CN113151006A - Trichoderma reesei strain capable of producing cellulase with improved activity and application thereof - Google Patents
Trichoderma reesei strain capable of producing cellulase with improved activity and application thereof Download PDFInfo
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Abstract
The invention discloses a Trichoderma reesei strain A2H capable of producing cellulase with improved activity, which is preserved in China general microbiological culture Collection center with the preservation number: CGMCC No.21470, the preservation time is as follows: on the day of 2021, 03 and 17, the address of the depository is: institute of microbiology, academy of sciences of china. The invention also discloses a method for producing cellulase with improved activity. Cellulases and compositions are also disclosed. The invention also discloses application of the Trichoderma reesei strain A2H or cellulase in cellulose degradation or scientific research or cellulase preparation. The cellulase filter paper enzyme activity of the trichoderma reesei A2H strain is improved by 46%, the extracellular protein secretion is improved by more than 30%, and the conversion rate of hydrolyzed steam-exploded corn straws is improved by more than 13%.
Description
Technical Field
The invention belongs to the technical field of cellulose degradation, and relates to a trichoderma reesei strain A2H capable of producing cellulase with improved activity, a method for producing the cellulase with improved activity, cellulase, a composition, and application of the trichoderma reesei strain A2H or the cellulase in cellulose degradation or scientific research or cellulase preparation.
Background
Cellulase is one of the main factors influencing the comprehensive utilization of biomass, particularly the cost of cellulosic ethanol at present. The enzyme cost accounts for about 20-30% of the production cost of cellulosic ethanol. The addition amount of the cellulase is usually 3-10% of the mass of the lignocellulose raw material, and the conversion rate of the raw material is over 85% after enzymolysis for 72 hours or longer. Compared with amylase, the cellulase efficiency is more than 1000 times different according to g sugar/(g enzyme h), and the cost is more than 30 times different. Therefore, improving the performance of the cellulase and reducing the use cost of the cellulase become important problems to be solved urgently in China for developing the cellulose ethanol industry.
The international largest enzyme preparation companies Novozymes, Genencor, DSM, etc. have a great development effort in the production of dedicated cellulase preparations from cellulosic ethanol, and several commercial cellulase preparations were introduced one after the other (Novozymes2012, Genecor 2012). Novozymes corporation has claimed second generation cellulase preparations
The enzyme cost of CTec2 is about 50 cents per gallon of ethanol. The company has newly developed a third-generation complex cellulase
CTec3, claiming that the amount of enzyme required per ton of ethanol produced is 50 kg, the enzyme cost accounts for 20% of the total cost, and is comparable to any enzyme preparation for cellulosic ethanol on the market in terms of substrate conversion efficiency (Novozymes 2012).
The method is an important method for greatly reducing the proportion of the cellulase in the cost of the cellulosic ethanol by developing a higher-yield cellulase production strain and optimizing a fermentation enzyme production process to further carry out on-site production. For example: an exemplary factory for annual production of 1000 tons of cellulose ethanol, established by Clariant, germany in bavaria, has been subjected to detailed comparative analysis on the advantages of integrated on-site enzyme production in years of experimental operation, and has found that the proportion of cellulase in the total production cost of ethanol can be reduced from 35% of that of commercially available enzyme when purchased from outsources or 28% of that of enzyme production in different places to 10% of that of integrated on-site enzyme production, and that cost and risk can be controlled, so that the cellulose ethanol has market competitiveness (Losordo Z2017).
Because the enzyme activity of the strain with the independent intellectual property rights in China is relatively low, the cost of the enzyme preparation in China is 2 times higher than that of the foreign advanced technology (such as Novitin) at present, and therefore, the development of the high-activity cellulase system is particularly critical.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and/or disadvantages and to provide at least the advantages described hereinafter.
It is a further object of the present invention to provide a strain of trichoderma reesei A2H capable of producing cellulase with increased activity.
It is another object of the present invention to provide a method for producing cellulase with improved activity.
It is another object of the present invention to provide a cellulase.
It is a further object of the present invention to provide compositions for providing a degrading effect in the degradation of cellulose.
It is a further object of the present invention to provide the use of Trichoderma reesei strain A2H or a cellulase in the degradation of cellulose or in scientific research or in cellulase preparations.
Therefore, the technical scheme provided by the invention is as follows:
trichoderma reesei strain A2H capable of producing cellulase with increased activity, Trichoderma reesei strain A2H, assigned by the taxonomic name: trichoderma reesei, strain Trichoderma reesei A2H was deposited in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the following deposit numbers: CGMCC No.21470, the preservation time is as follows: on the day of 2021, 03 and 17, the address of the depository is: xilu No. 1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.
A method for producing a cellulase with increased activity comprising the steps of:
1) inoculating Trichoderma reesei strain A2H in a culture medium for culturing;
2) harvesting cellulase from the culture medium in the form of a culture broth of Trichoderma reesei strain A2H grown in step 1).
Preferably, in the method for producing cellulase with improved activity, the culture conditions are as follows: the temperature is 24-28 ℃, the pH value is 4.8-5.2, the rotating speed is 250-300rpm, the dissolved oxygen in the fermentation liquor is 25-35% (v/v), and the culture is carried out for a certain time.
Preferably, in the method for producing cellulase with improved activity, the culture time is 24 to 120 hours.
Preferably, in the method for producing cellulase with improved activity, the culture conditions are as follows: the temperature is 26 ℃, the pH value is 5.0, and the dissolved oxygen in the fermentation liquor is 30% (v/v).
Preferably, in the method for producing cellulase with improved activity, the culture medium comprises the following components in the culture: 20-30 g/L glucose, 2-6 g/L corn steep liquor dry powder, 1.60-1.72 g/L KOH, (NH)4)2SO42.6-3.0 g/L and MgSO4 0.4～0.8g/L。
Preferably, in the method for producing cellulase with improved activity, the culture medium comprises the following components in the culture: 25g/L glucose, 4g/L corn steep liquor dry powder, 1.66g/L KOH, (NH)4)2SO42.8g/L and MgSO40.6g/L。
A cellulase derived from said trichoderma reesei strain A2H or said method.
A composition comprising a cellulase as described, said composition providing a degrading function in cellulose degradation.
The Trichoderma reesei strain A2H or the cellulase in cellulose degradation or scientific research or cellulase preparation application.
The invention at least comprises the following beneficial effects:
the invention utilizes the filamentous fungus microfluidic high-throughput screening technology, breaks through the traditional strain breeding technology, accurately and directionally promotes the cellulase activity of the strain with the independent intellectual property rights, and greatly improves the efficiency and the success rate of strain breeding. The cellulase strain with the independent intellectual property rights is created for the first time, and the monopoly of foreign enzyme preparations is broken.
The cellulase filter paper enzyme activity of the trichoderma reesei A2H strain is improved by 46%, the extracellular protein secretion is improved by more than 30%, and the conversion efficiency of hydrolyzed steam-exploded corn straws is improved by more than 13%.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is an electron micrograph of the hyphal morphology of Trichoderma reesei RUT-C30 and DES-15 strains according to the present invention;
FIG. 2 is a graph of a standard curve of glucose in one embodiment of the present invention;
FIG. 3 is a graph of a standard curve of bovine serum albumin in one embodiment of the present invention;
FIG. 4 is a graph showing cellulase filter paper enzyme activities of Trichoderma reesei RUT-C30, DES-15 and A2H strains in one embodiment of the present invention;
FIG. 5 is a graph showing the amount of extracellular secreted proteins of Trichoderma reesei RUT-C30, DES-15 and A2H strains in one embodiment of the present invention;
FIG. 6 is a graph comparing the cellulase hydrolysis efficiencies of Trichoderma reesei RUT-C30, DES-15 and A2H strains in one embodiment of the present invention.
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
The invention provides a trichoderma reesei strain A2H capable of producing cellulase with improved activity, the trichoderma reesei strain A2H is classified and named as follows: trichoderma reesei, strain Trichoderma reesei A2H was deposited in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the following deposit numbers: CGMCC No.21470, the preservation time is as follows: on the day of 2021, 03 and 17, the address of the depository is: xilu No. 1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.
The present invention also provides a method for producing a cellulase with increased activity, comprising the steps of:
1) inoculating Trichoderma reesei strain A2H into a culture medium for culture;
2) harvesting cellulase from the culture medium in the form of a culture broth of Trichoderma reesei strain A2H grown in step 1). Preferably, the culture medium after fermentation is centrifuged to obtain the supernatant, thus obtaining the product containing the cellulase.
In some of the embodiments of the present invention, preferably, the culture conditions are: the temperature is 24-28 ℃, the pH value is 4.8-5.2, the rotating speed is 250-300rpm, the dissolved oxygen in the fermentation liquor is 25-35% (v/v), and the culture is carried out for a certain time.
In some embodiments of the present invention, preferably, the culturing is performed for 24 to 120 hours.
In one embodiment of the present invention, preferably, the culture conditions are: the temperature is 26 ℃, the pH value is 5.0, and the dissolved oxygen in the fermentation liquor is 30% (v/v).
In some embodiments of the invention, it is preferred that the culture medium comprises the following components: 20-30 g/L glucose, 2-6 g/L corn steep liquor dry powder, 1.60-1.72 g/L KOH, (NH)4)2SO42.6-3.0 g/L and MgSO4 0.4～0.8g/L。
In one embodiment of the present invention, preferably, the culture medium comprises the following components: 25g/L glucose, 4g/L corn steep liquor dry powder, 1.66g/L KOH, (NH)4)2SO42.8g/L and MgSO4 0.6g/L。
The invention also provides cellulase derived from the Trichoderma reesei strain A2H or the method.
The invention also provides a composition comprising the cellulase, wherein the composition provides a degradation function in cellulose degradation.
The invention also provides application of the Trichoderma reesei strain A2H or the cellulase in cellulose degradation or scientific research or cellulase preparation.
In order to make the technical solution of the present invention better understood by those skilled in the art, the following examples are now provided for illustration:
the starting strain is Trichoderma reesei RUT-C30, and as shown in figure 1, Trichoderma reesei RUT-C30 is used as a base for mutagenesis to obtain a Trichoderma reesei DES-15 strain, and the biggest characteristic is that the hypha form is changed and the cellulase activity is improved. The strain with high cellulase yield is successfully screened by combining the strain breeding modification technology of the mutagenesis and high-throughput screening technology, compared with the original strain, the target strain DES-15 has the advantages that the hyphae is 0.6 times, the diameter is 1.6 times, the branch number is about 2 times, and the enzyme activity is improved by about 31% after fermentation optimization.
Based on a strain DES-15, the inventor utilizes ARTP mutagenesis, optimizes conditions such as trichoderma reesei droplet generation, a sorting chip, droplet size and the like by means of a droplet microfluidic high-throughput platform, overcomes the defect that the growth process of trichoderma reesei mycelia can cause the piercing of wrapped droplets to cause the pollution of cross metabolites among the droplets, screens and marks a combined fluorogenic substrate to achieve the aim of sorting the trichoderma reesei micro droplets, firstly creates a trichoderma reesei droplet microfluidic high-throughput screening technology, the screening throughput can reach 10000/h, reduces the screening period of the traditional method from 7-15 days to 24 hours to complete the screening period, breaks through the technical bottleneck of the traditional filamentous fungus screening method, accurately and directionally promotes the cellulase activity of the strains with intellectual property rights, greatly improves the efficiency and success rate of strain breeding, and compares the hypha form of the trichoderma reesei A2H with the strain DES-15, the change is not obvious, but the Trichoderma reesei A2H strain realizes that the enzyme activity of the cellulase filter paper is improved by 46 percent, and the extracellular protein secretion is improved by more than 30 percent.
The cellulase activity and extracellular protein secretion of the three strains are evaluated as follows:
the culture method comprises the following steps: the fermentation medium and the seed medium are both: glucose 25g/L, 4g/L of corn steep liquor dry powder, KOH1.66g/L, (NH)4)2SO4 2.8g/L，MgSO4 0.6g/L。
Fermentation tank control conditions: pH5.0, temperature 26 ℃, 250-. Sampling for 12, 24, 36, 48, 60, 72, 84, 96 and 120 hours of fermentation time, centrifuging to obtain supernatant, and performing enzyme activity and protein content determination. The filter paper enzyme activity and the protein secretion of the strain A2H which has high yield of cellulase bred in the whole fermentation process are obviously higher than those of the original strains RUT-C30 and DES-15.
Determination of cellulase filter paper enzyme activity: a piece of folded Whatman filter paper strip (50 +/-1 mg) is placed in a colorimetric tube, 1mL of 0.05M pH4.8 acetic acid-sodium acetate buffer solution and 0.5mL of diluted enzyme solution are sequentially added, then the mixture is placed in a water bath at 50 ℃ for reaction for 1 hour, and then the generated amount of generated glucose is determined by a DNS method, and the specific steps are as follows: and after the reaction is finished, adding 3mL of DNS reagent, boiling for 5min, absorbing 200 microliters of reaction solution, adding 1mL of distilled water for dilution, taking 200 microliters of the diluted solution, measuring the light absorption value of OD540, and comparing with the prepared glucose standard curve to obtain the content of glucose, thereby calculating the enzyme activity of the filter paper. The filter paper enzyme activity is defined as the amount of enzyme required to convert the substrate to 1 micromole of glucose per minute as 1 activity unit.
1. glucose-DNS Standard Curve
Drawing a glucose standard curve
Glucose was prepared in distilled water at a concentration of 100g/L and diluted as shown in the following table. After taking 0.5mL of the diluted glucose solution and adding 1mL of 0.05mol/L, pH4.8 citric acid buffer, and adding 3mL of DNS reaction solution with 1.5mL of 0.05mol/L, pH4.8 citric acid buffer as a zero point control, heating in a boiling water bath for 5min, diluting with distilled water as appropriate (generally, 0.2mL is added to 1mL of distilled water for dilution), taking 0.2mL to a 96-well plate, and measuring the absorbance at 540 nm.
TABLE 1 glucose standards
When the glucose concentration is in the range of 10g/L-34g/L, there is a good linear relationship between the absorbance at 540nm and the glucose concentration, and the correlation coefficient R20.997, and the formula is y-3.005 x +0.145, where y is the absorbance at 540nm and x is the glucose concentration (%). As shown in fig. 2.
② filter paper enzyme activity calculation formula ═ glucose release amount (mu mol/L)/mL enzyme solution/min
The raw data are as follows:
the results of calculating the filter paper enzyme activity using the formula are as follows, and a graph as shown in fig. 4 is plotted:
2. determination of protein concentration in fermentation broth
Protein concentration was determined using the Bradford method: the standard curve was plotted using bovine serum albumin as the standard. Adding 100 mu L of diluted enzyme solution into a test tube, then adding 1mL of Coomassie brilliant blue working solution, keeping the volume ratio of the Coomassie brilliant blue working solution to the enzyme solution to be about 10:1, uniformly mixing, standing for 5min, sucking 200 microlitres of the mixed solution, measuring the OD595 value in a 96-well plate, and calculating the protein concentration through a standard curve.
Drawing a bovine serum albumin standard curve
As shown in FIG. 3, the curve of bovine serum albumin is shown in FIG. 3, and it is known from FIG. 3 that when the protein concentration is in the range of 0.02g/L-0.2g/L, there is a good linear relationship between the absorbance at 595nm and the protein concentration, the correlation coefficient R2 is 0.992, and the formula is y 1.679x +0.014, where y is the absorbance at 595nm and x is the protein concentration (g/L). The original data of the protein concentration measurement of the fermentation broth
Calculating the protein content in the fermentation broth by using a bovine serum albumin standard curve, and drawing to obtain a curve chart shown in figure 5.
In order to further illustrate the superiority of the high-yield strain A2H in the aspect of producing cellulase, the highest value of the enzyme activity and the highest value of the protein secretion of the high-yield strain A2H of the cellulase are compared with the original strain, and the improvement rate of the filter paper enzyme activity is calculated. The Trichoderma reesei strain A2H is fermented for 120h, the filter paper enzyme activity can reach 29.00FPU/ml, and is improved by 46.5% compared with DES-15; the Trichoderma reesei strain A2H is fermented for 120h, the extracellular protein secretion amount reaches 13.5g/L, and is increased by 31.1% compared with DES-15; from the aspect of cellulase production efficiency, the efficiency of producing cellulase by the strain A2H is higher and is improved by 46.5 percent compared with DES-15.
3. Steam-exploded corn stalk saccharification hydrolysis experiment
To further evaluate the differences in cellulase production performance of different strains, the inventors selected lignocellulose hydrolysis conditions and compared the cellulase hydrolysis efficiency.
The saccharification and hydrolysis experiment of the steam exploded corn straws is carried out in a 20mL reaction system, the dosage of enzyme is 10FPU/g substrate, the substrate is the corn straws subjected to steam explosion treatment, 2.0g substrate (10 percent of the total volume), pH is 4.8, saccharification is carried out for 72h at 50 ℃, and three groups of parallel experiments are carried out simultaneously. Samples were taken at 24, 48 and 72 hours respectively, and after centrifugation, 50. mu.l of supernatant was taken for determination of glucose content. And (3) diluting the glucose solution by 10-100 times with water, and then determining the yield of the glucose by using a biosensor analyzer.
The cellulose conversion was calculated using the amount of glucose produced. As is apparent from FIG. 6 and the following table, the cellulase produced by the high-yield strain A2H has more excellent performance, and the conversion rate of the cellulose in the steam-exploded pretreated straws exceeds 95 percent after 72 hours of hydrolysis. After 72 hours of straw pretreatment by hydrolysis and steam explosion of cellulase produced by Trichoderma reesei strain DES-15, the cellulose conversion rate is 82.4%. The hydrolysis efficiency of the cellulase preparation produced by the strain A2H is improved by about 13 percent, and the hydrolysis performance is better.
The number of modules and the processing scale described herein are intended to simplify the description of the invention. The use, modifications and variations of the Trichoderma reesei strains of the invention capable of producing cellulase with increased activity and uses thereof will be apparent to those skilled in the art.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
Claims (10)
1. Trichoderma reesei strain A2H capable of producing cellulase with increased activity, Trichoderma reesei strain A2H, assigned by the taxonomic name: trichoderma reesei, strain Trichoderma reesei A2H was deposited in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the following deposit numbers: CGMCC No.21470, the preservation time is as follows: on the day of 2021, 03 and 17, the address of the depository is: xilu No. 1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.
2. A method for producing a cellulase with increased activity, comprising the steps of:
1) inoculating Trichoderma reesei strain A2H into a culture medium for culture;
2) harvesting cellulase from the culture medium in the form of a culture broth of Trichoderma reesei strain A2H grown in step 1).
3. The method for producing cellulase with increased activity according to claim 2, wherein the culture conditions are: the temperature is 24-28 ℃, the pH value is 4.8-5.2, the rotating speed is 250-300rpm, the dissolved oxygen in the fermentation liquor is 25-35% (v/v), and the culture is carried out for a certain time.
4. The method for producing a cellulase having an improved activity according to claim 3, wherein the culturing is carried out for 24 to 120 hours.
5. The method for producing cellulase with increased activity according to claim 3, wherein the culture conditions are: the temperature is 26 ℃, the pH value is 5.0, and the dissolved oxygen in the fermentation liquor is 30% (v/v).
6. The method for producing cellulase with increased activity according to claim 2, wherein the culture medium comprises the following components in the culture: 20-30 g/L glucose, 2-6 g/L corn steep liquor dry powder, 1.60-1.72 g/L KOH, (NH)4)2SO42.6-3.0 g/L and MgSO4 0.4～0.8g/L。
7. The method for producing cellulase with increased activity according to claim 6, wherein the culture medium comprises the following components in the culture: 25g/L glucose, 4g/L corn steep liquor dry powder, 1.66g/L KOH, (NH)4)2SO42.8g/L and MgSO4 0.6g/L。
8. A cellulase derived from trichoderma reesei strain A2H as claimed in claim 1 or the process as claimed in claim 2.
9. A composition comprising the cellulase enzyme of claim 8, said composition providing a degrading function in cellulose degradation.
10. Use of the cellulase of Trichoderma reesei strain A2H according to claim 1 or claim 8 in cellulose degradation or in scientific research or in cellulase preparation.
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| CN113729110A (en) * | 2021-09-26 | 2021-12-03 | 中国科学院天津工业生物技术研究所 | Efficient low-cost pretreatment and solid-state fermentation combined method for biomass material and application of method in production of single-cell protein feed |
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| CN114958623B (en) * | 2022-06-23 | 2024-03-15 | 中国科学院微生物研究所 | Trichoderma viride for high-yield cellulase and application thereof |
| CN115261238A (en) * | 2022-07-14 | 2022-11-01 | 大理大学 | Fermentation method of bacterial strain with high cellulase yield |
| CN117286039A (en) * | 2023-11-27 | 2023-12-26 | 中国科学院天津工业生物技术研究所 | Trichoderma reesei strain with optimized mycelium morphology and application thereof |
| CN117286039B (en) * | 2023-11-27 | 2024-03-12 | 中国科学院天津工业生物技术研究所 | Trichoderma reesei strain with optimized mycelium morphology and application thereof |
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