CN111944788A - Method for producing cellulase by inducing trichoderma reesei - Google Patents
Method for producing cellulase by inducing trichoderma reesei Download PDFInfo
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- CN111944788A CN111944788A CN202010823783.9A CN202010823783A CN111944788A CN 111944788 A CN111944788 A CN 111944788A CN 202010823783 A CN202010823783 A CN 202010823783A CN 111944788 A CN111944788 A CN 111944788A
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
Abstract
The invention discloses a method for promoting trichoderma reesei to produce cellulase, which comprises the steps of after trichoderma reesei is fermented for 24-36 hours, detecting that the dissolved oxygen of fermentation liquor is 1.5-2.0mg/mL and feeding a composite type induction carbon source when the fermentation liquor has a continuous descending trend, wherein the composite type induction carbon source is obtained by mixing straw lignocellulose enzymolysis liquid and glucose enzymolysis liquid according to the mass ratio of 3: 7-9. The fed-batch method can be set in a programmed manner, saves manpower and material resources, better conforms to the growth and propagation rules of trichoderma reesei compared with a constant-speed supplementing method, and is favorable for the production of cellulase. In addition, the invention pretreats the straws, which not only can increase the steam explosion degree of the straws, but also can remove the difficultly volatile substances in the straws influencing the enzymolysis efficiency, thereby effectively improving the enzymolysis efficiency of the straws as the non-soluble carbon source.
Description
Technical Field
The invention belongs to the technical field of biological fermentation, and particularly relates to a method for producing cellulase by inducing trichoderma reesei through feeding a carbon source.
Background
With the ever-increasing population today, the world is facing crisis in all respects, food, feed, chemicals and energy supplies, and the search for new renewable energy has been a field of high worldwide interest. Lignocellulose is a cheap renewable biomass material in nature, and has the characteristics of large total amount, low price, environmental protection and the like. Lignocellulose materials can be degraded into sugars through the synergistic effect of cellulose degrading enzymes, the sugars can be used as basic raw materials in bioengineering industrial production, the sugars are converted into chemical products with high added values such as ethanol, lactic acid and hydrogen through the fermentation effect of microorganisms, and the lignocellulose biomass is expected to replace petroleum to become an important renewable raw material for producing fuels and chemicals in the future. However, the existing biorefinery industry utilizing lignocellulose biomass in China still has the problems of low yield and high cost, and the main reason is that the cost of the cellulase is too high, which accounts for about 25-30% of the cost of the whole industry. Therefore, the development of low-cost and high-enzyme-activity cellulase is the primary task of industrial production of the biorefinery industry.
At present, Trichoderma reesei RUT-C30 is one of the most widely used cellulase producing strains. Exact research evidence already shows that the change of proper culture conditions can obviously improve the enzyme activity of the cellulase produced by trichoderma reesei fermentation, wherein carbohydrates and derivatives thereof play an important role in inducing the expression of cellulase genes. The inducers for producing cellulase from trichoderma reesei can be mainly divided into two categories, one category is insoluble inducers represented by lignocellulose and microcrystalline cellulose, and the other category is soluble inducers represented by oligosaccharide substances such as cellobiose, lactose and sophorose. Wherein, inducers such as microcrystalline cellulose and sophorose have higher cost and are not suitable for being added in large quantities in industrial production. Therefore, it is important to select an appropriate inducing carbon source to promote the high production of cellulase by trichoderma reesei.
Patent document CN201510962762.4 discloses a method for producing a high-efficiency cellulase mixture and its application, wherein non-soluble carbon source is used for fermentation, and reducing sugar control measures are adopted to reduce the repression effect of carbohydrate and improve the cellulase production capacity. The production method specifically comprises the following steps: fermenting by using trichoderma reesei or penicillium oxalicum as a cellulase production strain, wherein the pH is always maintained between 3.5 and 6.5 during the whole fermentation period, feeding is started when the pH is kept between 48 and 60 hours, the content of reducing sugar is controlled to be less than or equal to 2.5mg/mL after feeding, and a feeding rate adjusting stage is performed every 4 to 5 hours until the fermentation is finished. The feed culture medium comprises: the mixed solution of the glucose mother liquor or the xylose mother liquor, magnesium sulfate and zinc sulfate.
Patent document CN201010040047.2 discloses a method for producing cellulase, comprising: inoculating the trichoderma reesei into a fermentation culture medium, starting fed-batch culture after fermenting for 60-90 hours, wherein in the fed-batch culture process, the culture medium is fed-batch when the pH value of the fermentation broth is higher than 4.8, the culture medium is stopped being fed-batch when the pH value of the fermentation broth is lower than 4.5, and the fermentation is stopped when the total fermentation time reaches 192-. The method organically combines an insoluble carbon source and a soluble carbon source to synergistically induce the expression of the cellulase genes. The insoluble carbon source is at least one of cellulose powder, paper pulp and plant cellulose raw materials subjected to steam explosion, and the soluble carbon source consists of 87-94% of glucose and 6-13% of sophorose.
Patent document CN201810076624.X discloses a method for inducing Trichoderma reesei to produce cellulase by using pure straw solid feed supplement, which comprises the following steps: s1, plate culture; s2, seed tank culture; s3, fermentation culture: in the process of enzyme-producing culture after inoculation, when the pH value in the enzyme-producing fermentation tank is reduced to 4.5, ammonia water is used for regulating the pH value to be not lower than 4.5, and after the pH value is reduced for the second time, the material is supplemented when the pH value begins to rise; alternatively, feeding is performed when the dissolved oxygen saturation in the fermentor first begins to rise.
The prior art discloses that non-soluble carbon sources such as straws, bran and the like are used as initial carbon sources in the early stage, and soluble sugars such as glucose, sophorose and the like are added in a fermentation feed medium in the later stage to be used as supplementary carbon sources. In fact, the inventors of the present application found that the selection, the addition time and the addition mode of the late-stage carbon source supplement are important factors influencing the production of cellulase by trichoderma reesei.
Disclosure of Invention
On the basis of the background, the invention aims to provide a method for inducing trichoderma reesei to produce cellulase, and particularly, the method utilizes a non-soluble carbon source and a soluble carbon source for mixed fermentation, induces the trichoderma reesei to produce the cellulase in a high yield by optimizing and supplementing the carbon source, preferably, reduces the time cost and the economic cost for producing the cellulase by regulating and controlling the material supplementing speed and the material supplementing amount of the supplemented carbon source, and the cellulase produced by the method has high enzyme system composition complete activity.
The purpose of the invention is realized by the following technical scheme:
in a first aspect, the invention provides a method for promoting trichoderma reesei to produce cellulase, which comprises the step of feeding a composite type induced carbon source when the dissolved oxygen of fermentation liquor is detected to be 1.5-2.0mg/mL and the fermentation liquor has a continuous descending trend after fermentation for 24-36 hours in the trichoderma reesei fermentation stage, wherein the composite type induced carbon source is obtained by mixing straw lignocellulose enzymolysis liquid and glucose enzymolysis liquid according to the mass ratio of 2-6 (8-4), preferably, the straw lignocellulose enzymolysis liquid accounts for 20-50 parts, and most preferably 30-40 parts, based on 100 parts of the total mass of the straw lignocellulose enzymolysis liquid and the glucose enzymolysis liquid.
The preparation method of the straw lignocellulose enzymolysis liquid comprises the following steps: dissolving 20-25% (w/v) of straw materials subjected to steam explosion pretreatment in a crude enzyme solution of Trichoderma reesei which is not fermented by a compound induction carbon source, and reacting for 20-24h under the conditions of 50-60 ℃, pH 4.8-5.0 and 180-200 rpm. The straw material pretreated by steam explosion is obtained by crushing straws into 1-2cm coarse particles, mixing the coarse particles with water according to the proportion of 1:1-1.5, putting the mixture into a steam explosion tank, heating the mixture to 185-1.5 ℃, maintaining the temperature for 10-20min, then releasing pressure instantly, cooling, taking out, squeezing, drying and crushing.
The preparation method of the glucose enzymolysis liquid comprises the following steps: using 50-60% (w/v) glucose solution, adding 20-25CBU/g beta-glucosidase, reacting at 60-65 deg.C, pH 4.8-5.0, and 200rpm for 20-24 h.
The addition amount of the compound type induced carbon source is 50-60% of the mass of the basic carbon source in the trichoderma reesei fermentation culture medium.
The feeding mode of the compound type induced carbon source flow is as follows: adding 11-14% of induced carbon source in uniform flow for 24-36h, adding 15-17% of induced carbon source in uniform flow for 36-48h, adding 15-20% of induced carbon source in uniform flow for 48-60h, adding 20-24% of induced carbon source in uniform flow for 60-72h, adding 15-16% of induced carbon source in uniform flow for 72-84h, and adding 10-12% of induced carbon source in uniform flow for 84-96 h.
Through long-term experiments of the inventor, the most preferable feeding mode of the compound type induced carbon source flow is as follows: adding 12% of induced carbon source in uniform flow for 24-36h, adding 16% of induced carbon source in uniform flow for 36-48h, adding 20% of induced carbon source in uniform flow for 48-60h, adding 24% of induced carbon source in uniform flow for 60-72h, adding 16% of induced carbon source in uniform flow for 72-84h, and adding 12% of induced carbon source in uniform flow for 84-96 h. The feeding mode of the composite induced carbon source flow obtained by optimization can be completely set as a programmed feeding mode, so that manpower and material resources are saved, accurate quantification can be realized, and the method better conforms to the growth and propagation rules of trichoderma reesei compared with the mode of feeding the composite induced carbon source flow at a constant speed, and is more favorable for production of cellulase.
In a preferred embodiment of the present invention, the method for promoting the production of cellulase by trichoderma reesei specifically comprises the following steps:
(1) culturing the trichoderma reesei culture dish: adding the seed solution of Trichoderma reesei to the culture medium plate, wherein the total number of inoculated spores in each culture dish is 1 × 105-1×106Culturing in an incubator with temperature of 30-32 deg.C and humidity of 60-65% for 5-7 days, adding ultrapure water into the culture dish after the green villiform spores are covered on the whole plate, scraping off hyphae and spores, and storing at 4 deg.C;
(2) and (3) culturing trichoderma reesei in a seeding tank: inoculating the spore liquid preserved in the step (1) into a seeding tank to ensure that the spore concentration is 1 multiplied by 109-1×1010Culturing at normal temperature for 20-24 hours at the seed tank rotation speed of 180-;
(3) and (3) fermenting and culturing trichoderma reesei: and (3) inoculating the Trichoderma reesei seed solution obtained in the step (2) into a fermentation tank, controlling the rotation speed of the fermentation tank to be 180-350rpm/min, controlling the ventilation volume to be 1-1.5vvm, enabling the dissolved oxygen of the fermentation liquor to be 1.2-3.5mg/L, controlling the pH value of the fermentation liquor to be not lower than 4.0 by feeding 10% ammonia water, and after fermenting for 24-36h, detecting that the dissolved oxygen of the fermentation liquor is 1.5-2.0mg/mL and performing compound type induced carbon source feeding when the dissolved oxygen of the fermentation liquor continues to decline.
Preferably, the culture medium of the trichoderma reesei culture dish in the step (1) comprises the following components: 2 percent of anhydrous glucose, 4 percent of concentrated solution of macroelement nutrient salt producing enzyme, 0.04 percent of concentrated solution of microelement producing enzyme, 0.2 percent of corn steep liquor, 2 percent of Tween 80, 2 percent of agar and the balance of water. Wherein, the concentrated solution of the enzyme-producing macroelement nutrient salt comprises the following components: 7% KH2PO4、9.8％(NH4)2SO4、2.1％MgSO4The balance being water; the enzyme-producing trace element concentrated solution comprises the following components: 3.5% FeSO4·7H2O、1.09％MnSO4·H2O、0.98％ZnSO4·7H2O、1.4％CoCl2·6H2O and the balance of water.
Preferably, the components of the culture medium of the trichoderma reesei seed tank in the step (2) are as follows: 1% of glucose, 1.4% of concentrated solution of seed macroelement nutrient salt, 0.14% of concentrated solution of seed microelement, 0.07% of corn steep liquor and the balance of water. The seed macroelement nutrient salt concentrated solution comprises the following components: 14% KH2PO4、9.8％(NH4)2SO4、2.1％MgSO4The balance being water; the seed microelement concentrate comprises the following components: 3.5% FeSO4·7H2O、1.09％MnSO4·H2O、0.98％ZnSO4·7H2O、1.4％CoCl2·6H2O and the balance of water.
Preferably, the components of the fermentation medium of trichoderma reesei in the step (3) are as follows: 3.5-4.5% of non-soluble carbon source, 1-2% of glucose, 3-4% of concentrated solution of macroelement nutrient salt capable of producing enzyme, 0.01-0.04% of concentrated solution of microelement capable of producing enzyme, 0.1-0.2% of corn steep liquor and the balance of water.
Preferably comprising: 3.5-4.5% of non-soluble carbon source, 1% of glucose, 4% of concentrated solution of macroelement nutrient salt producing enzyme, 0.04% of concentrated solution of microelement producing enzyme, 0.2% of corn steep liquor and the balance of water. Wherein, the concentrated solution of the enzyme-producing macroelement nutrient salt comprises the following components: 7% KH2PO4、9.8％(NH4)2SO4、2.1％MgSO4The balance being water; the enzyme-producing trace element concentrated solution comprises the following components: 3.5% FeSO4·7H2O、1.09％MnSO4·H2O、0.98％ZnSO4·7H2O、1.4％CoCl2·6H2O and the balance of water.
More preferably, the content of the non-soluble carbon source in the trichoderma reesei fermentation medium is 4.5%.
The preparation of the non-soluble carbon source comprises the following steps:
(1) using H before steam explosion treatment2SO4Soaking the straws with mixed acid of fumaric acid;
(2) drying the soaked straws, and fumigating with alcohol.
The non-soluble carbon source used in the invention is prepared by the following method:
(1) soaking the straws with dilute acid before steam explosion treatment, wherein the dilute acid is H2SO4And preparing an acid solution from fumaric acid, wherein H is contained in the acid solution2SO4The mass concentration of the fumaric acid is 0.1-1 percent respectively;
(2) drying the soaked straws, crushing the straws into 1-2cm coarse grains, and fumigating the coarse grains with alcohol for 2-4 hours, wherein the alcohol is selected from 75-95% ethanol solution;
(3) adding water according to the volume ratio of 1:1-1.5 after fumigation, mixing, putting into a steam explosion tank, heating to 185-195 ℃, maintaining for 10-20min, then releasing pressure instantaneously, cooling, taking out, squeezing, drying and crushing to obtain the non-soluble carbon source.
In the most preferred embodiment of the present invention, H in the acid solution in the step (1)2SO4The mass concentration of the fumaric acid was 1%.
Preferably, the alcohol in step (3) is selected from 75% ethanol solution, and the fumigation is performed under ventilation condition.
The invention uses dilute acid to soak the straws, the dilute acid can ensure that the straw materials are soaked from a solid state to a solid-liquid mixed state, the contact area with steam in the steam explosion process is increased, and secondly, the acid solution can hydrolyze the bonds linked by cellulose and lignin, so that the lignin and the cellulose in the materials before steam explosion are primarily separated, the steam explosion degree is increased, and the enzymolysis efficiency of the straws as a carbon source is improved. In addition, because the straws contain nonvolatile substances, such as acetic acid, furfural and the like, which can affect the enzymolysis efficiency of the straws, the straws are preferably eliminated before steam explosion, and ethanol gas can carry nonvolatile acidic substances and aldehyde substances to volatilize out when the straws are fumigated by alcohol.
The straws are selected from one of corn straws, wheat straws, rice straws, wood chips, energy plants or forestry wastes, and are preferably the corn straws.
The trichoderma reesei is selected from trichoderma reesei RUT-C30 strain and comes from China general microbiological culture collection center.
The detection method of the filter paper enzyme activity (FPA) is determined according to an international standard method recommended by IUPAC, and one International Unit (IU) of the filter paper enzyme activity is equal to the enzyme amount required for generating 1.0 mu mol of glucose (calculated as reducing sugar) per minute under the standard enzymatic reaction condition and is expressed by IU/ml.
The invention has the following technical advantages: (1) the method uses corn straws as a non-soluble carbon source, and uses a dilute acid solution to pretreat the straws, so that the contact area of the straws and steam in the steam explosion process is increased, the dilute acid solution can hydrolyze cellulose and bonds linked with the lignin, so that the lignin and the cellulose in the material before steam explosion are primarily separated, the steam explosion degree is increased, and the difficultly volatile substances influencing the enzymolysis effect in the straws are removed in an alcohol fumigation mode; (2) according to the invention, cheap straw lignocellulose materials and glucose are used for generating a polysaccharide mixture through an enzymolysis reaction to serve as a composite type induction carbon source, and the polysaccharide mixture can play a role in inducing trichoderma reesei to synthesize cellulase in a large quantity; (3) the method adopts an original programmed feeding mode of inducing a carbon source, so that manpower and material resources are saved, compared with the mode of feeding the inducing carbon source at a constant speed, the feeding mode provided by the invention has better inducing effect on trichoderma reesei than the mode of feeding the inducer at a constant speed, and the activity of the cellulase obtained by fermentation is obviously improved.
Drawings
FIG. 1 shows the monitoring of the fermentation process of a programmed variable-speed fed-batch composite type induced carbon source;
FIG. 2 is a view showing the monitoring of the fermentation process of constant velocity fed-batch composite type induced carbon source;
FIG. 3 is a graph showing the relationship between the amount of the compound type induced carbon source and the enzyme activity of the crude enzyme solution.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Preparation example 1 preparation of non-soluble carbon Source
S1: respectively preparing H with the mass concentration of 2%2SO4Mixing the solution and 2% fumaric acid solution in equal volume to obtain H solution with mass concentration of 1%2SO4And a dilute acid solution of fumaric acid;
s2: soaking corn straws in a dilute acid solution for 24 hours, taking out and drying, crushing the dried corn straws into coarse grains of 1-2cm, and fumigating the coarse grains with 75% ethanol for 4 hours in a ventilation environment;
s3: adding the steamed straw coarse grains into equal volume of water, mixing, putting into a steam explosion tank, heating to 190 ℃, maintaining for 10min, releasing pressure instantly, cooling, taking out, squeezing, drying, and pulverizing to obtain non-soluble carbon source.
Preparation example 2 preparation of Complex type inducible carbon Source
S1: dissolving 20% (w/v) of straw materials subjected to steam explosion pretreatment in a crude enzyme solution of trichoderma reesei without adding a compound induction carbon source for fermentation, and reacting for 24h under the conditions of 50 ℃, pH 4.8 and 180rpm to obtain a straw lignocellulose enzymolysis solution.
S2: adding 20-25CBU/g beta-glucosidase into 50% (w/v) glucose solution, and reacting at 60 deg.C, pH 4.8 and 180rpm for 24h to obtain glucose enzymatic hydrolysate.
S3: mixing the straw lignocellulose enzymolysis liquid and the glucose enzymolysis liquid according to the mass ratio of 3:7 to obtain a composite induced carbon source for later use.
Comparative preparation example 1 preparation of non-soluble carbon Source
The specific preparation method is the same as that of preparation example 1, except that in step S1, H is prepared at a mass concentration of 2%2SO4Mixing the solution and 2% acetic acid solution in equal volume to obtain H solution with mass concentration of 1%2SO4And acetic acid, Steps S2-S3 are the same as in preparation example 1.
Comparative preparation example 2 preparation of non-soluble carbon Source
The specific preparation method is the same as that of preparation example 1, except that H with a mass concentration of 1% is prepared in step S12SO4The solution was diluted acid solution, and Steps S2-S3 were the same as in preparation example 1.
Comparative preparation example 3 preparation of non-soluble carbon Source
The specific preparation method is the same as that of preparation example 1, except that the coarse corn straw particles crushed in step S2 are directly added with equal volume of water for steam explosion without being fumigated by alcohol, and steps S1 and S3 are the same as that of preparation example 1.
EXAMPLE 1 production of cellulase by culturing Trichoderma reesei
S1: the seed liquid of the trichoderma reesei is sucked from a glycerol tube by a pipette and added to a culture medium plate, and the total number of the inoculated spores in each culture dish is about 1 × 105Placing the culture dish in a mould incubator, culturing at 30 ℃ and humidity 60% for 5 days, observing that the green villiform spores are spread on the whole flat plate, adding ultrapure water into the culture dish, scraping the grown hypha and spores by using a glass rod, sucking the hypha and spores into a centrifuge tube or an EP tube by using a liquid transfer gun, and storing in a refrigerator at 4 ℃;
s2: sterilizing the seeding tank at 121 deg.C for 20min, placing into a clean bench, sucking spore liquid stored at 4 deg.C with a liquid-transferring gun, inoculating in the seeding tank with spore concentration of about 1 × 109seed/L, rotation speed of seed tank 180rpm, ventilation 1vvm, culturing at normal temperature for 24h, and controlling seed by adding 2M NaOH solutionCulturing at pH of 4.8-5.0, sampling to measure absorbance to 7-7.2, and inoculating into fermentation tank for subsequent fermentation;
s3: sterilizing the fermentation tank at 121 ℃ for 20min, setting the ventilation volume to be 1.0vvm after the sterilization is finished, inoculating the seed solution in the step (2) into the fermentation tank, wherein the inoculation amount is 10%, the ventilation volume is kept unchanged in the fermentation process, regulating and controlling the stirring speed according to the change of dissolved oxygen in the fermentation tank, controlling the stirring speed to be in the range of 180-200rpm/min, controlling the dissolved oxygen of the fermentation liquor to be in the range of 1.2-2.5mg/L, controlling the pH value of the fermentation liquor to be not less than 4.0 by adding 10% ammonia water in a flowing manner during the period, detecting that the dissolved oxygen of the fermentation liquor is less than 1.5mg/mL and has a continuous reduction trend in the 30-hour fermentation period, adding a composite type induced carbon source in a flowing manner for 30-36 hours, adding 12% of an induced carbon source in a flowing manner for 36-48 hours, adding 16% of an induced carbon source in a flowing manner for 48-60 hours, adding 20% of an induced carbon source in a, adding 12% of induced carbon source in the fermentation liquor at constant speed for 84-96h, filtering and centrifuging the fermentation liquor to obtain cellulase liquor with enzyme activity of 88 IU/ml. The basic parameters of the fermentation process are monitored in the process of feeding the composite type induced carbon source, and the specific parameter change is shown in figure 1.
The culture dish culture medium, the seed tank culture medium and the fermentation culture medium used in the experiment are the most preferred modes disclosed in the specification, and are not described in detail herein.
EXAMPLE 2 production of cellulase by culturing Trichoderma reesei
The culture methods and medium components of the culture method of the culture dish of trichoderma reesei in the step S1 and the culture method of the culture tank of trichoderma reesei in the step S2 are the same as those of the example 1, except that the non-soluble carbon source of the basic medium component in the fermentation medium of trichoderma reesei in the step S3 is prepared by the comparative preparation example 1, and the dilute acid solution of the soaked corn stalks is 1% of H2SO4And acetic acid solution, the fermentation method of S3 is the same as that of example 1, and the feeding mode of the compound type induced carbon source is the same as that of example 1. After the fermentation is finished, cellulase liquid with the enzyme activity of 39IU/ml is obtained.
EXAMPLE 3 production of cellulase by culturing Trichoderma reesei
Step S1 culture of Trichoderma reesei culture dish and step S2 culture method and culture of Trichoderma reesei seed tankThe basic composition was the same as in example 1 except that the non-soluble carbon source as the basic medium composition in the Trichoderma reesei fermentation medium of step S3 was prepared in comparative preparation example 2, and the diluted acid solution in which the corn stover was soaked was 1% H2SO4Solution, S3 fermentation method is the same as example 1, and feeding manner of composite type induced carbon source is the same as example 1. After the fermentation is finished, cellulase liquid with the enzyme activity of 46IU/ml is obtained.
EXAMPLE 4 production of cellulase by culturing Trichoderma reesei
The culture methods and the culture medium components of the culture dish of trichoderma reesei in the step S1 and the seed tank of trichoderma reesei in the step S2 are the same as those in the example 1, except that the non-soluble carbon source of the basic culture medium component in the fermentation culture medium of trichoderma reesei in the step S3 is prepared in the comparative preparation example 3, the corn stalks are not subjected to alcohol fumigation, the fermentation method of S3 is the same as that in the example 1, and the feeding mode of the compound type induced carbon source is also the same as that in the example 1. After the fermentation is finished, cellulase liquid with the enzyme activity of 33IU/ml is obtained.
Compared with the results of example 1, the finally obtained cellulase activity is obviously reduced enough to prove that the pretreatment mode of the corn straws really has influence on the quality of the finally obtained cellulase, the corn straws are soaked by using a dilute acid solution, the contact area of the straws and steam in the steam explosion process is increased, the bonds linked with cellulose and lignin are hydrolyzed, the lignin and the cellulose in the material before the steam explosion are primarily separated, and the steam explosion degree is increased. A preferred dilute acid combination is H2SO4And fumaric acid, although acetic acid can be used as an acid solution, acetic acid is not favorable for enzymolysis of the corn stalks. The cellulase produced in example 4 has the lowest activity, which shows that after the straw coarse grains are fumigated by alcohol, most of the substances which are difficult to volatilize can be taken away by the alcohol gas, and the influence of the substances which are difficult to volatilize on the enzymolysis of the corn straws is reduced. In addition, ethanol remaining in the fumigated straw coarse grain may have a positive effect on cellulase production.
EXAMPLE 5 production of cellulase by culturing Trichoderma reesei
The culture methods and the culture medium components of the culture of the trichoderma reesei petri dish in the step S1 and the culture of the trichoderma reesei seed tank in the step S2 are the same as those in the example 1, except that the compound type induced carbon source starts to flow at a constant speed at the 30 th hour of fermentation in the fermentation culture of the trichoderma reesei in the step S3, and the addition amount of the induced carbon source is the same as that in the example 1. After the fermentation is finished, cellulase liquid with 35IU/ml of enzyme activity is obtained. The basic parameters of the fermentation process were monitored during feeding of the compound induced carbon source, and the specific parameter changes are shown in FIG. 2.
Compared with the difference between the graph 1 and the graph 2, the difference is mainly embodied in that the crude enzyme activity is different, the mode of inducing the carbon source by utilizing the programmed variable-speed fed-batch method is more favorable for the production of the cellulase, and the fed-batch method provided by the invention is more consistent with the growth rule of the cellulase.
Optimization example 1 influence of mixture ratio of composite type induced carbon source on enzyme activity
The culture of trichoderma reesei in petri dishes and the seed culture method were the same as in example 1, 11 sets of experiments were performed in the fermentation culture process, and mixtures of straw lignocellulose hydrolysate and glucose hydrolysate in different ratios were respectively set as composite induction carbon sources, as shown in table 1. The total amount of the compound type induced carbon source added was 50% of the basic carbon source of the medium in the fermenter, fed-batch was started from 30h of the fermentation, the procedure of fed-batch was the same as that in example 1, after the end of the fermentation, the crude enzyme solution was obtained by centrifugation, and the filter paper enzyme activity of the crude enzyme solution was measured, as shown in table 1.
TABLE 1 Induction Effect of inducers with different ratios on Trichoderma reesei
As can be seen from Table 1, the experimental group 4 is the group with the highest filter paper enzyme activity, the optimal proportion of the composite induced carbon source is 30% of straw lignocellulose enzymatic hydrolysate and 70% of glucose enzymatic hydrolysate, and the highest induced enzyme activity can reach 86 IU/mL.
Optimization example 2 influence of addition amount of Compound type Induction carbon Source on enzyme Activity
And (3) adjusting the addition amount of the induced carbon source according to the optimal proportion of the induced carbon source obtained in the optimization example 1, and observing whether the addition amount of the induced carbon source has influence on the cellulase activity. The culture method of the trichoderma reesei culture dish and the seed culture method are the same as example 1, and the compound induced carbon source proportion and the fed-batch procedure in the fermentation culture process are the same as example 1, except that 5 groups of experiments are set, and the amount of the fed-batch induced carbon source is respectively 30%, 40%, 50%, 60% and 70% of the basic carbon source of the culture medium in the fermentation system. The procedure of example 1 was followed in which crude enzyme solution was obtained by centrifugation after completion of fermentation in the same manner as in example 1, and the filter paper enzyme activity of the crude enzyme solution was measured, and the results are shown in FIG. 3.
As can be seen from FIG. 3, when the addition amount of the induced carbon source is most suitable for 50% of the basic carbon source of the culture medium, the addition amount of the induced carbon source is too small, the induction effect of Trichoderma reesei is not enough, and the yield of cellulase is not high; when too much carbon metabolism repression effect is generated, the trichoderma reesei can not be continuously and efficiently induced to synthesize a large amount of cellulase.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A method for promoting Trichoderma reesei to produce cellulase comprises the steps of after Trichoderma reesei is fermented for 24-36h, detecting that the dissolved oxygen of fermentation liquor is 1.5-2.0mg/mL and feeding a composite type induction carbon source in a flowing mode when the fermentation liquor continuously descends, wherein the composite type induction carbon source is obtained by mixing straw lignocellulose enzymolysis liquid and glucose enzymolysis liquid according to the mass ratio of 2-6 (8-4), and preferably, the straw lignocellulose enzymolysis liquid accounts for 20-50 parts by taking the total mass of the straw lignocellulose enzymolysis liquid and the glucose enzymolysis liquid as 100 parts.
2. The method as claimed in claim 1, wherein the preparation method of the straw lignocellulose enzymolysis solution is as follows: dissolving 20-25% (w/v) of straw materials subjected to steam explosion pretreatment in a crude enzyme solution of Trichoderma reesei which is not fermented by a compound induced carbon source, and reacting for 20-24h at 50-60 ℃, pH 4.8-5.0 and 200rpm at 180-; the preparation method of the glucose enzymolysis liquid comprises the following steps: using 50-60% (w/v) glucose solution, adding 20-25CBU/g beta-glucosidase, reacting at 60-65 deg.C, pH 4.8-5.0, and 200rpm for 20-24 h.
3. The method according to claim 1, wherein the addition amount of the composite type induction carbon source is 50-60% of the mass of the basic carbon source in the Trichoderma reesei fermentation medium.
4. The method of claim 1, wherein the complex induced carbon source stream is fed in a manner that: adding 11-14% of induced carbon source in uniform flow for 24-36h, adding 15-17% of induced carbon source in uniform flow for 36-48h, adding 15-20% of induced carbon source in uniform flow for 48-60h, adding 20-24% of induced carbon source in uniform flow for 60-72h, adding 15-16% of induced carbon source in uniform flow for 72-84h, and adding 10-12% of induced carbon source in uniform flow for 84-96 h.
5. The method of claim 3, wherein the Trichoderma reesei fermentation medium comprises the following components: 3.5-4.5% of non-soluble carbon source, 1-2% of glucose, 3-4% of concentrated solution of macroelement nutrient salt capable of producing enzyme, 0.01-0.04% of concentrated solution of microelement capable of producing enzyme, 0.1-0.2% of corn steep liquor and the balance of water.
6. The method according to claim 5, wherein the preparation of the non-soluble carbon source comprises the steps of:
(1) using H before steam explosion treatment2SO4Soaking the straws with mixed acid of fumaric acid;
(2) drying the soaked straws, and fumigating with alcohol.
7. The method according to claim 6, wherein the preparation of the non-soluble carbon source comprises the steps of:
(1) soaking the straws with dilute acid before steam explosion treatment, wherein the dilute acid is H2SO4And preparing an acid solution from fumaric acid, wherein H is contained in the acid solution2SO4The mass concentration of the fumaric acid is 0.1-1 percent respectively;
(2) drying the soaked straws, crushing the straws into 1-2cm coarse grains, and fumigating the coarse grains with alcohol for 2-4 hours, wherein the alcohol is selected from 75-95% ethanol solution;
(3) adding water according to the volume ratio of 1:1-1.5 after fumigation, mixing, putting into a steam explosion tank, heating to 185-195 ℃, maintaining for 10-20min, then releasing pressure instantaneously, cooling, taking out, squeezing, drying and crushing to obtain the non-soluble carbon source.
8. The method of claim 6, wherein the straw is selected from one of corn stover, wheat straw, rice straw, wood chips, energy plants, or forestry waste.
9. The method as claimed in claim 6, wherein the straw lignocellulose enzymolysis solution accounts for 30-40 parts based on 100 parts of the total mass of the straw lignocellulose enzymolysis solution and the glucose enzymolysis solution.
10. The method according to any one of claims 1 to 9, wherein the method for promoting the production of cellulase by trichoderma reesei specifically comprises the following steps:
(1) adding the seed solution of Trichoderma reesei to the culture medium plate, wherein the total number of inoculated spores in each culture dish is 1 × 105-1×106Culturing in an incubator with temperature of 30-32 deg.C and humidity of 60-65% for 5-7 days, adding ultrapure water into the culture dish after the green villiform spores are covered on the whole plate, scraping off hypha and spores, and storing for use;
(2) will be provided withInoculating the spore liquid preserved in the step (1) into a seeding tank to ensure that the concentration of the spores is 1 multiplied by 109-1×1010Culturing at normal temperature for 20-24 hours at the seed tank rotation speed of 180-;
(3) and (3) inoculating the Trichoderma reesei seed solution obtained in the step (2) into a fermentation tank, controlling the rotation speed of the fermentation tank to be 180-350rpm/min, controlling the ventilation volume to be 1-1.5vvm, enabling the dissolved oxygen of the fermentation liquor to be 1.2-3.5mg/L, controlling the pH value of the fermentation liquor to be not lower than 4.0 by adding ammonia water in a flowing manner, and after fermenting for 24-36h, detecting that the dissolved oxygen of the fermentation liquor is 1.5-2.0mg/mL and adding a compound type induced carbon source in a flowing manner when the dissolved oxygen of the fermentation liquor continues to decline.
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