Protein production method using Cornhull

Abstract

According to the present invention, it is possible to efficiently produce proteins by culturing a filamentous fungus belonging to the genus Trichoderma using a medium containing a corn hull pulverization product having a relative particle mass peak in a particle size range of 70-150 µm in a volume-based particle size distribution obtained by a measuring method based on a laser diffraction and scattering method.

Description

Technical Field

[0001] The present invention relates to a method for producing a protein using Trichoderma filamentous fungi with corn hulls.

Background Art

[0002] Trichoderma filamentous fungi are known to have a high protein production ability, and studies on protein production using these filamentous fungi have been conducted. Trichoderma filamentous fungi are known for their high ability to produce cellulase, which is classified as a saccharifying enzyme among proteins, using substances such as cellulose, lactose, and cellobiose as inducers. To enhance the protein production amount, many studies have been carried out on modifying genes that control cellulase production and optimizing culture conditions since ancient times.

[0003] Patent Document 1 discloses that when culturing Trichoderma filamentous fungi, adding soybean hulls, pulp, corn hulls, etc. to the culture medium improves the productivity of proteins, especially the productivity of cellulase.

[0004] In addition, as a method for improving the production amount of cellulase of Trichoderma filamentous fungi, Non-Patent Document 1 describes a method of culturing Trichoderma filamentous fungi in a culture medium added with glucose or lactose.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Non-Patent Documents

[0006]

Non-Patent Document 1

[0007] The objective is to provide a method for further increasing protein productivity using filamentous fungi of the genus Trichoderma. [Means for solving the problem]

[0008] The inventors focused particularly on Cornhull as a substance that improves the protein productivity of Trichoderma filamentous fungi and conducted intensive research. As a result, they found that when Trichoderma filamentous fungi were cultured with Cornhull pulverized materials of various particle sizes, which were prepared by grinding Cornhull, and which had a relative particle amount peak in the range of 70 μm to 150 μm in volume-based particle size distribution obtained by laser diffraction and scattering measurement, were added to the culture medium, the effect of improving protein productivity was high, leading to the completion of the present invention.

[0009] In other words, the present invention consists of the following (1) to (9). (1) A method for producing protein, comprising the step of culturing Trichoderma filamentous fungi using a culture medium containing Cornhull pulverized material having relative particle amount peaks in the range of particle diameter 70 μm to 150 μm in a volume-based particle size distribution obtained by a measurement method using laser diffraction and scattering. (2) The method for producing a protein according to (1), wherein the culture medium comprises glucose and / or lactose. (3) The method for producing a protein according to (1) or (2), wherein the culture method in the culture step is deep culture. (4) A method for producing a protein according to any one of (1) to (3), comprising the steps of: maintaining the culture medium obtained in the culture step at a temperature of 25°C or higher and 55°C or lower; and separating the culture medium after the holding step from a solid-liquid fraction to recover the protein from the liquid fraction. (5) A method for producing a protein according to any one of (1) to (4), wherein the protein is cellulase. (6) The method for producing the protein according to (5), wherein the cellulase exhibits β-glucosidase activity that degrades at least 4-nitrophenyl-β-D-glucopyranoside. A method for producing sugar, comprising the steps of producing cellulase by the protein production method described in (7)(5) or (6), and saccharifying cellulose-containing biomass using the cellulase obtained in the first step. (8) Cornhull milled material having a relative particle quantity peak in the range of particle diameters between 70 μm and 150 μm in the volume-based particle size distribution obtained by the laser diffraction-scattering method. A culture medium containing the cornhull pulverized material described in (9)(8). [Effects of the Invention]

[0010] This invention makes it possible to efficiently produce proteins by culturing Trichoderma filamentous fungi using Cornhull pulverized material that has a relative particle amount peak in the range of particle diameter 70 μm to 150 μm in the volume-based particle size distribution obtained by a laser diffraction-scattering measurement method. [Brief explanation of the drawing]

[0011] [Figure 1] Particle size distribution results for volume-based Conehull pulverized materials A-E obtained by laser diffraction and scattering measurement method. [Figure 2] Particle size distribution results of volume-based cornhull pulverized material F obtained by the laser diffraction and scattering method. [Modes for carrying out the invention]

[0012] This invention is characterized by the efficient production of proteins by culturing Trichoderma filamentous fungi, which are microorganisms with excellent protein production capabilities, together with Cornhull pulverized material that has a relative particle amount peak in the range of 70 μm to 150 μm in particle size, as measured by a volume-based particle size distribution obtained by laser diffraction and scattering methods.

[0013] The filamentous fungi of the genus Trichoderma used in this invention are also called the genus Hypocrea, but are referred to as the genus Trichoderma in this specification. Specifically, the filamentous fungi of the genus Trichoderma used in this invention are preferably Trichoderma reesei, Trichoderma viride, Trichoderma atroviride, and Trichoderma longibrachiatum, and more preferably Trichoderma reesei. Furthermore, the filamentous fungi of the genus Trichoderma are not limited to wild-type strains, and mutant strains of Trichoderma that have been improved to enhance protein production capacity can also be preferably used. For example, mutant strains of Trichoderma can be used that have been subjected to mutation treatment with mutagens or ultraviolet irradiation, resulting in a decrease in the viscosity of the culture medium when cultured or an improvement in protein productivity. In addition, genetically modified strains that have a decrease in the viscosity of the culture medium when cultured, or genetically modified strains with improved protein productivity, can also be used using genetic engineering technology. Alternatively, mutant strains obtained by combining the aforementioned mutagens or mutation treatments such as ultraviolet irradiation with genetic recombination technology may be used.

[0014] Specific examples of Trichoderma reesei include Trichoderma parareesei (ATCC MYA-4777), which is the ancestor of Trichoderma reesei, the QM6a strain (NBRC31326), the QM9123 strain (ATCC24449), the QM9414 strain (NBRC31329), the PC-3-7 strain (ATCC66589), the QM9123 strain (NBRC31327), the RutC-30 strain (ATCC56765), the CL-847 strain (Enzyme. Microbiol. Technol., 10, 341-346 (1988)), the MCG77 strain (Biotechnol. Bioeng. Symp., 8, 89 (1978)), the MCG80 strain (Biotechnol. Bioeng., 12, 451-459 (1982)), and their derivative strains. The QM6a strain, QM9414 strain, and QM9123 strain can be obtained from NBRC (NITE Biological Resource Center), and the PC-3-7 strain and RutC-30 strain can be obtained from ATCC (American Type Culture Collection).

[0015] Corn hull refers to the seed coat covering corn seeds and is part of corn seeds. Corn hull is also called corn seed coat, corn fiber, corn feed, etc., but in this specification, it is referred to as corn hull. The corn hull used in the present invention preferably has few impurities other than corn hull, such as protein and starch. The corn hull may be directly prepared from corn seeds and used, or it may be obtained as a by-product in the manufacturing process of corn starch and used.

[0016] The ground corn hull used in the present invention has a relative particle amount peak in the range of particle diameter of 70 μm or more and 150 μm or less in the volume-based particle size distribution obtained by the measurement method using laser diffraction / scattering method. Hereinafter, the ground corn hull having the relative particle amount peak of this characteristic may be described as the ground corn hull of the present invention in this specification.

[0017] The corn hull pulverized product of the present invention can be prepared by crushing corn hulls. The pulverization conditions may be either wet or dry conditions. A well-known pulverizer may be used for pulverizing the corn hulls. Specifically, a roller mill or a jet mill classified as a fine pulverizer, a hammer mill or a pin mill classified as a high-speed rotary pulverizer, a rotary mill, a vibration mill or a planetary mill classified as a container-driven mill, an attritor or a bead mill classified as an ultrafine pulverizer may be used. The pulverization conditions of the pulverizer may be adjusted so that the corn hull pulverized product pulverized by the pulverizer is measured using the measurement method shown below, and a pulverized product having a target particle size is obtained.

[0018] The particle size of the corn hull pulverized product of the present invention uses the value measured based on the laser diffraction / scattering method. Ethanol is used as the dispersant for the sample used in the measurement, and a semiconductor laser with a wavelength of 680 nm is used as the light source. The specific measurement method is as follows. First, the corn hull pulverized product of the measurement sample is mixed with ethanol used as the dispersion medium, and the mixture is treated with ultrasonic waves for 3 minutes or more until it becomes uniform to obtain a measurement sample. Then, using this sample, the intensity distribution at each scattering angle is measured at the sensor part of the device, and the particle content of each particle size is calculated from the scattering pattern of the standard sample that is closest to the actually measured scattering pattern using the scattering patterns of various standard samples calculated in advance, and the particle size distribution is obtained.

[0019] The measuring instrument for measuring based on the laser diffraction / scattering method used in the present invention may be any measuring instrument capable of detecting particle sizes of 0.01 μm or more and 1 mm or less. For example, a laser diffraction type particle size distribution measuring device “SALD-2000J” manufactured by Shimadzu Corporation may be used. Also, the measurement of the particle size is performed at least twice or more, and the average value is taken as the measured value.

[0020] The Cornhull pulverized material of the present invention is a Cornhull pulverized material having a relative particle amount peak in the range of particle diameters of 70 μm to 150 μm in the volume-based particle size distribution obtained by the laser diffraction-scattering measurement method, preferably in the range of particle diameters of 70 μm to 140 μm, more preferably in the range of particle diameters of 70 μm to 130 μm, even more preferably in the range of particle diameters of 70 μm to 120 μm, and particularly preferably in the range of particle diameters of 75 μm to 120 μm. In the present invention, having a relative particle amount peak in the above range means that the highest relative particle amount peak among the detected peaks is in the above range. Here, there are several indicators of particle size other than the relative particle amount peak, but whether or not it corresponds to the Cornhull pulverized material of the present invention is determined by whether or not it has a relative particle amount peak in the range of 70 μm to 150 μm in the volume-based particle size distribution obtained by the laser diffraction-scattering measurement method. For example, particle size is sometimes expressed as average particle diameter (sometimes referred to as average particle size, which is synonymous with average particle diameter), but the average particle diameter does not necessarily coincide with the particle diameter at which the relative particle amount peaks. Even if a Cornhull pulverized material has an average particle diameter of 70 μm or more and 150 μm or less, if it does not have a relative particle amount peak in that range, it does not qualify as a Cornhull pulverized material of the present invention.

[0021] The method for culturing Trichoderma filamentous fungi using the culture medium to which the Cornhull pulverized material of the present invention has been added is not particularly limited, and can be used for example, liquid culture using centrifuge tubes, flasks, jar fermenters, tanks, etc., or solid culture using plates, etc. Trichoderma filamentous fungi need to be cultured under aerobic conditions, and among these culture methods, deep culture in which aeration and stirring are performed in a jar fermenter or tank is particularly preferred. The aeration rate is preferably 0.1 vvm to 2.0 vvm, more preferably 0.3 vvm to 1.5 vvm, and particularly preferably 0.5 vvm to 1.0 vvm. The culture temperature is preferably 25°C to 35°C, more preferably 25°C to 31°C. The pH conditions in culture are preferably pH 3.0 to 7.0, and more preferably pH 4.0 to 6.0. The incubation period is carried out under conditions that allow for protein production, until a recoverable amount of protein is accumulated. This is typically between 24 and 288 hours, with 36 to 240 hours being more preferable.

[0022] The protein produced by the present invention is not particularly limited, but it is possible to efficiently produce a protein that is secreted outside the bacterial cell, and among these, an enzyme is preferred, more preferably a saccharifying enzyme such as cellulase, amylase, invertase, chitinase, or pectinase, and even more preferably cellulase.

[0023] To recover proteins from the culture medium obtained in this invention, the culture medium can be separated into solid and liquid components, and the proteins contained in the liquid fraction can be recovered. Solid-liquid separation can be performed by any method, such as filtration or centrifugation, and a filter press, screw decanter, or continuous centrifuge can be used.

[0024] When culturing Trichoderma filamentous fungi in a medium containing Cornhull, the resulting culture solution may not yield a clear liquid fraction during solid-liquid separation. Furthermore, when using a filter membrane or filter cloth for solid-liquid separation, the filtration time may be prolonged due to clogging of the filter membrane or filter cloth. These problems can be solved by maintaining the culture solution obtained in this invention at a constant temperature as a pretreatment for solid-liquid separation. In this specification, the process of maintaining the culture solution at a constant temperature as a pretreatment for solid-liquid separation may be referred to as a heat retention treatment.

[0025] The temperature maintained during the heat retention treatment is preferably 25°C to 55°C, more preferably 30°C to 55°C, and particularly preferably 40°C to 50°C.

[0026] The heat retention treatment should ideally be carried out for approximately 5 to 30 hours.

[0027] The heat retention treatment may be carried out under static conditions, but if the volume of culture medium is large, it is preferable to stir the treatment to ensure that the temperature of the culture medium is uniform. When stirring, it is preferable to stir gently so as not to increase the dissolved oxygen concentration in the culture medium. For example, when using a 5L jar fermenter (manufactured by Biot Co., Ltd.), a stirring speed of 50 rpm to 700 rpm is preferable, 100 rpm to 600 rpm is more preferable, and 200 rpm to 500 rpm is particularly preferable. In addition to stirring, it is preferable to perform the heat retention treatment under conditions that do not increase the dissolved oxygen concentration of the culture medium. For example, if aeration is performed during cultivation, the aeration function can be shut off and the heat retention treatment can be carried out.

[0028] When a culture medium is subjected to heat retention treatment, it is possible to obtain a clearer liquid fraction compared to a culture medium that has not been subjected to heat retention treatment. Furthermore, filtration resistance is reduced even with high-speed filtration methods such as vacuum filtration, enabling efficient solid-liquid separation. When performing vacuum filtration, it is recommended to use a plastic holder for vacuum filtration, such as a polysulfone holder (manufactured by Advantech Co., Ltd.).

[0029] When separating solids and liquids using a polysulfone holder, the filter cloth used can be, for example, a polypropylene filter cloth with an air permeability of 0.5 mL / (min*cm). 2 ) or more 500mL / (min*cm 2 A filter cloth of the following quality may be used. In this case, a pre-coat of the filter cloth with a filter aid may be applied before solid-liquid separation. Diatomaceous earth may be used as the filter aid, for example, "Radiolite" (manufactured by Showa Chemical Industry Co., Ltd.) can be used, and the grade of "Radiolite" should be between #100 and #3000. The filter aid may be mixed with the culture solution after the heat retention treatment before use. When the filter aid and the culture solution after the heat retention treatment are mixed beforehand, the filter aid should be added to the culture solution at a concentration of 1% to 20% by weight.

[0030] In addition, if the heat retention treatment is performed by leaving it undisturbed, or if the culture medium is left undisturbed after the heat retention treatment, the solid components will separate to the top and the liquid components to the bottom. Therefore, solid-liquid separation can also be performed by collecting the liquid fraction at the bottom.

[0031] The liquid fraction obtained by solid-liquid separation of the culture medium obtained in this invention can be further sterilized using an MF membrane or concentrated using a UF membrane, depending on its intended use.

[0032] The turbidity of the aforementioned liquid fraction is calculated in Nephelometric Turbidity Units (NTU). A commercially available turbidimeter can be used to measure turbidity, for example, the HACH 2100P portable turbidimeter. The turbidimeter should be calibrated in advance using a manufacturer-approved second standard solution such as formazin solution.

[0033] The turbidity of the liquid fraction is preferably 500 NTU or less, more preferably 300 NTU or less, even more preferably 200 NTU or less, and particularly preferably 100 NTU or less.

[0034] Cellulase, a protein preferably produced in the present invention, includes enzymes that have degrading activity against xylan, cellulose, and hemicellulose. Specific examples include cellobiohydrolase (EC 3.2.1.91) which produces cellobiose by hydrolysis of cellulose chains, endoglucanase (EC 3.2.1.4) which hydrolyzes from the central portion of cellulose chains, β-glucosidase (EC 3.2.1.21) which hydrolyzes cellooligosaccharides and cellobiose, xylanase (EC 3.2.1.8) which is characterized by its action on hemicellulose and especially xylan, and β-xylosidase (EC 3.2.1.37) which hydrolyzes xylooligosaccharides. When cellulase is produced according to the present invention, protein concentration and activity are improved, and this effect is particularly pronounced in β-glucosidase.

[0035] In this invention, the protein concentration is measured as follows: The culture solution obtained by culturing Trichoderma filamentous fungi using the method of this invention is centrifuged at 15,000 × g for 10 minutes, and the supernatant is used as the protein solution. 5 μL of the cellulase solution diluted in 250 μL of the Cellulase Quick Start Bradford Protein Assay (Bio-Rad) is added, and the absorbance used at 595 nm is measured after standing at room temperature for 15 minutes. Bovine serum albumin solution is used as the standard solution, and the protein concentration contained in the saccharifying enzyme solution is calculated based on the calibration curve. When measuring the protein concentration of cellulase, the protein solution can be treated as the cellulase solution.

[0036] β-glucosidase activity is measured by the following method. First, 10 μL of enzyme diluent is added to 90 μL of 50 mM acetate buffer containing 1 mM 4-nitrophenyl-β-glucopyranoside (manufactured by Sigma-Aldrich Japan Co., Ltd.), and the mixture is allowed to react at 30°C for 10 minutes. Next, 10 μL of 2 M sodium carbonate is added and the reaction is stopped by thorough mixing, and the increase in absorbance at 405 nm is measured. Finally, the activity is calculated with 1 U representing the activity that releases 1 μmol of 4-nitrophenol per minute.

[0037] The composition of the culture medium for culturing Trichoderma filamentous fungi used in this invention is not particularly limited as long as it is a culture medium composition that enables Trichoderma filamentous fungi to produce protein, other than the corn hull pulverized material of this invention, and well-known culture medium compositions for Trichoderma filamentous fungi can be used. As a nitrogen source, for example, polypeptone, meat juice, CSL, soybean meal, etc. In addition, an inducer substance for protein production may be added separately to the culture medium.

[0038] The concentration of the cornhull pulverized material of the present invention added to the culture medium should be approximately 1% by weight or more and 50% by weight or less as the final concentration in the culture medium, preferably 5% by weight or more and 30% by weight or less, and more preferably 5% by weight or more and 25% by weight or less.

[0039] Furthermore, the culture medium may contain glucose and / or lactose, and it is preferable that it contains both glucose and lactose. The amount of glucose and / or lactose added to the culture medium should be about 1 g / L each.

[0040] Glucose and / or lactose may be added during the culture using the corn hull pulverized product of the present invention. In this case, the glucose and / or lactose may be in liquid or solid form, but it is preferable to dissolve them in water or the like to prepare a sugar solution and use that. When adding glucose and / or lactose during the culture using the corn hull pulverized product of the present invention, it is preferable to add 10 g or more of glucose and / or 1 g or more of lactose per 1 L of culture medium per 24 hours of culture, more preferably 25 g or more of glucose and / or 2.5 g or more of lactose, and particularly preferably 50 g or more of glucose and / or 5 g or more of lactose.

[0041] Furthermore, the ratio of glucose to lactose in the mixed sugar added during the culture using the corn hull pulverized material of the present invention is preferably equal or greater in the amount of glucose than lactose. The timing for starting the addition of liquid sugar is preferably up to 144 hours from the start of culture, more preferably up to 72 hours from the start of culture, and particularly preferably up to 48 hours from the start of culture. The sugar may be added once, multiple times, or continuously.

[0042] When producing cellulase according to the present invention, cellulose and other inducers that induce cellulase production, such as xylan, may be added to the culture medium. Alternatively, cellulose and xylan may be used, or biomass containing cellulose and xylan may be added as inducers. Specific examples of biomass containing cellulose and xylan include plants such as seed plants, ferns, mosses, algae, and aquatic plants, as well as waste construction materials. Seed plants are classified into gymnosperms and angiosperms, and both can be preferably used. Angiosperms are further classified into monocots and dicots. Specific examples of monocots include bagasse, switchgrass, napier grass, eryanthus, corn stover, corn cob, rice straw, and wheat straw, while specific examples of dicots include beet pulp, eucalyptus, oak, and birch.

[0043] Furthermore, pre-treated biomass containing cellulose or xylan may be used. The pre-treatment method is not particularly limited, but known methods such as acid treatment, sulfuric acid treatment, dilute sulfuric acid treatment, alkali treatment, hydrothermal treatment, subcritical treatment, pulverization treatment, and steaming treatment can be used. Pulp may be used as such pre-treated cellulose or xylan-containing biomass.

[0044] Furthermore, while it is possible to use the culture medium containing the Trichoderma filamentous fungi as an enzyme solution without removing the fungal cells from the culture medium obtained by culturing the corn hull pulverized material of the present invention and Trichoderma filamentous fungi, it is preferable to treat the culture medium in a way that prevents the growth of Trichoderma filamentous fungi when using it as a protein lysis solution. Methods for treating the culture medium in a way that prevents the growth of fungal cells include heat treatment, chemical treatment, acid / alkali treatment, and UV treatment.

[0045] Furthermore, if the protein to be produced is cellulase, the cellulase can be used to saccharify cellulose-containing biomass and produce sugar. In addition, the cellulase obtained by culturing the mutant strain has particularly high β-glucosidase activity compared to the cellulase obtained by culturing the parent strain before the mutation was introduced. Therefore, it can efficiently decompose cellulose-containing biomass to obtain a sugar solution with a high glucose concentration, and thus produce more sugar.

[0046] Furthermore, biomass similar to the cellulose-containing biomass described above as an inducer, or pre-treated biomass, may be used as the target for saccharification.

[0047] The conditions for the saccharification reaction are not particularly limited, but the temperature of the saccharification reaction is preferably in the range of 25°C to 60°C, and more preferably in the range of 30°C to 55°C. The duration of the saccharification reaction is preferably in the range of 2 hours to 200 hours. The pH of the saccharification reaction is preferably in the range of pH 3.0 to pH 7.0, and more preferably in the range of pH 4.0 to pH 6.0. In the case of cellulase derived from Trichoderma filamentous fungi, the optimal pH for the reaction is 5.0. Furthermore, since a change in pH occurs during the hydrolysis process, it is preferable to add a buffer to the reaction solution or to carry out the reaction while maintaining a constant pH using an acid or alkali.

[0048] When separating and recovering enzymes from the saccharification solution, the solution can be filtered using an ultrafiltration membrane or similar method, and the enzymes can be recovered on the impermeable side. If necessary, solid components may be removed from the saccharification solution as a pre-filtration step. The recovered enzymes can be reused in the saccharification reaction. [Examples]

[0049] The present invention will be specifically described below with reference to examples.

[0050] <Reference Example 1> Protein concentration measurement conditions Protein concentration measurement reagent: Quick Start Bradford Protein Assay (Bio-Rad) Measurement conditions Measurement temperature: room temperature Protein concentration measurement reagent: 250 μL Culture medium for filamentous fungi: 5 μL Reaction time: 5 minutes Absorbance: 595nm Standard product: BSA.

[0051] <Reference Example 2> Measurement conditions for cellulase activity (Measurement conditions for β-glucosidase activity) Substrate: 4-nitrophenyl-β-glucopyranoside (manufactured by Sigma-Aldrich Japan Co., Ltd.) Reaction solution: 90 μL of 50 mM acetate buffer containing 1 mM 4-nitrophenyl-β-glucopyranoside Enzyme diluent: 10 μL Reaction temperature: 30℃ Reaction time: 10 minutes Reaction stopper: 10 μL of 2M sodium carbonate Absorption: 405nm.

[0052] <Reference Example 3> Measurement conditions for particle size distribution (Pre-processing) Dispersion medium: Primary ethanol Dispersion medium usage: 10mL Cell: Quantity Cell Ultrasonic treatment conditions: Oscillation frequency 40kHz, 3 minutes.

[0053] (Particle size distribution measurement) Equipment: Laser diffraction particle size distribution analyzer SALD-2000J (manufactured by Shimadzu Corporation) Light source: Semiconductor laser (wavelength: 680nm) Sensor: 76-element diffraction / scatter light sensor Measurement temperature: 22~25℃ Complex refractive index of particles: 2.00~0.20i (standard refractive index value in the instrument manual) Particle measurement range: 0.03~700μm Measurement sample size: 2 (measurements were taken twice using different measuring solutions, and the average value was used).

[0054] <Test Example 1> Measurement of volume-based particle size distribution of Cornhull pulverized materials A to F by laser diffraction and scattering method. Corn hull (manufactured by Taihuangdao Liuyong Agricultural Products Processing Co., Ltd.) was crushed by Materis Co., Ltd., and corn hull pulverized material was prepared. The particle size of five types of corn hull pulverized material was measured using the method of Reference Example 3, and relative particle size peaks were confirmed in the volume-based particle size distribution obtained by the laser diffraction-scattering method at particle sizes of 34.3 μm, 41.9 μm, 114.5 μm, 171.2 μm, and 256.0 μm.

[0055] Hereafter, the Cornhull pulverized material with a relative particle amount peak at a particle size of 34.3 μm will be referred to as Cornhull pulverized material A, the Cornhull pulverized material with a relative particle amount peak at a particle size of 41.9 μm will be referred to as Cornhull pulverized material B, the Cornhull pulverized material with a relative particle amount peak at a particle size of 114.5 μm will be referred to as Cornhull pulverized material C, the Cornhull pulverized material with a relative particle amount peak at a particle size of 171.2 μm will be referred to as Cornhull pulverized material D, the Cornhull pulverized material with a relative particle amount peak at a particle size of 256.0 μm will be referred to as Cornhull pulverized material E, and the Cornhull pulverized material with a relative particle amount peak at a particle size of 76.6 μm will be referred to as Cornhull pulverized material F. The particle size distribution of Cornhull pulverized materials A to E is shown in Figure 1, and the particle size distribution of Cornhull pulverized material F is shown in Figure 2.

[0056] Furthermore, Cornhull pulverized material D is the same pulverized material with an average particle size of 100 μm disclosed in Comparative Example 1 of International Publication No. 2018 / 159573.

[0057] <Example 1> Batch culture using Cornhull pulverized material C (preculture) Spores of Trichoderma reesei PC-3-7 strain (ATCC#66589) are 1.0 × 10 7 The solution was diluted with physiological saline to a concentration of 1 mL / mL, and 1 mL of this diluted spore solution was inoculated into 100 mL of pre-culture medium in a 500 mL baffled flask as shown in Table 1. The culture was then performed in a shaking incubator at 28°C and 120 rpm for 72 hours.

[0058] [Table 1]

[0059] (main culture) The main culture medium was prepared with the composition shown in Table 2 to contain 100 g / L of the corn hull pulverized material C prepared in Test Example 1, and deep culture studies were conducted using a microjar fermenter (Bio-Jr.8, manufactured by Biot Co., Ltd.).

[0060] This culture was performed by inoculating 10 mL of pre-culture medium of Trichoderma reesei PC-3-7 strain into 100 mL of the main culture medium.

[0061] The culture conditions were as follows: after inoculating the culture medium with the pre-culture solution, deep culture was performed for 96 hours at 28°C, 900 rpm, and an aeration rate of 100 mL / min, while maintaining a controlled pH of 5.

[0062] [Table 2]

[0063] (Collection of culture medium) 1 mL of culture medium was collected 96 hours after the start of culture. The culture medium was centrifuged at 15,000 × g at 4°C for 10 minutes to obtain the supernatant. The supernatant was filtered through a 0.22 μm filter, and the resulting filtrate was used as the protein solution and cellulase solution in the following experiments.

[0064] (Measurement of protein concentration) Using the method described in Reference Example 1, the cellulase protein concentration in the culture medium was measured 96 hours after the start of culture. As a result, the protein concentration in the culture medium was 9.6 g / L.

[0065] (Measurement of β-glucosidase activity) Under the conditions of Reference Example 2, the activity of β-glucosidase was measured in the culture medium collected during the culturing process. Activity was calculated by measuring the increase in absorbance at 405 nm, with 1 U representing the activity that releases 1 μmol of substrate per minute. The activity of the culture medium obtained from culturing with Cornhull pulverized material C was 2.5 U / mL. The results are shown in Table 3.

[0066] <Comparative Example 1> Batch culture using Cornhull pulverized materials A, B, D, and E (Measurement of protein concentration) Batch culture tests were conducted under the same conditions as in Example 1, except that Cornhull pulverized materials A, B, and E prepared in Test Example 1 were used. The culture time was 96 hours for cultures using Cornhull pulverized material A or B, and extended to 120 hours for cultures using Cornhull pulverized material D or E because the protein concentration in the culture medium was low. Subsequently, using the method described in Reference Example 1, the cellulase protein concentration in the culture medium was measured at 96 hours from the start of culture for cultures using Cornhull pulverized material A and B, and at 120 hours from the start of culture for cultures using Cornhull pulverized material D and E. As a result, the protein concentration in the culture medium obtained using the medium containing Cornhull pulverized material A was 9.0 g / L, the protein concentration in the culture medium obtained using the medium containing Cornhull pulverized material B was 9.1 g / L, the protein concentration in the culture medium obtained using the medium containing Cornhull pulverized material D was 8.0 g / L, and the protein concentration in the culture medium obtained using the medium containing Cornhull pulverized material E was 7.2 g / L. The results are shown in Table 3.

[0067] (Measurement of β-glucosidase activity) Under the conditions of Reference Example 2, the activity of β-glucosidase was measured in the culture medium collected during the culturing process. Activity was calculated by measuring the increase in absorbance at 405 nm, with 1 U representing the activity that releases 1 μmol of substrate per minute. As a result, the activity in the culture medium of Cornhull pulverized material A was 2.1 U / mL, the activity in the culture medium of Cornhull pulverized material B was 2.0 U / mL, the activity in the culture medium of Cornhull pulverized material D was 1.7 U / mL, and the activity in Cornhull pulverized material E was 1.2 U / mL. The results are shown in Table 3.

[0068] [Table 3]

[0069] <Example 2> Batch culture using Cornhull pulverized material C and culture with added glucose and lactose (preculture) Spores of Trichoderma reesei PC-3-7 strain (ATCC#66589) are 1.0 × 10 7 Dilute with physiological saline to a concentration of / mL, and inoculate 2.5 mL of this diluted spore solution into 250 mL of pre-culture medium in a 1 L baffled flask as shown in Table 1. The cells were incubated in a shaking incubator at 28°C and 120 rpm for 72 hours.

[0070] (main culture) Using the corn hull pulverized material C prepared in Test Example 1 at a concentration of 100 g / L, the culture medium with the composition shown in Table 2 was prepared, and deep culture was performed using a 5 L jar fermenter (manufactured by Biot Co., Ltd.).

[0071] 200 mL of pre-culture solution of Trichoderma reesei PC-3-7 strain was inoculated into 2 L of the main culture medium, each supplemented with Cornhull pulverized material C. Pre-culture was performed in the same manner as in Test Example 1. For the main culture, after inoculating the pre-culture medium into the main culture medium, deep culture was performed for 240 hours at 28°C, 700 rpm, and an aeration rate of 2 L / min, while maintaining a pH of 5.0.

[0072] (Addition of glucose and lactose during the culture process) Ten hours after the start of the culture, 250 mL of the glucose and lactose liquid sugar medium shown in Table 4 was added to the culture medium daily until 240 hours after the start of the culture.

[0073] [Table 4]

[0074] (Collection of culture medium) 20 mL of culture medium was collected at each time point following the start of cultivation. A portion of the collected culture medium was centrifuged at 15,000 × g at 4°C for 10 minutes to obtain the supernatant. The supernatant was filtered through a 0.22 μm filter, and the resulting filtrate was used as cellulase solution in the following experiments.

[0075] (Measurement of protein concentration) The protein concentration was measured over time from the culture medium cultured using Cornhull pulverized material C under the conditions of Reference Example 1. The relative value of the maximum protein concentration in Example 2 to the protein concentration in the batch culture of Example 1 was determined, and the protein concentration was increased by 3.3 times. The results are shown in Table 5.

[0076] <Example 3> Batch culture using Cornhull pulverized material F and culture with added glucose and lactose A batch culture test was performed under the same conditions as in Example 1, except that Cornhull pulverized material F was used. After the culture test, samples were prepared in the same manner as in Example 1 and measured under the conditions of Reference Examples 1 and 2. As a result, in the batch culture using Cornhull pulverized material F, the cellulase protein concentration in the culture medium at 96 hours after the start of culture was 9.4 g / L, and the β-glucosidase activity was 2.5 U / mL. The results are shown in Table 3.

[0077] A culture test with glucose and lactose was conducted under the same conditions as in Example 2, except that corn hull pulverized material F was used. After the culture test, samples were prepared in the same manner as in Example 2 and measured under the conditions of Reference Example 1. The relative value of the maximum protein concentration in the culture with glucose and lactose added to the protein concentration in the batch culture was determined, and it was found that the protein concentration increased by 3.4 times. The results are shown in Table 5.

[0078] <Comparative Example 2> Batch culture using Cornhull pulverized materials A, B, and D, and culture with added glucose and lactose. The culture test was performed under the same conditions as in Example 2, except that Cornhull pulverized materials A, B, and D were used. After the culture test, samples were prepared in the same manner as in Example 2 and measured under the conditions of Reference Example 1 to determine the relative value of the maximum protein concentration during liquid sugar-added culture to the protein concentration in the batch culture of Comparative Example 1. As a result, the value was 2.6 when Cornhull pulverized material A or B was used, and 1.8 when Cornhull pulverized material D was used. The results are shown in Table 5.

[0079] [Table 5]

[0080] <Example 4> Solid-liquid separation using heat-treated culture medium (Heat retention treatment) The culture was carried out under the conditions of Example 2, and 96 hours after the start of culture, the pH control and aeration of the 5L jar fermenter were turned off. A portion of the culture solution was taken from the culture tank and subjected to solid-liquid separation treatment. The remaining culture solution was kept warm for 12 hours with the culture tank's stirring speed set to 400 rpm and the temperature set to 40°C or 50°C.

[0081] (Solid-liquid separation treatment) A polypropylene filter cloth (manufactured by Hitachi Zosen Corporation) was placed in a polysulfone holder KP-47S (manufactured by Advantec Corporation). RO water containing 10% (w / v) "Radiolite" #700 (manufactured by Showa Chemical Industry Co., Ltd.) was added, and the pressure was reduced to form a precoat layer. Then, a solution made by adding 1 g of "Radiolite" #700 to 20 mL of a heat-treated culture medium and mixing thoroughly was added, and solid-liquid separation was performed under reduced pressure.

[0082] (Turbidity measurement) The turbidity of 20 mL of the filtrate obtained by solid-liquid separation was measured using a portable turbidimeter, model 2100P (HACH). As a result, the NTU was 804 when no heat retention treatment was performed, compared to 303 when heat retention treatment was performed at 40°C and 90 when heat retention treatment was performed at 50°C. This indicates that heat retention treatment significantly reduced the turbidity of the filtrate, resulting in a clearer liquid fraction.

[0083] <Summary> From the results of the examples and comparative examples shown in Tables 3 and 5, when Trichoderma filamentous fungi were cultured using the Cornhull pulverized material of the present invention, which has a relative particle amount peak in the range of particle diameters from 70 μm to 150 μm in the volume-based particle size distribution obtained by the laser diffraction-scattering method, the activity of β-glucosidase and protein concentration in the culture medium were higher, and protein productivity was improved compared to cultures using Cornhull pulverized material of other particle sizes. Furthermore, in culture tests in which glucose and lactose were added during the culture process, the effect of adding glucose and lactose on improving the protein concentration in the culture medium was particularly high when using the Cornhull pulverized material of the present invention.

Claims

1. A method for producing protein, comprising the step of culturing Trichoderma filamentous fungi using a culture medium containing Cornhull pulverized material having a relative particle amount peak in the range of particle diameter 70 μm to 120 μm in a volume-based particle size distribution obtained by a laser diffraction / scattering measurement method.

2. The method for producing a protein according to claim 1, wherein the culture medium comprises glucose and / or lactose.

3. The method for producing a protein according to claim 1 or 2, wherein the culture method in the culture step is deep culture.

4. A method for producing a protein according to any one of claims 1 to 3, comprising the steps of: maintaining the culture solution obtained in the culture step at a temperature of 25°C or higher and 55°C or lower; and separating the culture solution after the holding step from a solid-liquid fraction to recover the protein from the liquid fraction.

5. A method for producing a protein according to any one of claims 1 to 4, wherein the protein is cellulase.

6. The method for producing a protein according to claim 5, wherein the cellulase exhibits β-glucosidase activity that degrades at least 4-nitrophenyl-β-D-glucopyranoside.

7. A method for producing sugar, comprising the steps of producing cellulase by the protein production method described in claim 5 or 6, and saccharifying cellulose-containing biomass using the cellulase obtained in the first step.

8. Cornhull pulverized material having a relative particle quantity peak in the range of particle diameters between 70 μm and 120 μm, as measured by a volume-based particle size distribution obtained by laser diffraction and scattering methods.

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