WO2021100737A1

WO2021100737A1 - Method for producing cellulase

Info

Publication number: WO2021100737A1
Application number: PCT/JP2020/042903
Authority: WO
Inventors: 岳史坂本; 宏幸小西; 俊陽新井
Original assignee: 花王株式会社
Priority date: 2019-11-18
Filing date: 2020-11-18
Publication date: 2021-05-27
Also published as: JP2021078392A; JP7446089B2; BR112022009617A2

Abstract

Provided is a method for inexpensive and highly efficient microbiological protein production. Provided is a method for producing a cellulase, the method comprising culturing a filamentous fungus in the presence of a derived carbon substrate and a non-derived carbon substrate, wherein the ratio R (R= (feeding rate of non-derived carbon substrate)/(feeding rate of derived carbon substrate)) is 100 or less in a period where the rate of change in respiratory activity of the filamentous fungus is 0.1 or more.

Description

Method for producing cellulase

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

Filamentous fungi are attracting attention as degrading bacteria of plant polysaccharides because they produce various types of cellulase and xylanase. Among them, Trichoderma has been studied as a microorganism for producing a cellulase-based biomass-degrading enzyme because it can produce cellulase and xylanase at the same time and in a large amount.

In the culture of microorganisms, glucose has conventionally been widely used as a carbon source. In addition to carbon sources, inducers may be required for the production of proteins such as enzymes by microorganisms. For example, the expression of the α-amylase gene of Aspergillus oryzae is induced by starch, maltose, and the like.

Patent Document 1 describes the first step for the growth of the bacterium in the presence of the cellulosic growth substrate in the batch phase, and the growth and enzyme of the bacterium in the presence of the inducible cellulosic substrate in the feed phase. A method for producing cellulase using filamentous fungi, which comprises a second step for production, and ethanol as a monomeric sugar of an enzyme hydrolyzate of lactose, glucose, xylose, and cellulosic biomass as the carbonaceous growth substrate. It is selected from the residue obtained after fermentation and the crude extract of water-soluble pentose derived from the pretreatment of cellulosic biomass, and the derived carbonaceous substrate is simply an enzymatic hydrolyzate of lactose, cellobiose, sophorose, and cellulosic biomass. It is disclosed that it is selected from the residue obtained after ethanol fermentation of cellulosic sugar and the crude extract of water-soluble pentose derived from the pretreatment of cellulosic biomass. Patent Document 2 describes ethanol as a carbon source for enzyme production and an enzyme hydrolyzate of a cellulosic or ligno-cellulosic material as an induced carbon source in a method for producing cellulose or hemicellulose-degrading enzyme by a fungus such as trichoderma. It is disclosed to use the residue from fermentation (including glucose, xylose, etc.). In Patent Document 3, the carbon addition rate 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 in the fed batch or continuous fermentation medium. A method is disclosed, and it is disclosed that the method is used to cause a microorganism to produce a protein such as an enzyme. The expression of trichoderma's major cellulase genes cbh1, cbh2, egl1 and egl2 is induced by cellulose, cellobiose, etc. (Non-Patent Document 1).

(Patent Document 1) International Publication No. 2013/0269664 (Patent Document 2) Japanese Patent Application Laid-Open No. 2006-217916 (Patent Document 3) International Publication No. 2013/124351 (Non-Patent Document 1) Curr. Genomics, 14: 230-249 (2013)

The present invention is a method for producing cellulase.
Including culturing filamentous fungi in the presence of inducible and non-inducible carbon substrates, including
The ratio R represented by the following formula A is 100 or less during the period when the rate of change in the respiratory activity of the filamentous fungus is 0.1 or more.
Formula A: R = supply rate of non-inducible carbon substrate / supply rate method of derived carbon substrate is provided.

Relative cellulase productivity of Comparative Example 1, Example 1, Example 2 and Example 3 in 1 to 7 days of culture. Let the cellulase productivity of Comparative Example 1 on the 7th day of culturing be 100%.

Detailed description of the invention

Development of culture technology for inexpensively and efficiently producing proteins such as enzymes by microorganisms is required. In the presence of glucose, a control mechanism called catabolite repression causes a decrease or saturation of substance productivity by microorganisms. For example, catabolite repression has been reported in filamentous fungi such as Trichoderma, and its mechanism is being analyzed. Fed-batch culture in which glucose is repeatedly or continuously added to the culture may suppress catabolite repression and improve the protein productivity of microorganisms. However, in protein production by glucose-batch culture, precise control of glucose concentration in the culture is required in order to prevent catabolite repression.

In the production of cellulase using microorganisms, the present inventors select an inducible carbon substrate such as cellulose to induce cellulase production and a non-inducible carbon substrate such as glucose, and the ratio thereof depends on the state of respiratory activity of the microorganism. It has been found that the cellulase productivity of microorganisms is improved by supplying the culture medium with adjustment.

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

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

Examples of cellulase-producing microorganisms cultivated by the method of the present invention include bacteria, yeast, filamentous fungi, and the like, of which filamentous fungi are preferable. As filamentous fungi, for example, the genus Acremonium, the genus Aspergillus, the genus Aureobaside, the genus Bjerkandera, the genus Ceripoliopsis, the genus Chrysosporium, the genus Coprinus, the genus Coriolus, the genus Cryptococcus, the genus Cryptococcus, genus, Neocallimastix spp, Neurospora spp, Paecilomyces spp, Penicillium spp, Phanerochaete spp., Phlebia spp, Piromyces spp, Pleurotus spp, Rhizopus spp, Schizophyllum sp, Talaromyces sp, Thermoascus sp., Thielavia genus Tolypocladium sp, Trametes sp, and the genus Trichoderma Filamentous fungi, of which the genus Acremonium, the genus Aspergillus, the genus Chrysosporium, the genus Fusarium, the genus Humicola, the genus Mycelioph The genus Neurospora, the genus Penicillium, the genus Piromyces, the genus Talaromyces, the genus Thermoascus, the genus Thielavia, and the genus Trichoderma. From the viewpoint of cellulase productivity and the biomass saccharification performance of the obtained cellulase, the genus Trichoderma is more preferable, and Trichoderma reesei and its mutants are even more preferable. Examples of Trichoderma Risei and its variants include Trichoderma Risei QM9414 strain, PC-3-7 strain, and variants thereof. Examples of the mutant strain include a mutant strain generated by modification such as gene mutation and gene recombination.

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

The non-inducible carbon substrate used for culturing the microorganism is a carbon substrate that does not induce cellulase expression of the microorganism and generally causes catabolite repression (stop of cellulase production) of the microorganism. Examples of the non-inducible carbon substrate include at least one selected from the group consisting of saccharides that do not induce cellulase production of microorganisms, such as glucose, fructose, sucrose, maltose, and maltooligosaccharides. Of these, at least one selected from the group consisting of glucose, maltose, and maltooligosaccharide is preferable from the viewpoint of versatility and assimilation in microbial culture.

The derived carbon substrate is preferably added in batches to the culture tank. More preferably, the derived carbon substrate is added to the initial medium. The initial concentration of the derived carbon substrate in the culture tank (eg, the concentration of the derived carbon substrate in the initial medium) is preferably 1 to 15% by weight / volume. On the other hand, the addition of the non-inducible carbon substrate to the culture tank is preferably continuous addition (for example, fed-batch) from the viewpoint of avoiding catabolite repression of microorganisms. More preferably, the non-inducible carbon substrate is fed into the culture tank. For example, an aqueous solution of the non-inducible carbon substrate may be poured into the culture tank. The concentration of the non-inducible carbon substrate in the aqueous solution to be fed is preferably 2 to 90% by mass / volume, more preferably 5 to 80% by mass / volume. If the concentration of the non-inducible carbon substrate in the aqueous solution to be fed is too low, a large amount of the aqueous solution will be fed into the culture, which imposes a burden on the culture equipment. On the other hand, if the concentration of the non-inducible carbon substrate in the aqueous solution is too high, it becomes difficult to control the flow of the non-inductive carbon group into the culture.

The derived carbon substrate and non-inducible carbon substrate used in the culture are preferably sterilized. When the carbon substrate is added in batch, it may be sterilized after being introduced into the culture tank together with the initial medium or the like, or may be added to the culture tank after being sterilized in advance. When the carbon substrate is fed, it may be sterilized in advance and then fed into the culture tank. For sterilization of the carbon substrate, heat sterilization, pressure heat sterilization using a high-pressure steam sterilizer (autoclave), sterilization by spraying saturated steam, or the like can be generally adopted. Sterilization conditions include conditions in which the number of spores of Geobacillus stearothermophilus, which is a thermostable bacterium, ^{decreases up to 10 to 12} times (for example, equivalent to 20 minutes at 120 ° C.) or more severe conditions.

In an example of the method of the present invention, an initial medium containing the derived carbon substrate is introduced into a culture tank, and then the culture tank is sterilized. After seeding the microorganism in the sterilized culture tank, the microorganism is cultured while pouring an aqueous solution of the non-inducible carbon substrate.

The initial medium used for the culture may be a medium usually used for the microorganism to be cultured. For example, the initial medium is a carbon source containing the above-mentioned derived carbon substrate, a nitrogen source, a metal salt such as a magnesium salt or a zinc salt, a sulfate, a phosphate, a pH adjuster, a surfactant, a microorganism such as an antifoaming agent. It can contain various components generally contained in the medium of. The composition of the components in the medium can be appropriately selected. 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.

The feeding of the non-inducible carbon substrate into the culture tank can be carried out according to the usual fed-batch culture procedure. For example, an aqueous solution of a non-inducible carbon substrate may be fed into the culture tank while controlling the flow rate using a general feed controller or the like. Fed-batch of the non-inducible carbon substrate into the culture tank may be started at the same time as the start of the culture, or after the rate of change in the respiratory activity of the microorganism described later reaches a certain value (for example, 0.1 or more). May be good. It is convenient to start the feeding of the non-inducible carbon substrate at the same time as the start of the culture. The initial value of the flow acceleration 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 the cell concentration. is there.

In the method of the present invention, the amount of the derived carbon substrate and the non-inducible carbon substrate supplied to the culture medium during culturing of the microorganism is adjusted depending on the state of respiratory activity of the culturing microorganism. More specifically, the ratio of the supply rate of the non-inducible carbon substrate to the supply rate of the inductive carbon substrate during the period when the rate of change in the respiratory activity of the microorganism in culture is 0.1 or more R [R = non-inducible carbon substrate Supply rate / induction carbon substrate supply rate] is adjusted to 100 or less, preferably 50 or less, and more preferably 10 or less. On the other hand, from the viewpoint of reducing the consumption of the derived carbon substrate, the ratio of the supply rate of the non-induced carbon substrate to the supply rate of the derived carbon substrate is preferably 1 or more.

From the viewpoint of controlling the metabolism of a microorganism, in the method of the present invention, the rate of change in the respiratory activity of the microorganism during culture is 0.01, following a period in which the rate of change in the respiratory activity of the microorganism is 0.1 or more. During the above period, it is preferable to adjust the ratio R to 100 or less, preferably 50 or less, and more preferably 10 or less.

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

In the present specification, the respiratory activity of a microorganism is _{calculated by dividing the CO 2} excretion rate from the culture derived from the respiration of the cells by the cell concentration of the culture. For example, the respiratory activity of a microorganism can be calculated according to the following formula (1). The CO ₂ emission rate can be calculated by obtaining the volume of the culture and the _{rate of change in the CO 2} _{emission per hour from the total CO 2} emission [g-CO ₂ ] per unit time of the culture. For example, the _{total CO 2} emission amount [g-CO ₂ ] per unit time of the culture can _{be calculated according to the following formula (2) based on the CO 2} concentration [vol%] of the culture and the culture aeration amount.
Respiratory activity [g-CO ₂ / g-dry cell / h]
= (CO ₂ emission rate [g-CO ₂ / L-culture / h]) ÷ (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 the culture (vol%)
g-dry cell: CO ₂ concentration [vol%] Dry mass (g) of cells contained in the culture at the time of measurement
L-Culture: CO ₂ concentration [vol%] Culture volume (L) at the time of measurement

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

In the method of the present invention, the respiratory activity of microorganisms can be calculated hourly according to the above formulas (1) and (2). The rate of change in respiratory activity can be determined from the respiratory activity measured over time. That is, the rate of change in respiratory activity is calculated by dividing the difference in respiratory activity calculated at two consecutive measurement time points by the respiratory activity calculated at an earlier point in time, as shown in the following formula (3).
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 supply rate of the induced carbon substrate when added in batch is defined as the change in concentration (disappearance rate) of carbon derived from the induced carbon substrate in the culture per unit time. The rate of carbon loss 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 removes the non-inducible carbon substrate that dissolves 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 elementally analyzed to determine the total carbon content in the dry solid content. The amount of carbon derived from the derived carbon substrate is obtained by subtracting the amount of carbon derived from the microorganism from the total amount of carbon. That is, assuming that the C / N ratio of the microbial cells in the culture is constant, the C / N ratio of the microbial cells calculated by the measurement of the microbial body sample at the time of culturing the inoculum and the amount of nitrogen in the dry solid content. From, the amount of carbon derived from the microorganism in the dry solid content is calculated. The difference between the total carbon content of the dry solids and the carbon content derived from the microbial body is calculated as the carbon content derived from the derived carbon substrate, and by dividing this by the amount of the culture, the carbon derived from the derived carbon substrate in the culture is calculated. The concentration is required.
(2) Next, the difference in carbon concentration derived from the derived carbon substrate calculated at two different time points is obtained. The difference value reflects the change in concentration of carbon from the derived 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 feeding rate of the non-inducible carbon substrate to be fed is defined as the amount of carbon derived from the non-inducible carbon substrate to be fed into the culture per unit time. For example, based on the fed-batch set by a feed controller and the concentration of the non-inducible carbon substrate in the aqueous solution to be fed, the supply rate of the non-inducible carbon substrate [g-carbon / L-culture derived from the non-inductive carbon substrate]. Liquid / h] can be determined.

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

In the method of the present invention, various conditions for culturing a microorganism can be appropriately set according to a conventional method according to the species of the microorganism, the scale of the culture, and the like, except for the above-mentioned supply rate of the carbon substrate. For example, as the culture tank used for culturing, conventionally known ones can be appropriately adopted. Specific examples thereof include a flask, an aeration-stirring type culture tank, a bubble tower type culture tank, a fluidized bed type culture tank, and the like, and an aeration-stirring type culture tank is preferable. 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 3-7, more preferably pH 3.5-6 when the microorganism is a filamentous fungus, for example. The pH of the culture can be adjusted with a conventional pH adjuster 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 provided in the culture tank. The culture period is preferably 4 to 10 days.

After culturing, recover the desired cellulase from the culture. In the case of secretory cellulase such as filamentous fungal cellulase, cellulase can be recovered from the culture supernatant. When cellulase is contained in the cell, the cell can be destroyed to remove the cellulase-containing fraction and recover the cellulase. Cellulase can be recovered by methods usually used in the art, for example, tilting method, membrane separation, centrifugation, electrodialysis method, use of ion exchange resin, distillation, salting out, etc., or a combination thereof. it can. The recovered cellulase may be further isolated or purified.

In the case of a secretory cellulase in which the target cellulase is secreted into the culture supernatant, the microorganism used for producing the cellulase in the present invention can be used repeatedly. That is, cellulase can be produced again by collecting the microbial cells separated from the culture supernatant and culturing them in a new medium in the presence of an inducible carbon substrate and a non-inducible carbon substrate.

The method for producing cellulase of the present invention may be a batch method in which the culture of microorganisms, the recovery of cellulase accumulated in the culture, and the replacement of the medium are alternately performed, or the medium is intermittent with some microorganisms. It may be a semi-batch method or a continuous method in which culturing of microorganisms and recovery of cellulase are carried out in parallel while being replaced in a target or continuous manner.

Hereinafter, the present invention will be described 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 the Trichoderma Riesei PC-3-7 strain in a cellulose-containing medium. As an inoculum culture, add 50 mL of medium to a 500 mL flask, ^{inoculate spores of the PC-3-7 strain to 1 × 10 5} cells / mL, and shake at 28 ° C. and 220 rpm (PRECI, PRXYg-98R). It was cultured. The medium composition is as shown in Table 1. The bacterial species culture was carried out for 2 days.

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

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

_{(2) Measurement of respiratory activity During culture, CO 2} concentration (v / w%) in the culture solution was used by an exhaust gas analyzer (DEX-1562A; Biot Co., Ltd.) and a flow meter with a valve (MODEL RK1200; Cofflock Co., Ltd.). ) And the culture aeration rate (L / min) were measured every hour. At the same time, 5 mL of the culture solution was collected and centrifuged (Hitachi Koki Co., Ltd., himac CF7D2, rotor model: RT3S3, rotor installation adapter: 50 mL 4 x 4, tube dimensions: diameter 36.5 mm, length 120 mm, 3,000 rpm. , 15 min) to separate the supernatant and the solid content. The supernatant was filtered through a 0.45 μm filter (material: cellulose acetate) and then subjected to the cellulase productivity measurement described later. The solid content was washed twice with 15 mL of ion-exchanged water by centrifugation and lyophilized. The solid content of the dried product 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 (Elemental). The total nitrogen concentration and total carbon concentration in the culture broth were obtained by dividing the measured nitrogen amount and carbon amount by the culture amount, respectively. Similarly, the filamentous cell sample at the time of inoculum culture was elementally analyzed, and the C / N ratio of the cell 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 dry solid content The amount of carbon derived from the cells was calculated (Equation (4)). Next, the cellulose-derived carbon concentration was determined according to the formula (5). Further, the cell concentration in the culture solution (converted to dry mass) was calculated by the formula (6), assuming that the amount of carbon in the dried cells was 45% by mass.
Carbon concentration derived from cells [gC / L]
= N concentration in dry solids [gN / L] x 5.7 [Bacterial cell C / N ratio [gC / gN]]… (4)
Cellulose-derived carbon concentration [gC / L]
= Total carbon concentration in dry solids [gC / L] -Carbon-derived carbon concentration in dry solids [gC / L]… (5)
Mycelium concentration [g-dry cell / L-culture] = carbon concentration derived from mycelium [gC / L] ÷ 0.45… (6)

The rate of change in 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 emission rate [g-CO ₂ / L-culture / h]) ÷ (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 the 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 Subsequently, the cellulose supply rate with respect to the culture solution was calculated based on the formula (7) using the carbon concentration derived from cellulose obtained by the formula (5).
Cellulose supply rate [carbon derived from g-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 set value [glucose aqueous solution / h] of the feed controller. The glucose concentration 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, and the molar mass of carbon in glucose is 12 × 6 = 72 g / mol. ), The glucose supply rate to the culture solution was calculated based on the formula (8).
Glucose supply rate [g-carbon derived from non-inducible 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 amount [L]
= Feed controller setting value [g / h] × 0.6 × 0.4 ÷ Culture amount [L]… (8)

(4) Measurement of cellulase productivity The protein concentration in the culture supernatant obtained in (2) was quantified by the Bradford method and used as the cellulase concentration. For quantification, 0.125 to 0.75 mg / mL bovine gamma globulin (BGG) was used as a standard substance, and a microplate reader (Molecular Devices) was used by Quick start protein assay (Bio-Rad). The absorbance at 595 nm was measured with VersaMax), and the protein concentration was 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, the cellulase productivity of filamentous fungi [g-Protein / L] / gC] was calculated. The relative cellulase productivity of Comparative Example 1, Example 1, Example 2 and Example 3 in 1 to 7 days of culture was determined when the cellulase productivity on the 7th day of culture of Comparative Example 1 was 100%. .. Table 3 shows the changes in the respiratory activity change rate and the carbon substrate supply rate ratio of the cells of Comparative Examples 1 and 1 to 3, and the relative cellulase productivity on the 7th day of culturing. The relative cellulase productivity on days 1 to 7 of culture is shown in Table 4 and FIG.

Claims

It is a method for producing cellulase.
Including culturing filamentous fungi in the presence of inducible and non-inducible carbon substrates, including
The ratio R represented by the following formula A is 100 or less during the period when the rate of change in the respiratory activity of the filamentous fungus is 0.1 or more.
Formula A: R = supply rate of non-inducible carbon substrate / supply rate method of inductive carbon substrate.
The method according to claim 1, wherein the ratio R is 50 or less.
The method according to claim 1, wherein the ratio R is 10 or less.
The method according to claim 1, wherein the ratio R is 1 or more.
The claim that the ratio R is 100 or less in a period in which the rate of change in the respiratory activity of the filamentous fungus is 0.1 or more, followed by a period in which the rate of change in the respiratory activity of the filamentous fungus is 0.01 or more. The method according to any one of 1 to 4.
The method according to any one of claims 1 to 5, wherein the derived carbon substrate is at least one selected from the group consisting of lactose, cellobiose, sophorose, gentiobiose and cellulose.
The method according to any one of claims 1 to 6, wherein the non-inducible carbon substrate is at least one selected from the group consisting of glucose, fructose, sucrose, maltose and maltooligosaccharide.
The method according to any one of claims 1 to 7, wherein the derived carbon substrate is batch-added to the culture tank.
The method according to claim 8, wherein the supply rate of the derived carbon substrate is the rate of disappearance of carbon derived from the derived carbon substrate in the culture per unit time.
The method according to claim 8 or 9, wherein the initial concentration of the derived carbon substrate in the culture tank is 1 to 15% by mass / volume%.
The method according to any one of claims 1 to 10, wherein the non-inducible carbon substrate is fed into a culture tank.
The method according to claim 11, wherein the supply rate of the non-inducible carbon substrate is the amount of carbon derived from the non-inducible carbon substrate to be fed into the culture per unit time.
The method according to claim 11 or 12, wherein an aqueous solution of the non-inducible carbon substrate is poured into the culture tank, and the concentration of the non-inducible carbon substrate in the aqueous solution is 2 to 90 mass / volume%.
The method according to any one of claims 1 to 13, wherein the initial rate of fed-batch of the non-inducible carbon substrate is 0.15 to 0.50 g / L-initial medium / h in terms of carbon concentration.
The method according to any one of claims 1 to 14, wherein the filamentous fungus belongs to the genus Trichoderma.
The method according to claim 15, wherein the filamentous fungus is Trichoderma reesei or a mutant strain thereof.
The method according to any one of claims 1 to 16, comprising adjusting the ratio R to be 100 or less during a period in which the rate of change in respiratory activity of the filamentous fungus is 0.1 or more.