JP7446089B2 - Method for producing cellulase - Google Patents

Description

The present invention relates to a method for producing cellulase using microorganisms.

Filamentous fungi are attracting attention as fungi that degrade plant polysaccharides because they produce a wide variety of cellulases and xylanases. Among them, Trichoderma is being researched as a microorganism for producing cellulase-based biomass-degrading enzymes because it can simultaneously produce cellulase and xylanase in large quantities.

Glucose has conventionally been widely used as a carbon source in the cultivation of microorganisms. Furthermore, in addition to a carbon source, an inducer may be required for the production of proteins such as enzymes by microorganisms. For example, expression of the α-amylase gene of Aspergillus oryzae is induced by starch, maltose, and the like.

Patent Document 1 describes a first step for bacterial growth in the presence of a carbonaceous growth substrate in a batch phase, and a first step for bacterial growth in the presence of an induced carbonaceous substrate and an enzyme in a fed-batch phase. A method for producing cellulase using filamentous fungi, comprising a second step for production, and the carbonaceous growth substrate is ethanol of monomeric sugars such as lactose, glucose, xylose, and enzymatic hydrolysates of cellulosic biomass. The derivatized carbonaceous substrate is selected from the residue obtained after fermentation and the crude extract of water-soluble pentoses derived from the pretreatment of cellulosic biomass, and the derivatized carbonaceous substrate is a monomer of lactose, cellobiose, sophorose, enzymatic hydrolyzate of cellulosic biomass. and the crude extract of water-soluble pentoses derived from the pretreatment of cellulosic biomass. Patent Document 2 describes a method for producing cellulose- or hemicellulose-degrading enzymes using fungi such as Trichoderma, in which ethanol of an enzymatic hydrolyzate of a cellulosic or ligno-cellulosic material is used as a carbon source and a derived carbon source for enzyme production. The use of residues from fermentation (including glucose, xylose, etc.) is disclosed. Patent Document 3 discloses that in a fed-batch or continuous fermentation medium, the rate of carbon addition to the fermentation medium is controlled based on the amount of carbon added to the medium and the amount of oxygen consumed or the amount of carbon lost to carbon dioxide. A method is disclosed, and the use of the method to cause microorganisms to produce proteins, such as enzymes, is disclosed. The expression of major cellulase genes cbh1, cbh2, egl1, and egl2 of Trichoderma is induced by cellulose, cellobiose, etc. (Non-Patent Document 1).

International Publication No. 2013/026964 JP2006-217916A International Publication No. 2013/124351

Curr. Genomics, 14:230-249 (2013)

There is a need for the development of culture techniques for producing proteins such as enzymes inexpensively and efficiently using microorganisms. In the presence of glucose, a control mechanism called catabolite suppression causes a decrease or saturation of material productivity by microorganisms. For example, catabolite suppression has been reported in filamentous fungi such as Trichoderma, and the mechanism is being analyzed. Fed-batch culture, in which glucose is repeatedly or continuously fed into the culture, may inhibit catabolite suppression and improve microbial protein productivity. However, protein production by glucose fed-batch culture requires precise control of the glucose concentration in the culture to prevent catabolite suppression.

In the production of cellulase using microorganisms, the present inventors determined the ratio of induced carbon substrates such as cellulose that induce cellulase production and non-induced carbon substrates such as glucose depending on the state of respiratory activity of the microorganisms. It has been found that cellulase productivity of microorganisms can be improved by adjusting the supply to the culture medium.

The present invention is a method for producing cellulase, comprising:
culturing the filamentous fungus in the presence of an induced carbon substrate and a non-induced carbon substrate;
During the period when the rate of change in the respiratory activity of the filamentous fungus is 0.1 or more, the ratio R represented by the following formula A is 100 or less,
Formula A: R=Feed Rate of Underivatized Carbon Substrate/Feed Rate of Derived Carbon Substrate Methods are provided.

The present invention can improve the yield of cellulase from microorganisms.

Relative cellulase productivity of Comparative Example 1, Example 1, Example 2, and Example 3 on days 1 to 7 of culture. The cellulase productivity of Comparative Example 1 on the 7th day of culture is defined as 100%.

The present invention provides a method for producing cellulase using a microorganism. The method for producing cellulase according to the present invention includes culturing a cellulase-producing microorganism in the presence of an induced carbon substrate and a non-induced carbon substrate.

Examples of cellulase-producing microorganisms to be cultured in the method of the present invention include bacteria, yeast, filamentous fungi, and among these, filamentous fungi are preferred. Examples of filamentous fungi include Acremonium genus, Aspergillus genus, Aureobasidium genus, Bjerkandera genus, Ceriporiopsis genus, Chrysosporium genus, Coprinus genus, Coriolus genus, Cryptococcus Us, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora Genus, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Rhizopus, Schizophrenia phyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, and Trichoderma Among these, Acremonium, Aspergillus, Chrysosporium, Fusarium, Humicola, Myceliophthora, and B Neurospora, Penicillium, Piromyces, Talaromyces, Thermoascus, Thielavia, and Trichoderm a) is preferred. From the viewpoint of cellulase productivity and biomass saccharification performance of the obtained cellulase, Trichoderma is more preferred, and Trichoderma reesei and its mutants are even more preferred. Examples of Trichoderma reesei and mutant strains thereof include Trichoderma reesei QM9414 strain, PC-3-7 strain, and mutant strains thereof. Examples of the mutant strain include mutant strains produced by modifications such as gene mutation and genetic recombination.

The induced carbon substrate used for culturing the microorganism may be any carbon substrate that induces cellulase expression in the microorganism. Examples of the induced carbon substrate include saccharides that induce cellulase expression in microorganisms, such as at least one selected from the group consisting of lactose, cellobiose, sophorose, gentiobiose, and cellulose. These derivatized carbon substrates are essential carbon substrates in the method of the present invention in order to culture and produce cellulase, which is generally referred to as an inducible enzyme. From the viewpoint of cellulase inducibility and cost, preferably the derivatized carbon substrate is cellulose. The cellulose may be crystalline cellulose, cellulosic biomass, or pulverized products thereof.

A non-inducing carbon substrate used for culturing the microorganism is a carbon substrate that does not induce cellulase expression in the microorganism and generally results in catabolite suppression (cessation of cellulase expression) in the microorganism. Examples of the non-inducing carbon substrate include sugars that do not induce cellulase expression in microorganisms, such as at least one selected from the group consisting of glucose, fructose, sucrose, maltose, and maltooligosaccharides. Among these, at least one selected from the group consisting of glucose, maltose, and maltooligosaccharides is preferred from the viewpoint of versatility and assimilation in microbial culture.

The derivatized carbon substrate is preferably added in batches to the culture vessel. More preferably, the derivatized carbon substrate is added to the initial medium. The initial concentration of the induced carbon substrate in the culture vessel (eg the concentration of the induced carbon substrate in the initial medium) is preferably between 1 and 15% w/v. On the other hand, it is preferable that the non-induced carbon substrate be added to the culture tank continuously (for example, fed-batch) from the viewpoint of avoiding suppression of microbial catabolites. More preferably, the uninduced carbon substrate is fed to the culture tank. For example, an aqueous solution of the non-induced carbon substrate may be added to a culture tank. The concentration of the uninduced carbon substrate in the fed aqueous solution is preferably 2 to 90% by weight/volume, more preferably 5 to 80% by weight/volume. If the concentration of the non-induced carbon substrate in the aqueous solution to be fed is too low, a large amount of the aqueous solution will be fed to the culture, which will place a burden on the culture equipment. On the other hand, if the concentration of the non-induced carbon substrate in the aqueous solution is too high, it becomes difficult to control the amount of non-induced carbon groups added to the culture.

The induced carbon substrate and non-induced carbon substrate used in the culture are preferably sterilized. When adding the carbon substrate in batches, it may be sterilized after being introduced into the culture tank together with the initial medium etc., or it may be sterilized in advance and then added to the culture tank. When feeding the carbon substrate, it may be sterilized in advance and then fed into the culture tank. For sterilization of the carbon substrate, generally heat sterilization, pressure heat sterilization using a high-pressure steam sterilizer (autoclave), sterilization by spraying saturated steam, etc. may be employed. Sterilization conditions include conditions that reduce the number of spores of Geobacillus stearothermophilus, a heat-resistant bacterium, by 10 ^-12 times (e.g., equivalent to 20 minutes at 120° C.) or harsher conditions.

In one example of the method of the invention, a culture vessel is introduced with an initial medium containing the derivatized carbon substrate, and then the culture vessel is sterilized. After the microorganisms are seeded in the sterilized culture tank, the microorganisms are cultured while being fed with the aqueous solution of the non-induced carbon substrate.

The initial medium used for the culture may be any medium commonly used for the microorganism to be cultured. For example, the initial medium may contain microorganisms such as a carbon source including the above-mentioned induced carbon substrate, a nitrogen source, metal salts such as magnesium salts and zinc salts, sulfates, phosphates, pH adjusters, surfactants, and antifoaming agents. It can contain various components commonly included in culture media. The component composition in the medium can be selected as appropriate. The initial medium may be a synthetic medium, a natural medium, a semi-synthetic medium, or a commercially available medium. The initial medium is preferably a liquid medium.

Feeding the non-induced carbon substrate to the culture vessel may be carried out according to conventional fed-batch culture procedures. For example, an aqueous solution of a non-induced carbon substrate may be added to the culture tank while controlling the amount of addition using a general feed controller or the like. The addition of the non-inducing carbon substrate to the culture tank may be started at the same time as the start of the culture, or after the rate of change in the respiration activity of the microorganisms described below reaches a certain value (for example, 0.1 or more). Good too. It is convenient to start feeding the non-induced carbon substrate at the same time as the start of culture. The initial value of the flow rate of the non-induced carbon substrate is preferably 0.15 to 0.50 g/L-initial medium/h in terms of carbon concentration, but it can be adjusted depending on the induced carbon substrate concentration and bacterial cell concentration. be.

In the method of the present invention, the amount of the induced carbon substrate and the non-induced carbon substrate supplied to the culture medium during microbial culture is adjusted depending on the state of respiratory activity of the microorganism to be cultured. More specifically, during a period in which the rate of change in the respiratory activity of microorganisms during culture is 0.1 or more, the ratio R of the supply rate of the non-induced carbon substrate to the supply rate of the induced carbon substrate [R = non-induced carbon substrate feed rate of the derivatized carbon substrate] is adjusted to 100 or less, preferably 50 or less, more preferably 10 or less. On the other hand, from the viewpoint of reducing consumption of the induced carbon substrate, the ratio of the supply rate of the non-induced carbon substrate to the rate of supply of the induced carbon substrate is preferably 1 or more.

From the viewpoint of metabolic control of microorganisms, in the method of the present invention, the rate of change in the respiratory activity of the microorganism during culture is 0.01 or more following a period in which the rate of change in the respiratory activity of the microorganism is 0.01 or more. In the above period, it is preferable to adjust the ratio R to 100 or less, preferably 50 or less, more preferably 10 or less.

From the viewpoint of metabolic control of microorganisms, in the method of the present invention, the rate of change in the respiratory activity of the microorganism is 0.001 or more following a period in which the rate of change in the respiratory activity of the microorganism is 0.01 or more during culture. It is further preferable to adjust the ratio R to 100 or less, preferably 50 or less, and more preferably 10 or less during a certain period.

In this specification, the respiratory activity of a microorganism is calculated by dividing the rate of CO ₂ excretion from a culture derived from bacterial respiration by the bacterial cell concentration of the culture. For example, the respiratory activity of a microorganism can be calculated according to the following formula (1). Further, the CO ₂ emission rate can be calculated by determining the volume of the culture and the rate of change in the CO ₂ emission per unit time from the total CO ₂ emission per unit time [g-CO ₂ ] of the culture. For example, the total amount of CO ₂ discharged per unit time [g-CO ₂ ] of the culture can be calculated according to the following formula (2) based on the CO ₂ concentration [vol%] of the culture and the amount of culture aeration.
Respiratory activity [g-CO ₂ /g-dry cell/h]
= (CO ₂ emission rate [g-CO ₂ /L-culture/h]) ÷ (bacterial cell concentration [g-dry cell/L-culture]) …(1)
here,
g-CO ₂ = (CO ₂ concentration [vol%] ÷ 100 × culture aeration rate [L/min] × 60 [min])
÷ ([molar volume] × 44[CO ₂ molar mass]) …(2)
Here, CO ₂ concentration [vol%]: CO ₂ concentration in culture (vol%)
g-dry cell: Dry mass (g) of bacterial cells contained in the culture when measuring _CO2 concentration [vol%]
L-Culture: Culture volume (L) when measuring _CO2 concentration [vol%]

The CO ₂ concentration [vol%] of the culture can be measured using an exhaust gas analyzer for culture tanks. As the exhaust gas analyzer, a non-dispersive infrared absorption type exhaust gas analyzer (for example, DEX-1562A; manufactured by Biot Co., Ltd.) can be used, but the exhaust gas analyzer is not limited thereto. The amount of aeration in the culture can be measured using a flow meter (for example, a flow meter manufactured by Cofloc Co., Ltd.). For example, it is preferable to measure the flow rate while controlling the ventilation amount using a flow meter with a valve (for example, manufactured by COFLOC Co., Ltd.).

In the method of the present invention, the respiratory activity of microorganisms can be calculated hourly according to equations (1) and (2) above. From the respiratory activity measured over time, the rate of change in the respiratory activity can be determined. That is, the rate of change in respiratory activity is calculated by dividing the difference in respiratory activity calculated at two consecutive measurement points by the respiratory activity calculated at an earlier point in time, as shown in equation (3) below.
Rate of change in respiratory activity = {｜Respiratory activity at time t ₂ - Respiratory activity at time t ₁ |
÷ (respiratory activity at time t ₁ )} (t ₂ > t ₁ , | t ₁ − t ₂ | = 1)…(3)

In the present invention, the feed rate of the derivatized carbon substrate when added batchwise is defined as the concentration change per unit time (disappearance rate) of carbon from the derivatized carbon substrate in the culture. The rate of carbon disappearance from the derived carbon substrate can be measured, for example, by the following procedure:
(1) First, the solid content is separated from the sampled culture by centrifugation or the like. This substantially eliminates uninduced carbon substrates that dissolve in the medium. The obtained solid content is dried and the mass of the dry solid content is measured. Next, the dry solid content is subjected to elemental analysis to determine the total carbon content in the dry solid content. The amount of carbon derived from the induced carbon substrate is determined by subtracting the amount of carbon derived from microorganisms from the total carbon amount. That is, assuming that the C/N ratio of microorganisms during culture is constant, the C/N ratio of microorganisms calculated by measuring the microorganism sample during seed culture and the amount of nitrogen in the dry solid content. From this, the amount of carbon derived from microorganisms in the dry solid content is calculated. The difference between the total carbon content of the dry solid content and the carbon content derived from microorganisms is calculated as the carbon content derived from the induced carbon substrate, and by dividing this by the amount of the culture, the carbon derived from the induced carbon substrate in the culture is calculated. Concentration is required.
(2) Next, determine the difference between the carbon concentrations derived from the induced carbon substrate calculated at two different time points. The difference value reflects the concentration change in carbon concentration from the induced carbon substrate in the culture between the two time points. Therefore, by calculating the concentration change per unit time from the difference value, the supply rate of the induced carbon substrate to the culture [g-carbon derived from the induced carbon substrate/L-culture solution/h] can be determined. .

In the present invention, the feed rate of fed uninduced carbon substrate is defined as the amount of carbon from the uninduced carbon substrate fed into the culture per unit time. For example, the feed rate of the non-induced carbon substrate [g-carbon derived from the non-induced carbon substrate/L-culture liquid/h] can be determined.

Alternatively, in the present invention, the feed rate of the induced carbon substrate when fed-batch is defined as the amount of carbon from the induced carbon substrate that is fed into the culture per unit time. Furthermore, in the present invention, the feed rate of the batch-added non-induced carbon substrate is defined as the concentration change (disappearance rate) per unit time of carbon derived from the non-induced carbon substrate in the culture. The specific calculation procedure for the feeding rate of the carbon substrate added in fed-batch and batchwise manner is as described above.

In the method of the present invention, conditions for culturing the microorganisms, other than the above-mentioned supply rate of the carbon substrate, can be appropriately set according to the species of the microorganisms, the scale of the culture, etc. according to conventional methods. For example, conventionally known culture tanks can be used as appropriate for the culture tank. Specific examples include flasks, aeration-stirring type culture tanks, bubble column type culture tanks, and fluidized bed type culture tanks, with aeration-stirring type culture tanks being preferred. For example, when the microorganism is a filamentous fungus, the culture temperature is preferably 25 to 35°C, more preferably 28±2°C. For example, when the microorganism is a filamentous fungus, the pH of the culture is preferably maintained at pH 3 to 7, more preferably at pH 3.5 to 6. Adjustment of the pH of the culture can be carried out with conventional pH adjusting agents such as ammonia. The pH of the culture in the present invention refers to a value measured at a culture temperature of 28°C. The pH of the culture can be measured with an electrode attached to the culture tank. The culture period is preferably 4 to 10 days.

After culturing, the desired cellulase is collected from the culture. In the case of secreted cellulase such as filamentous fungal cellulase, the cellulase can be recovered from the culture supernatant. If cellulase is contained in the cell, the cellulase can be recovered by disrupting the cell and removing a fraction containing the cellulase. Cellulase can be recovered by methods commonly used in the field, such as gradient method, membrane separation, centrifugation, electrodialysis, use of ion exchange resins, distillation, salting out, etc., or a combination thereof. can. The recovered cellulase may be further isolated or purified.

When the target cellulase is a secreted cellulase that is secreted into the culture supernatant, the microorganism used to produce the cellulase in the present invention can be used repeatedly. That is, by collecting the culture supernatant and the separated microbial cells and culturing them in a new medium in the presence of an induced carbon substrate and a non-induced carbon substrate, cellulase can be produced again.

The method for producing cellulase of the present invention may be a batch method in which culturing of microorganisms, collection of cellulase accumulated in the culture and replacement of the medium are performed alternately, or a method in which some of the microorganisms and the medium are intermittent. It may be a semi-batch method or a continuous method in which microorganism culture and cellulase recovery are performed in parallel while changing the culture medium or continuously.

Hereinafter, the present invention will be explained in more detail based on Examples, but the present invention is not limited thereto.

(1) Culture conditions (bacterial species culture)
Cellulase was produced by culturing Trichoderma reesei PC-3-7 strain in a cellulose-containing medium. Add 50 mL of medium to a 500 mL flask for seed culture, inoculate with spores of PC-3-7 strain at 1 x 10 ⁵ cells/mL, and shake at 28°C and 220 rpm (PRECI, PRXYg-98R). Cultured. The medium composition is shown in Table 1. Bacterial species culture was carried out for 2 days.

(preculture)
The initial medium contained 2% powdered cellulose (KC Flock W400; Nippon Paper Industries) and other medium components shown in Table 2. A 1 L jar fermenter BMZ-P (Biot Co., Ltd.) containing 500 mL of the initial medium was inoculated with 10% (v/v%) of the seed culture solution and cultured for 1 day to obtain a preculture solution. . The settings of the jar fermenter were as follows: temperature 28°C, aeration rate 0.5 vvm, pH: 4.5 ± 0.1, and stirring frequency varied to maintain DO = 3.0 ppm. The pH of the culture solution was controlled to be maintained within the above predetermined range using a 10% (w/w%) ammonia aqueous solution.

(main culture)
The initial medium contained 4% powdered cellulose (KC Hook W400; Nippon Paper Industries) and other medium components shown in Table 2. A 2 L jar fermenter BMZ-P (Biot Co., Ltd.) containing 1000 mL of the initial medium was inoculated with 1% (v/v%) of the preculture solution and cultured for 7 days. The settings of the jar fermenter were as follows: temperature: 30° C., aeration rate: 1.0 vvm, pH: 4.5±0.1, stirring frequency was variable, and was set to maintain DO = 2.0 ppm. The pH of the culture solution was controlled to be maintained within the above predetermined range using a 10% (w/w%) ammonia aqueous solution. During the culture, the rate of change in the respiratory activity of the filamentous fungi and the cellulose supply rate were measured every hour according to the procedures (2) to (3) below. A 60% (w/w%) glucose aqueous solution was fed using a feed controller DFR (Biot Co., Ltd.). The feeding of the glucose aqueous solution is started at a carbon concentration of 0.15 to 0.50 g/L-initial medium/h, and then the ratio of the glucose feeding rate to the cellulose feeding rate [glucose (non-induced carbon) feeding rate] The feeding amount was controlled so that /[cellulose (induced carbon) supply rate] was the value shown in Table 3 below (Comparative Example 1, Examples 1 to 3).

(2) Measurement of respiratory activity During the culture, the CO ₂ concentration (v/w%) in the culture solution was measured using an exhaust gas analyzer (DEX-1562A; Biot Co., Ltd.) and a flow meter with a valve (MODEL RK1200; Cofloc Co., Ltd.). ) and culture aeration rate (L/min) were measured hourly. At the same time, 5 mL of culture solution was collected and centrifuged (manufactured by Hitachi Koki Co., Ltd., himac CF7D2, rotor model: RT3S3, rotor installation adapter: 50 mL 4 x 4 pieces, tube dimensions: diameter 36.5 mm, length 120 mm, 3,000 rpm) , 15 min) to separate the supernatant and solid content. The supernatant was filtered through a 0.45 μm filter (material: cellulose acetate) and then subjected to the cellulase productivity measurement described below. The solid content was washed twice with 15 mL of ion-exchanged water by centrifugation and freeze-dried. The solid content of the dried material was measured, and the dry solid content concentration of the culture solution was calculated.

The amount of elements (nitrogen and carbon) in the dry solid content of the culture solution was measured using an elemental analyzer vario EL cube (manufactured by Elemental). By dividing the measured amounts of nitrogen and carbon by the amount of culture, the total nitrogen concentration and total carbon concentration in the culture solution were obtained, respectively. Similarly, a filamentous fungal cell sample obtained during seed culture was subjected to elemental analysis, and the C/N ratio of the fungal cells was calculated. By multiplying the calculated C/N ratio (C: 45%, N: 7.9%, C/N ratio = 5.7) by the nitrogen concentration in the dry solid content, The amount of carbon derived from the bacterial cells was calculated (Equation (4)). Next, the cellulose-derived carbon concentration was determined according to equation (5). Furthermore, assuming that the amount of carbon in the dried bacterial cells was 45% by mass, the bacterial cell concentration in the culture solution (in terms of dry mass) was calculated using equation (6).
Carbon concentration derived from bacterial cells [gC/L]
= N concentration in dry solid content [gN/L] × 5.7 [Bacterial cell C/N ratio [gC/gN]] …(4)
Cellulose-derived carbon concentration [gC/L]
= Total carbon concentration in dry solid content [gC/L] - Bacterial cell-derived carbon concentration in dry solid content [gC/L] …(5)
Bacterial cell concentration [g-dry cell/L-culture] = Bacterial cell-derived carbon concentration [gC/L] ÷ 0.45...(6)

The rate of change in the respiratory activity of filamentous fungi was calculated based on the following formulas (1)' to (3)'.
Respiratory activity [g-CO ₂ /g-dry cell/h]
= (CO ₂ excretion rate [g-CO ₂ /L-culture/h]) ÷ (bacterial cell concentration [g-dry cell/L-culture] …(1)'
here,
g-CO ₂ = (CO ₂ concentration [vol%] ÷ 100 × culture aeration rate [L/min] × 60 [min])
÷(Vm × 44[CO ₂ molar mass]) …(2)'
Here, CO ₂ concentration [vol%]: CO ₂ concentration in culture (vol%)
Vm: Molar volume of ideal gas = 22.4
L-Culture: Culture volume (L)
Rate of change in respiratory activity = {｜Respiratory activity at time t ₂ - Respiratory activity at time t ₁ |
÷ (respiratory activity at time t ₁ )} (t ₂ > t ₁ , | t ₁ − t ₂ | = 1)...(3)'

(3) Measurement of carbon substrate supply rate Next, using the cellulose-derived carbon concentration determined by formula (5), the cellulose supply rate to the culture solution was calculated based on formula (7).
Cellulose feed rate [g-carbon derived from derived carbon substrate/L-culture solution/h]
= Cellulose-derived carbon concentration at time t ₂ - Cellulose-derived carbon concentration at time t ₁ (t ₂ > t ₁ , | t ₁ − t ₂ | = 1) …(7)

The glucose supply rate was calculated based on the setting value of the feed controller [glucose aqueous solution/h]. The concentration of glucose in the aqueous solution is 60 w/w%, the mass ratio of carbon in glucose is 0.4 (72/180, where the molar mass of glucose is 180 g/mol, the molar mass of carbon in glucose is 12 x 6 = 72 g/mol ), the supply rate of glucose to the culture solution was calculated based on equation (8).
Glucose feed rate [g-carbon from uninduced carbon substrate/L-culture/h]
= Feed controller setting value [g-glucose aqueous solution/h] × glucose concentration in aqueous solution (%) × mass ratio of carbon in glucose ÷ culture volume [L]
= Feed controller setting value [g/h] × 0.6 × 0.4 ÷ Culture volume [L] …(8)

(4) Measurement of Cellulase Productivity The protein concentration in the culture supernatant obtained in (2) was quantified by the Bradford method and determined as the cellulase concentration. For quantitative determination, bovine gamma globulin (BGG) of 0.125 to 0.75 mg/mL was used as a standard substance, using a quick start protein assay (standard assay) (manufactured by Bio-Rad) and a microplate reader (manufactured by Molecular Devices, Inc.). VersaMax) to measure the absorbance at 595 nm and calculate the protein concentration. The cellulase productivity of filamentous fungi [g-Protein/L] is calculated by dividing the protein concentration [g-Protein/L] of the obtained supernatant by the carbon concentration [gC/L] supplied up to the time of sampling the supernatant. /gC] was calculated. Relative cellulase productivity of Comparative Example 1, Example 1, Example 2, and Example 3 was determined on days 1 to 7 of culture when the cellulase productivity on day 7 of culture of Comparative Example 1 was taken as 100%. . Table 3 shows the rate of change in respiration activity of the bacterial cells and the change in the carbon substrate supply rate ratio of Comparative Example 1 and Examples 1 to 3, as well as the relative cellulase productivity on the 7th day of culture. Furthermore, the relative cellulase productivity on days 1 to 7 of culture is shown in Table 4 and FIG. 1.

Claims (7)

A method for producing cellulase, comprising:
culturing Trichoderma fungi in the presence of cellulose and glucose ; and
Measuring the respiratory activity of the Trichoderma bacteria over time;
including;
During the period in which the rate of change in the respiratory activity of the Trichoderma bacteria is 0.1 or more, the ratio R represented by the following formula A is 100 or less,
Formula A: R= Glucose Feed Rate/ Cellulose Feed Rate Method. 2. The method of claim 1, wherein the ratio R is 50 or less. 2. The method of claim 1, wherein the ratio R is 10 or less. The ratio R is 100 or less during a period in which the rate of change in the respiratory activity of the Trichoderma bacteria is 0.01 or more following a period in which the rate of change in the respiratory activity of the Trichoderma bacteria is 0.01 or more. The method according to any one of items 1 to 3. A method according to any one of claims 1 to 4 , wherein the cellulose is added to the culture vessel in batches. The method according to any one of claims 1 to 5 , wherein the glucose is fed into a culture tank. The method according to any one of claims 1 to 6 , wherein the Trichoderma bacterium is Trichoderma reesei or a mutant strain thereof.