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CN-122012570-A - Method for preparing cellulase and application thereof

CN122012570ACN 122012570 ACN122012570 ACN 122012570ACN-122012570-A

Abstract

The invention provides a method for preparing cellulase and application thereof. The method for preparing the cellulase comprises the steps of S1) improving the expression level of SNC1 genes in Trichoderma reesei to obtain Trichoderma reesei engineering bacteria, and S2) culturing the Trichoderma reesei engineering bacteria and collecting the cellulase. Can solve the problem that Trichoderma reesei is difficult to produce cellulase in a large quantity under the culture condition containing glucose in the prior art, and is suitable for the technical field of microorganisms.

Inventors

  • ZHANG YAN
  • NIU SHUO
  • XIAO MINGHUA
  • YU CHEN
  • ZHENG XIANLIANG
  • HE JUNFENG
  • CHEN SHI
  • YU HUASHUN

Assignees

  • 安琪酶制剂(宜昌)有限公司
  • 安琪酵母股份有限公司

Dates

Publication Date
20260512
Application Date
20260413

Claims (15)

  1. 1. A method of preparing a cellulase comprising: s1) improving the expression level of SNC1 genes in Trichoderma reesei to obtain Trichoderma reesei engineering bacteria; s2) culturing the Trichoderma reesei engineering bacteria and collecting to obtain the cellulase; The culturing comprises the step of culturing the trichoderma reesei engineering bacteria in a fermentation medium, wherein a carbon source in the fermentation medium comprises glucose, and the concentration of the glucose is 10-30g/L.
  2. 2. The method according to claim 1, wherein the sequence of the SNC1 gene comprises a sequence of SNC1 gene derived from Trichoderma reesei QM6a or the SNC1 gene of Trichoderma reesei itself.
  3. 3. The method according to claim 1, wherein the protein encoded by the SNC1 gene is an amino acid sequence shown in SEQ ID NO. 1 or a protein having a homology of 70% or more with the amino acid sequence.
  4. 4. The method according to claim 1 or 3, wherein the SNC1 gene is the nucleic acid sequence shown in SEQ ID NO. 2 or a sequence having a homology of 70% or more with the nucleic acid sequence.
  5. 5. The method of claim 1, wherein the fermentation medium comprises 1-3% glucose, 0.8-3% corn steep liquor, 0.1-1% ammonium sulfate, 0.05-0.6% dipotassium hydrogen phosphate, 0.02-0.18% magnesium sulfate, and 0.02-0.1% calcium chloride.
  6. 6. The method of claim 5, wherein glucose in the fermentation medium is added to the fermentation medium in the form of a fermentation syrup; the preparation method of the fermentation syrup comprises the steps of mixing glucose and 0.05-1.5% of beta-glucosidase to obtain a reaction system, and reacting the reaction system under the stirring condition to obtain the fermentation syrup.
  7. 7. The method according to claim 6, wherein the temperature of the reaction system is 50-60 ℃, the pH of the reaction system is 4.0-5.0, the time for carrying out the reaction under the stirring condition is 70-80 hours, and the rotation speed of the stirring is 80-150rpm.
  8. 8. The method of claim 6, wherein the concentration of glucose in the fermentation syrup is 400-600g/L and the concentration of beta-glucosidase is 10000-15000 IU/g.
  9. 9. The method of claim 1, wherein S1) comprises transferring an exogenous SNC1 gene into the Trichoderma reesei and/or increasing the expression level of an endogenous SNC1 gene in the Trichoderma reesei.
  10. 10. The method of claim 9, wherein transferring the exogenous SNC1 gene into the Trichoderma reesei comprises transferring an SNC1 expression cassette into the Trichoderma reesei, the SNC1 expression cassette comprising a promoter and the SNC1 gene linked in sequence from 5 'to 3', the promoter comprising Pcdna1.
  11. 11. The method of claim 10, wherein transferring exogenous SNC1 genes into Trichoderma reesei comprises transferring a plasmid into Trichoderma reesei, the plasmid comprising the SNC1 expression cassette, the plasmid comprising pPTRII.
  12. 12. A method for modifying a trichoderma reesei carbon catabolic repression effect, comprising increasing the expression level of SNC1 gene in trichoderma reesei.
  13. 13. The retrofitting method according to claim 12, wherein said retrofitting method steps include any one or more of: Transforming the SNC1 gene into the Trichoderma reesei, or increasing the copy number of the SNC1 gene in the Trichoderma reesei, or replacing the promoter of the SNC1 gene in the Trichoderma reesei with a strong promoter.
  14. 14. The method of engineering according to claim 12 or 13, wherein the SNC1 gene comprises the SNC1 gene of the method of producing cellulase according to any one of claims 2 to 4.
  15. 15. The method for producing cellulase according to any one of claims 1 to 11, or the method for modifying the effect of derepression of carbon metabolism of trichoderma reesei according to any one of claims 12 to 14, or the use of said SNC1 gene in trichoderma reesei culture, or cellulase production.

Description

Method for preparing cellulase and application thereof Technical Field The invention relates to the technical field of microorganisms, in particular to a method for preparing cellulase and application thereof. Background Cellulose is a homopolysaccharide formed by connecting glucose through beta-1, 4 glycosidic bonds, is a main component of plant cell walls, and is the most abundant renewable resource on the earth. Cellulases are a complex enzyme system capable of degrading cellulose into glucose, and can be classified into endoglucanases, exoglucanases and beta-glucosidases according to the site of action on cellulose. When the cellulose is used as a substrate, the endoglucanase can recognize and cut beta-1, 4 glucosidic bonds of an amorphous region in the cellulose, so that short-chain cellooligosaccharides and cellopolysaccharides with different lengths are released, and substrates are provided for exoglucanase and beta-glucosidase. Therefore, endoglucanases play an important role in the hydrolysis process of cellulose, and the activity of the endoglucanases often directly influences the hydrolysis efficiency of cellulose by cellulase. At present, trichoderma reesei (Trichoderma reesei) can produce cellulase to become an important enzyme preparation production strain in the industrial fields of biofuel, food and feed, papermaking and textile and the like, has abundant genome data and has the potential of synthetic biological transformation, and simultaneously has great application value in the fields of biological control and plant symbiosis. However, in the actual production process, the production of cellulase by Trichoderma reesei still faces challenges such as further improvement of enzyme yield and inhibition of cellulase synthesis by carbon catabolism. Carbon catabolism repression refers to a phenomenon in which microorganisms preferentially use readily decomposable carbon sources such as glucose in mixed carbon sources containing glucose and other saccharides, while inhibiting the expression of genes involved in the metabolism of other carbon sources. This repressing effect limits the efficient expression of cellulases in industrial production, especially when inexpensive and readily available carbon sources are used. Therefore, how to increase the enzyme activity of Trichoderma reesei cellulase and to alleviate the inhibition of carbon catabolism on cellulase synthesis is a problem to be solved in the current cellulase production field. Disclosure of Invention The invention mainly aims to provide a method for preparing cellulase and application thereof, and the method is used for constructing Trichoderma reesei engineering bacteria for producing cellulase by improving the expression of SNC1 genes in Trichoderma reesei so as to solve the problem that Trichoderma reesei is difficult to produce cellulase in a large quantity under the culture condition containing glucose in the prior art. In order to achieve the above object, according to a first aspect of the present invention, there is provided a method for producing cellulase, comprising S1) increasing the expression level of SNC1 gene in Trichoderma reesei to obtain Trichoderma reesei engineering bacteria, S2) culturing the Trichoderma reesei engineering bacteria and collecting the cellulase. Further, the sequence of the SNC1 gene includes a sequence of SNC1 gene derived from Trichoderma reesei QM6a or SNC1 gene of Trichoderma reesei itself. Further, the protein encoded by the SNC1 gene is an amino acid sequence shown in SEQ ID NO.1, or a protein having homology of 70% or more with the amino acid sequence. Further, the SNC1 gene is a nucleic acid sequence shown in SEQ ID NO. 2 or a sequence having a homology of 70% or more with the nucleic acid sequence. Further, the culturing comprises culturing the Trichoderma reesei engineering bacteria in a fermentation medium, wherein the carbon source in the fermentation medium comprises glucose, and the concentration of the glucose is 10-30g/L. Further, the fermentation medium comprises 1-3% of glucose, 0.8-3% of corn steep liquor, 0.1-1% of ammonium sulfate, 0.05-0.6% of dipotassium hydrogen phosphate, 0.02-0.18% of magnesium sulfate and 0.02-0.1% of calcium chloride. The culture medium of the application contains 1-3% (w/v) glucose in the mass-volume ratio. Further, glucose in the fermentation medium is added into the fermentation medium in the form of fermentation syrup, and the preparation method of the fermentation syrup comprises the steps of mixing glucose with 0.05-1.5% of beta-glucosidase to obtain a reaction system, and reacting the reaction system under the stirring condition to obtain the fermentation syrup. Further, the temperature of the reaction system is 50-60 ℃, the pH of the reaction system is 4.0-5.0, the reaction time under the stirring condition is 70-80h, and the stirring rotating speed is 80-150rpm. Further, the concentration of glucose in the fermentation syrup is 400-600g/L, and th