invention.

[0036] Culture of microorganisms capable of producing cellulase is performed using a medium commonly used in the field of cellulase production. Furthermore, cellulase production can be improved by culturing the microorganisms in a medium containing a cellulase inducer. Examples of cellulase inducers include cellulose decomposition intermediates including lactose, sophorose, gentiobiose, and cellobiose, crystalline cellulose, xylan, and biomass containing cellulose and xylan. Specific examples of biomass containing cellulose and xylan include pulp, bagasse, cassava pulp, switchgrass, napier grass, erianthus, corn stover, corn cob, corn hull, rice straw, wheat straw, beet pulp, eucalyptus, oak, and birch. Pulp or corn hull is preferred, and corn hull is more preferred. An example of a cellulase inducer made from corn hull is the corn hull pulp described in WO 2021 / 235419.

[0037] Pretreated biomass containing cellulose and xylan is also preferably used as a cellulase inducer. Known pretreatment methods such as acid treatment, sulfuric acid treatment, dilute sulfuric acid treatment, alkali treatment, hydrothermal treatment, subcritical treatment, fine pulverization treatment, steaming treatment, and pulverization treatment can be used.

[0038] Furthermore, liquid sugar or biomass may be added during the culture to further improve the cellulase accumulation concentration. Specific examples of liquid sugar to be added include cellulose decomposition intermediates including glucose, fructose, sucrose, maltose, lactose, sophorose, gentiobiose, and cellobiose, and specific examples of biomass include crystalline cellulose, xylan, and biomass containing cellulose and xylan. Specific examples of biomass containing cellulose and xylan include the biomass described above. When added to the culture tank, liquid sugar is preferred from the viewpoint of operability, and glucose and lactose are particularly preferred. The timing to start adding the liquid sugar used is preferably within 144 hours from the start of culture, more preferably within 72 hours from the start of culture, and particularly preferably within 24 hours from the start of culture. Sugar may be added once, multiple times, or continuously.

[0039] The culture method for culturing a microorganism capable of producing cellulase and accumulating cellulase in the culture medium is not particularly limited. For example, culture can be performed by liquid culture using centrifuge tubes, flasks, jar fermenters, tanks, etc., or solid culture using plates, etc. In addition, the aeration conditions for the microorganism capable of producing cellulase may be determined according to the conditions appropriate for the microorganism being cultured. However, when a filamentous fungus of the genus Trichoderma is used as the microorganism, culture under aerobic conditions is preferred. Among these culture methods, submerged culture in a flask, particularly a jar fermenter, or a tank in which culture is performed with aeration and stirring is preferred. The aeration rate is preferably approximately 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 approximately 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 continued under conditions that allow the cells to grow or the protein to be produced until a recoverable amount of cells is accumulated, which is usually about 24 to 288 hours, and more preferably 36 to 240 hours.

[0040] Furthermore, prior to the aforementioned culturing, it is preferable to pre-culture a microorganism capable of producing cellulase, and then subject the pre-culture solution to the aforementioned culturing. The carbon source used in the pre-culture may be a sugar that does not induce cellulase, such as glucose, fructose, sucrose, or maltose, or a cellulase inducer such as cellobiose, crystalline cellulose, xylan, or biomass containing cellulose and xylan. However, glucose is preferred from the viewpoint of culturing operations, such as ease of assimilation and liquid transfer to the main culture solution. The culture conditions are preferably those used for the culturing described above. When using a Trichoderma or Talaromyces fungus as the microorganism capable of producing cellulase, a portion of the pre-culture solution of the Trichoderma or Talaromyces fungus can be used as the Trichoderma or Talaromyces fungus culture solution, which is the main component of the filter aid of the present invention.

[0041] It is preferable to reduce the bacterial cell fraction of the cellulase-containing culture solution obtained by the above-mentioned method before filtration. By reducing the bacterial cell fraction in advance, it is possible to prevent the filter chamber of the device used in the filtration process from becoming filled with solids. Centrifugation is a preferred method for reducing the bacterial cell fraction. Devices used for centrifugation include, for example, bottom discharge types, continuous types, and separation plate types (DeLaval type). There are no particular limitations on the centrifugation conditions, as long as the bacterial cells can be precipitated and removed.

[0042] In the present invention, a cellulase-containing culture solution is filtered together with the filter aid of the present invention to filter out microbial cells and recover cellulase. The present invention is also characterized in that the filter aid is prepared independently of the filtration process. For example, in a filtration process for filtering cellulase from a cellulase-containing Trichoderma filamentous fungus culture solution, diatomaceous earth is body-fed as a filter aid, or diatomaceous earth is pre-coated on a filter cloth as a filter aid. The filtered solution becomes a mixture of diatomaceous earth and a Trichoderma filamentous fungus culture solution containing cellulase. However, such a mixture does not fall under the filter aid of the present invention, and such an embodiment does not fall under the definition of "preparing the filter aid of the present invention independently of the cellulase production process." The filtration method for a cellulase-containing culture solution using the filter aid of the present invention is as described above, and may be a filtration process in which the filter aid is body-fed, a filtration process in which the filter aid is pre-coated on a filter cloth, or both.

[0043] The cellulase obtained by filtering out the microbial cells may be used as a crude enzyme solution as it is, or may be formulated by a known method before use.

[0044] The cellulase obtained by the present invention can be widely used for the hydrolysis of cellulose-containing biomass. Cellulose-containing biomass contains at least cellulose or hemicellulose, and specific examples include pulp, bagasse, cassava pulp, switchgrass, napier grass, erianthus, corn stover, corn cob, corn hull, rice straw, wheat straw, beet pulp, eucalyptus, oak, and birch. These cellulose-containing biomass contain impurities such as the high molecular weight aromatic compound lignin and hemicellulose. However, cellulose-containing biomass obtained by partially decomposing the lignin and hemicellulose using acid, alkali, or hot compressed water as a pretreatment may be used as cellulose. The hydrolysis conditions may be set to the optimal reaction conditions for the fungal cellulase, with a treatment temperature of 40 to 60°C, a treatment pH of 3 to 7, and a cellulose-containing biomass solids concentration of 0.1 to 30% being preferred. Setting the optimal reaction conditions within the above ranges maximizes the biomass hydrolysis efficiency. This hydrolysis treatment may be performed batchwise or continuously. The hydrolysate obtained by such an enzyme treatment contains monosaccharide components such as glucose and xylose, and can therefore be used as a fermentation raw material sugar for the fermentation production of various chemical products.

[0045] When using the cellulase obtained by the present invention, an enzyme component having another function, such as an oxidase, a reductase, a hydrolase, a transferase, or an isomerase, may be added to the cellulase as an enzymatic preparation. In particular, such an enzyme component having another function is not limited to filamentous fungi, and enzyme components derived from any organism or produced by genetic recombination may be added and used.

[0046] The present invention will be specifically explained below by way of examples, but the present invention is not limited thereto.

[0047] Reference Example 1 Preparation of a filamentous fungus culture solution 1.0 × 10 spores of Trichoderma reesei PC-3-7 (ATCC 66589) (hereinafter simply referred to as "Trichoderma filamentous fungi") were added to the culture solution. ７Two 1 L baffled flasks containing 250 mL of the medium shown in Table 1 were prepared, and 2.5 mL of the diluted spore solution was inoculated. The flasks were cultured for 72 hours at 28 ° C. and 120 rpm in a shaking incubator. One of the flasks was inoculated with the main culture solution of Reference Example 2, and the other flask was used to prepare the filter aid of Reference Example 2. The culture was continued until the end of the main culture.

[0048] After completion of the culture, 2 mL of the Trichoderma culture was placed in a 2 mL Eppendorf tube and centrifuged at 20,000 × g at 4°C for 10 minutes. The supernatant was discarded, washed with distilled water, and then centrifuged again at 20,000 × g at 4°C for 10 minutes to recover a fungal cell pellet. The recovered fungal cell pellet was dried in a dry heat sterilizer at 105°C for 2 hours and its weight (g-cell / 2 mL) was measured using an electronic balance. The dry fungal cell weight (g-cell / L) of the Trichoderma culture was found to be 1.5 g / L.

[0049] Furthermore, when Talaromyces cellulolyticus C1 strain (FERM P-18508) (hereinafter also referred to simply as "Talaromyces filamentous fungi") was used as the filamentous fungus, cultivation was also carried out in a similar manner. The dry cell weight (g-cell / L) of the Talaromyces filamentous fungus culture solution was determined in the same manner as for the Trichoderma filamentous fungus culture solution, and was found to be 34.7 g / L.

[0050]

[0051] Reference Example 2: Preparation of crude enzyme solution derived from filamentous fungi When Trichoderma reesei PC-3-7 strain was used as the filamentous fungus, 2.5 L of the cellulase production medium shown in Table 2 was placed in a 5 L jar fermenter (manufactured by Biot) and sterilized in an autoclave at 121°C for 40 minutes. 250 mL of the culture solution of Trichoderma reesei PC-3-7 from Reference Example 1 was used as a preculture solution and inoculated into the cellulase production medium (inoculation amount 10% v / v). The main culture medium was inoculated with the culture solution prepared in Reference Example 1, and submerged culture was initiated under the following culture conditions: 28°C, 700 rpm, and an aeration volume of 1 vvm, while controlling the pH at 5.0. After 10 hours of cultivation, a sugar solution containing glucose and lactose shown in Table 3 was added at a rate of 225 mL / day, and cultivation was continued while adjusting the sugar solution feed rate so that the dissolved oxygen concentration was 20%.

[0052] When Talaromyces cellulolyticus C1 strain was used as the filamentous fungus, the conditions other than the cellulase inducer (lactose), culture temperature (30 ° C), pH conditions (pH 4.0), and fed-batch conditions (no fed-batch) were the same as those for the Trichoderma reesei PC-3-7 strain.

[0053]

[0054]

[0055] After completion of the culture, 80 mL of the culture medium was collected and dispensed in equal amounts into two 50 mL centrifuge tubes (manufactured by Corning). To remove the fungal cells, the centrifuge tubes were centrifuged at 3,000 × g and 4°C for 10 minutes, followed by decantation to obtain crude enzyme solutions derived from filamentous fungi of the genus Trichoderma and crude enzyme solutions derived from filamentous fungi of the genus Talaromyces. The protein concentration and β-xylosidase specific activity of these crude enzyme solutions were measured by the methods described in Reference Examples 4 and 5 below. Furthermore, the pectinase specific activity and galactanase specific activity of the crude enzyme solution derived from filamentous fungi of the genus Talaromyces were measured by the methods described in Reference Examples 6 and 7 below. These values ​​were set to 100% and compared with the values ​​in the following Examples and Comparative Examples.

[0056] Reference Example 3 Preparation of Alkali-Treated Bagasse 9.3 g of caustic soda was mixed with 100 g of dry bagasse, and the mixture was reacted at 121° C. for 30 minutes to prepare alkali-treated bagasse.

[0057] <Reference Example 4> Protein concentration measurement conditions Protein concentration measurement reagent: Quick Start (trademark) Bradford 1x Dye Reagent (manufactured by Bio-Rad) Protein concentration measurement reagent: 250 µL Crude enzyme dilution solution: 5 µL Measurement temperature: room temperature Reaction time: 5 minutes Absorbance: 595 nm Standard: BSA.

[0058] Reference Example 5: Measurement conditions for β-xylosidase specific activity Substrate: p-nitrophenyl-β-xylopyranoside (Sigma-Aldrich Japan) Reaction solution: 90 μL of 50 mM acetate buffer containing 1 mM p-nitrophenyl-β-xylopyranoside Crude enzyme dilution: 10 μL Reaction temperature: 30°C Reaction time: 10 minutes Reaction stopper: 10 μL of 2 M sodium carbonate Absorbance: 405 nm Enzyme reaction: The enzyme dilution was mixed with the substrate cooled to 4°C on a 96-well PCR plate and incubated at 30°C for 10 minutes using a thermal cycler. The reaction solution was then quickly cooled to 4°C, and the enzymatic reaction was stopped by adding 10 μL of 2 M sodium carbonate. Activity quantification: The absorbance of the reaction solution at 405 nm was measured using a microplate reader, and the amount of p-nitrophenol released in the reaction solution was calculated from the value of a calibration curve based on a standard. β-Xylosidase activity was calculated from the amount of p-nitrophenol contained in the diluted enzyme solution, with 1 U of activity defined as the amount of enzyme that liberates 1 μmol of p-nitrophenol per minute. The activity per mg of protein was calculated by dividing the β-xylosidase activity contained in the diluted enzyme solution by the protein concentration of the diluted enzyme solution.

[0059] Reference Example 6: Conditions for measuring pectinase specific activity Substrate: 90 μL of 100 mM sodium acetate (pH 5.0) containing 5.5 g / L polygalacturonic acid Enzyme diluent: 10 μL (diluted to a protein concentration of 0.25 g / L) Standard: 0.25 to 4 g / L galacturonic acid Color-developing reagent: DNS reagent (5 g / L 3,5-dinitrosalicylic acid (DNS) and 300 g / L sodium potassium tartrate dissolved in 0.4 M sodium hydroxide) Enzyme reaction: The enzyme diluent was mixed with the substrate cooled to 4°C on a 96-well PCR plate and incubated at 50°C for 10 minutes using a thermal cycler. The reaction solution was then quickly cooled to 4°C, and the enzymatic reaction was stopped by adding 10 μL of 1 M sodium hydroxide. Color reaction: 40 μL of the enzyme reaction solution and 80 μL of DNS reagent were mixed on a new 96-well PCR plate and heated at 95°C for 5 minutes using a thermal cycler. The color solution was cooled to room temperature and mixed with 120 μL of water, after which 180 μL was transferred to a new 96-well microplate. Activity quantification: The absorbance of the color solution at 540 nm was measured using a microplate reader, and the amount of reducing sugar released in the reaction solution was calculated from the value of a calibration curve based on a standard. 1 U of activity was defined as the amount of enzyme liberating 1 μmol of reducing sugar per minute, and pectinase activity was calculated from the amount of reducing sugar contained in the enzyme dilution. Activity per mg of protein was calculated by dividing the pectinase activity contained in the enzyme dilution by the protein concentration of the enzyme dilution.

[0060] Reference Example 7: Conditions for Measuring Galactanase Specific Activity Substrate: 90 μL of 100 mM sodium acetate (pH 5.0) containing 5.5 g / L potato-derived galactan. Enzyme Diluent: 10 μL (diluted to a protein concentration of 0.25 g / L). Standard: 0.25-4 g / L galactose. Color Reagent: DNS Reagent. Enzyme Reaction: The enzyme diluent was mixed with the substrate cooled to 4°C on a 96-well PCR plate and incubated at 50°C for 10 minutes using a thermal cycler. The reaction solution was then quickly cooled to 4°C, and the enzymatic reaction was terminated by adding 10 μL of 1 M sodium hydroxide. Color Reaction: 40 μL of the enzyme reaction solution and 80 μL of DNS Reagent were mixed on a new 96-well PCR plate and heated at 95°C for 5 minutes using a thermal cycler. The color solution was cooled to room temperature and mixed with 120 μL of water, and 180 μL of the solution was transferred to a new 96-well microplate. Quantification of activity: The absorbance of the color-developing solution at 540 nm was measured using a microplate reader, and the amount of reducing sugar released in the reaction solution was calculated from the value of a calibration curve based on a standard. 1 U of activity was defined as the amount of enzyme that releases 1 μmol of reducing sugar per minute, and galactanase activity was calculated from the amount of reducing sugar contained in the enzyme dilution. The activity per mg of protein was calculated by dividing the galactanase activity contained in the enzyme dilution by the protein concentration of the enzyme dilution.

[0061] Reference Example 8 Measurement of sugar concentration A solution was prepared by adding 20 μL of 1N NaOH to 180 μL of the crude enzyme solution as a reaction stop solution, and xylose was quantitatively analyzed using ACQUITY UPLC (Waters, Milford, MA, USA) under the following conditions to measure the concentration. Column: ACQUITY UPLC BEH Amide column (2.1 × 100 mm, 1.7 μm, manufactured by Waters Corporation) Mobile phase: 80% (v / v) acetonitrile and 0.2% (v / v) trimethylamine (TEA) mixed solution Flow rate: Elution with a gradient of acetonitrile from 80% to 75% at 0.3 mL / min Temperature: 50°C Detection method: ELSD (evaporative light scattering detector system, manufactured by Waters Corporation).

[0062] Comparative Example 1: Solid-liquid separation of a crude enzyme solution derived from Trichoderma fungi using diatomaceous earth as a filter aid (precoat agent). Diatomaceous earth "Celite 512" was added to distilled water to a concentration of 10% (w / v), and the mixture was cultured at room temperature for 1 hour with shaking at 120 rpm to prepare a precoat filter aid. 10 mL of this filter aid was dripped onto a filter cloth in a polysulfone holder, followed by suction filtration with a vacuum pump to form a precoat layer of the filter aid. The eluted water was discarded.

[0063] Next, 20 mL of the crude enzyme solution derived from Trichoderma fungi prepared in Reference Example 2 was gently dropped onto the polysulfone holder on which the precoat layer had been formed, and the solution was subjected to suction filtration using a vacuum pump. The resulting filtrate was recovered, and the protein concentration and β-xylosidase specific activity were measured using the methods described in Reference Examples 4 and 5.

[0064] As shown in Table 4, the protein concentration decreased slightly before and after suction filtration, which is thought to be due to dilution by the water remaining during precoat formation. The β-xylosidase specific activity was 53.9% compared to that of the unfiltered crude enzyme solution derived from Trichoderma fungi (Reference Example 2).

[0065] Example 1: Solid-liquid separation of a crude enzyme solution derived from Trichoderma fungi using a mixture of diatomaceous earth and culture broth containing Trichoderma fungi as a filter aid (precoat agent). The entire amount of Trichoderma fungi culture broth prepared by the method described in Reference Example 1 was centrifuged at 3,000 × g and 4°C for 10 minutes to separate the supernatant and fungi. The fungal fraction was collected, suspended in distilled water, and then centrifuged at 3,000 × g and 4°C for 10 minutes to wash the fungal fraction. The washed fungal fraction was suspended in an equal amount of distilled water to the separated supernatant fraction, and diatomaceous earth "Celite 512" was added to make a 10% (w / v) concentration. The suspension was cultured at room temperature for 1 hour with shaking at 120 rpm to prepare a filter aid for precoat. The crude enzyme solution derived from Trichoderma fungi prepared in Reference Example 2 was subjected to suction filtration in the same manner as in Comparative Example 1, except that this filter aid was used, and the filtrate was recovered.The protein concentration and β-xylosidase specific activity of the recovered filtrate were measured using the methods described in Reference Examples 4 and 5.

[0066] As shown in Table 4, the protein concentration decreased slightly before and after suction filtration, which is thought to be due to dilution by the water remaining during precoat formation. On the other hand, the β-xylosidase specific activity was 74.7% compared to that of the unfiltered crude enzyme solution derived from Trichoderma fungi (Reference Example 2).

[0067] Example 2: Solid-liquid separation of a crude cellulase enzyme solution using a mixture of diatomaceous earth and the supernatant of a Trichoderma fungus culture solution as a filter aid (precoat agent). The entire Trichoderma fungus culture solution prepared by the method described in Reference Example 1 was centrifuged at 3,000 × g and 4°C for 10 minutes to separate the supernatant from the fungus cells. Diatomaceous earth "Celite 512" was added to the resulting supernatant to a concentration of 10% (w / v), and the mixture was cultured with shaking at 120 rpm at room temperature for 1 hour to prepare a precoat filter aid. The crude enzyme solution derived from Trichoderma fungus prepared in Reference Example 2 was subjected to suction filtration using the same procedure as in Comparative Example 1, except that this filter aid was used, and the filtrate was recovered. The protein concentration and β-xylosidase specific activity of the recovered filtrate were measured using the methods described in Reference Examples 4 and 5.

[0068] As shown in Table 4, the protein concentration decreased slightly before and after suction filtration, which is thought to be due to dilution by the water remaining during precoat formation. On the other hand, the β-xylosidase specific activity was 68.1% compared to that of the unfiltered crude enzyme solution derived from Trichoderma fungi (Reference Example 2).

[0069] Comparative Example 2: Solid-liquid separation of a crude enzyme solution derived from Trichoderma fungi using diatomaceous earth as a filter aid (precoat agent and body feed agent). Diatomaceous earth "Celite 512" was added to distilled water to a concentration of 10% (w / v), and the mixture was cultured at room temperature for 1 hour with shaking at 120 rpm to prepare a filter aid for precoat and body feed. 10 mL of this filter aid was dripped onto a filter cloth in a polysulfone holder, followed by suction filtration with a vacuum pump to form a precoat layer of the filter aid. The eluted water was discarded.

[0070] Next, 10 mL of the aforementioned filter aid was added to 10 mL of the crude enzyme solution derived from Trichoderma fungi prepared in Reference Example 2 (body feed), and the mixture was gently dropped onto a polysulfone holder on which a precoat layer had been formed. The filtrate was then collected by suction filtration using a vacuum pump, and the protein concentration and β-xylosidase specific activity of the collected filtrate were measured using the methods described in Reference Examples 4 and 5.

[0071] As shown in Table 4 and Figure 1, the protein concentration decreased slightly before and after suction filtration, which is thought to be due to dilution by the water remaining during precoat formation. The specific activity of β-xylosidase was 22.5% compared to that of the unfiltered crude enzyme solution derived from Trichoderma fungi (Reference Example 2).

[0072] Comparative Example 3: Solid-liquid separation of a crude enzyme solution derived from Trichoderma fungi using a mixture of diatomaceous earth and a fungal cell fraction from a Trichoderma fungal culture as a filter aid (precoat agent and body feed agent). The entire amount of Trichoderma fungal culture prepared by the method described in Reference Example 1 was centrifuged at 3,000 × g and 4°C for 10 minutes to separate the supernatant and fungal cells. The fungal cell fraction was collected, suspended in distilled water, and then centrifuged at 3,000 × g and 4°C for 10 minutes to wash the fungal cell fraction. The washed fungal cell fraction was suspended in an equal amount of distilled water to the separated supernatant fraction, and diatomaceous earth "Celite 512" was added to make a 10% (w / v) concentration. The suspension was cultured at room temperature for 1 hour with shaking at 120 rpm to prepare a filter aid for precoat and body feed. The crude enzyme solution derived from Trichoderma fungi was subjected to suction filtration and the filtrate was recovered in the same manner as in Comparative Example 2, except that this filter aid was used. The protein concentration and β-xylosidase specific activity of the recovered filtrate were measured using the methods described in Reference Examples 4 and 5.

[0073] As shown in Table 4, the protein concentration decreased slightly before and after suction filtration, which is thought to be due to dilution by the water remaining during precoat formation. On the other hand, the β-xylosidase specific activity was 26.2% compared to the unfiltered crude enzyme solution derived from Trichoderma fungi (Reference Example 2), which was found to be equivalent to that of Comparative Example 2.

[0074] Example 3 Solid-Liquid Separation of a Crude Enzyme Solution Derived from a Filamentous Fungus of the Genus Trichoderma Using a Mixture of Diatomaceous Earth and Culture Solution Containing Filamentous Fungus Cells of the Genus Trichoderma as a Filter Aid (Precoat Agent and Body Feed Agent) Diatomaceous earth "Celite 512" was added to the total volume of a culture solution of a filamentous fungus of the genus Trichoderma prepared by the method described in Reference Example 1 so as to give a concentration of 10% (w / v), and the mixture was cultured with shaking at 120 rpm at room temperature for 1 hour to prepare a filter aid for precoat and body feed. The crude enzyme solution derived from a filamentous fungus of the genus Trichoderma was subjected to suction filtration and the filtrate was recovered in the same manner as in Comparative Example 2, except that this filter aid was used. The protein concentration and β-xylosidase specific activity of the recovered filtrate were measured using the methods described in Reference Examples 4 and 5.

[0075] As shown in Table 4 and Figure 1, the protein concentration decreased slightly before and after suction filtration, which is thought to be due to dilution by the water remaining during precoat formation. On the other hand, the β-xylosidase specific activity was 88.4% compared to that of the unfiltered crude enzyme solution derived from Trichoderma fungi (Reference Example 2).

[0076] Example 4 Solid-Liquid Separation of a Crude Enzyme Solution Derived from a Fungus of the Genus Trichoderma Using a Mixture of Diatomaceous Earth and the Supernatant of a Culture Solution of a Fungus of the Genus Trichoderma as a Filter Aid (Precoat Agent and Body Feed Agent) The entire amount of a culture solution of a fungus of the genus Trichoderma prepared by the method described in Reference Example 1 was centrifuged at 3,000 × g and 4°C for 10 minutes to separate the supernatant from the fungus cells. A mixture of the supernatant fraction and diatomaceous earth "Celite 512" was then prepared. The crude enzyme solution derived from a fungus of the genus Trichoderma was subjected to suction filtration in the same manner as in Comparative Example 2, except that this mixture was used as the filter aid for the precoat and body feed. The protein concentration and β-xylosidase specific activity of the recovered filtrate were measured by the methods described in Reference Examples 5 and 6.

[0077] As shown in Table 4, the protein concentration decreased slightly before and after suction filtration, which is thought to be due to dilution by the water remaining during precoat formation. On the other hand, the β-xylosidase specific activity was 67.8% compared to that of the unfiltered crude enzyme solution derived from Trichoderma fungi (Reference Example 2).

[0078] Example 5 Solid-Liquid Separation of a Crude Enzyme Solution Derived from a Filamentous Fungus of the Genus Trichoderma Using a Mixture of Diatomaceous Earth and Culture Solution Containing Filamentous Fungus Cells of the Genus Talaromyces as a Filter Aid (Precoat Agent and Body Feed Agent) Diatomaceous earth "Celite 512" was added to the total volume of a culture solution of a filamentous fungus of the genus Talaromyces prepared by the method described in Reference Example 1 so as to give a concentration of 10% (w / v), and the mixture was cultured with shaking at 120 rpm at room temperature for 1 hour to prepare a filter aid for precoat and body feed. The crude enzyme solution derived from a filamentous fungus of the genus Trichoderma was subjected to suction filtration and the filtrate was recovered in the same manner as in Comparative Example 2, except that this filter aid was used. The protein concentration and β-xylosidase specific activity of the filtrate recovered by the methods described in Reference Examples 4 and 5 were measured.

[0079] As shown in Table 4, the protein concentration decreased slightly before and after suction filtration, which is thought to be due to dilution by the water remaining during precoat formation. On the other hand, the β-xylosidase specific activity was 81.2% compared to that of the unfiltered crude enzyme solution derived from Trichoderma fungi (Reference Example 2).

[0080] Comparative Example 4: Solid-liquid separation of crude enzyme solution derived from Talaromyces filamentous fungi using diatomaceous earth as a filter aid (precoat agent and body feed agent). Diatomaceous earth "Celite 512" was added to distilled water to a concentration of 10% (w / v), and the mixture was cultured at room temperature for 1 hour with shaking at 120 rpm to prepare a filter aid for precoat and body feed. 10 mL of this filter aid was dropped onto a filter cloth in a polysulfone holder, and then suction filtered with a vacuum pump to form a precoat layer of the filter aid. The separated water was discarded.

[0081] Next, 10 mL of the above-mentioned filter aid was added to 10 mL of the crude enzyme solution derived from filamentous fungi of the genus Talaromyces prepared in Reference Example 2 (body feed), and the mixture was gently dropped onto a polysulfone holder on which a precoat layer had been formed. The filtrate was then collected by suction filtration using a vacuum pump, and the protein concentration, β-xylosidase specific activity, pectinase specific activity, and galactanase specific activity of the filtrate collected by the methods described in Reference Examples 4 to 7 were measured.

[0082] As shown in Table 4, the β-xylosidase specific activity was reduced to 18.8% compared to the unfiltered crude enzyme solution derived from filamentous fungi of the genus Talaromyces (Reference Example 2). Furthermore, the pectinase specific activity and galactanase specific activity were 85.6% and 73.8%, respectively, compared to the unfiltered crude enzyme solution derived from filamentous fungi of the genus Talaromyces (Reference Example 2).

[0083] Example 6 Solid-Liquid Separation of a Crude Enzyme Solution Derived from Filamentous Fungi of the Genus Talaromyces Using a Mixture of Diatomaceous Earth and Culture Solution Containing Filamentous Fungi of the Genus Talaromyces as a Filter Aid (Precoat Agent and Body Feed Agent) Diatomaceous earth "Celite 512" was added to the total volume of a culture solution of filamentous fungi of the genus Talaromyces prepared by the method described in Reference Example 1 so as to give a concentration of 10% (w / v), and the mixture was cultured with shaking at 120 rpm at room temperature for 1 hour to prepare a filter aid for precoat and body feed. Except for using this filter aid, the crude enzyme solution derived from filamentous fungi of the genus Talaromyces was subjected to suction filtration according to the procedure of Comparative Example 4 to recover the filtrate, and the protein concentration, β-xylosidase specific activity, pectinase specific activity, and galactanase specific activity of the recovered filtrate were measured using the methods described in Reference Examples 4 to 7.

[0084] As shown in Table 4, the β-xylosidase specific activity was 84.7% compared to the unfiltered crude enzyme solution derived from filamentous fungi of the genus Talaromyces (Reference Example 2). Furthermore, the pectinase specific activity and galactanase specific activity were 102% and 97.9%, respectively, compared to the unfiltered crude enzyme solution derived from filamentous fungi of the genus Talaromyces (Reference Example 2).

[0085]

[0086] Example 7 Cellulose-Containing Biomass Saccharification Test Using the unfiltered crude enzyme solution derived from Trichoderma fungi obtained in Reference Example 2, and the filtrates obtained in Comparative Examples 2 and 3, and Examples 3 and 4, a saccharification test was carried out on alkali-treated bagasse.

[0087] Alkali-treated bagasse prepared according to the method described in Reference Example 4 was added to 5% (w / v) biomass and incubated in a rotating rotor at 50°C for 7 hours, after which the reaction supernatant was recovered by centrifugation at 20,000 x g and 4°C for 10 minutes. 20 μL of 1 N NaOH, a reaction stop solution, was added to 180 μL of the reaction supernatant, and the amount of released xylose was quantified under the analytical conditions shown in Reference Example 7.

[0088] As shown in Table 5, when an unfiltered crude enzyme solution derived from Trichoderma fungi (Reference Example 2) was used, the amount of xylose released was 10.1 g / L. When the filtrates of Comparative Examples 2 and 3 were used, the amounts of xylose released were 7.87 g / L and 8.04 g / L, respectively, which were 22.7% and 20.3% less than those of Reference Example 2. On the other hand, when the filtrate of Example 3 was used, the amount of xylose released was 11.5 g / L, which was a 13.9% increase compared to Reference Example 2. Furthermore, when the filtrate of Example 4 was used, the amount of xylose released was 9.55 g / L, which was a 5.44% decrease compared to Reference Example 2, but the decrease was less than 10%.

[0089]

Claims

1. A filter aid that is a mixture of an inorganic filter aid and a filamentous fungal culture liquid (excluding a filamentous fungal culture liquid that is the liquid to be filtered).

2. The filter aid according to claim 1, wherein the inorganic filter aid is diatomaceous earth.

3. The filter aid according to claim 1, wherein the filamentous fungus is a filamentous fungus of the genus Trichoderma or a filamentous fungus of the genus Talaromyces.

4. The filter aid according to claim 1, wherein the culture medium is cultured until the dry cell weight (g-cell / L) of the filamentous fungus cells becomes at least 1.0 g / L or more.

5. The filter aid according to claim 1, wherein the culture medium contains cultured filamentous fungal bodies.

6. A filtration treatment method comprising a step of performing filtration together with the filter aid according to any one of claims 1 to 5.

7. The filtration treatment method according to claim 6, wherein the filtration treatment step is a filtration treatment step of adding the filter aid according to any one of claims 1 to 5 to the liquid to be filtered.

8. The filtration treatment method according to claim 6, wherein the filtration treatment step is a filtration treatment step in which the liquid to be filtered passes from the precoat side of a filter medium precoated with the filter aid according to any one of claims 1 to 5. The filtration treatment method.

9. A method for producing cellulase, comprising: a step (1) of culturing a microorganism capable of producing cellulase; and a step (2) of filtering the culture solution containing the microbial cells after the culture obtained in the step (1) together with the filter aid according to any one of claims 1 to 5 prepared independently of the step (1), and recovering the cellulase from which the microbial cells have been filtered out.

10. The method for producing cellulase according to claim 9, wherein the microorganism in step (1) is a microorganism capable of producing cellulase having β-xylosidase activity, and step (2) is a step of recovering the cellulase having β-xylosidase activity.

11. The filtration treatment in the step (2) is a filtration treatment in which the filter aid according to any one of claims 1 to 5 prepared independently of the step (1) is added to the culture solution containing the microbial cells after culture obtained in the step (1). The method for producing cellulase according to claim 9.

12. The filtration treatment in the step (2) is a filtration treatment in which the culture solution containing the microbial cells after culture obtained in the step (1) is passed through a filter medium precoated with the filter aid according to any one of claims 1 to 5, which is prepared independently of the step (1), from the precoat side. The method for producing cellulase according to claim 9.

13. The method for producing cellulase according to claim 9, wherein the filtration treatment in step (2) is performed using a filter press.

14. The method for producing cellulase according to claim 9, wherein the microorganism capable of producing cellulase cultured in step (1) is a filamentous fungus of the genus Trichoderma or a filamentous fungus of the genus Talaromyces.

15. The method for producing cellulase according to claim 14, comprising a step of mixing a preculture solution of the filamentous fungus to be cultured in the step (1) with an inorganic filter aid to prepare the filter aid according to any one of claims 1 to 5. The method for producing cellulase according to claim 14,

16. A method for producing sugar, comprising: a step of producing cellulase by the method according to any one of claims 9 to 15; and a step of hydrolyzing cellulose-containing biomass with the cellulase obtained in the step.