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Method for increasing yield of cellulase produced by Trichoderma reesei

A technology of cellulase and microorganisms, applied in the field of microorganisms, can solve the problems of decreased production of cellulase, decreased biomass of Trichoderma reesei, unfeasible cellulase, etc., to achieve increased enzyme production, increased production and activity, The effect of good economic sense

Active Publication Date: 2020-03-17
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Otherwise, the loss of Cre1 protein will lead to a large decrease in Trichoderma reesei biomass, resulting in a severe decrease in the final cellulase production
Therefore, the inventors believe that the method of achieving high expression of cellulase by knocking it out is not feasible

Method used

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  • Method for increasing yield of cellulase produced by Trichoderma reesei
  • Method for increasing yield of cellulase produced by Trichoderma reesei
  • Method for increasing yield of cellulase produced by Trichoderma reesei

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Construction of Trichoderma reesei Cre1 mutant strain

[0051] 1. Use Phanta high-fidelity enzyme to amplify the target gene, the system is as follows:

[0052]

[0053]

[0054] PCR program setting:

[0055]

[0056] 2. Gel configuration and electrophoresis:

[0057] ①The configuration of 0.8% (mass volume fraction) agarose gel:

[0058] Add 0.4g agarose to 50mL 1×TAE electrophoresis buffer, heat it in a microwave oven to completely dissolved / completely transparent liquid state, add 3-4μL Genegreen nucleic acid dye, pour it into the gel making plate, and wait for the gel to solidify;

[0059] ②Sample preparation: mix 5μL of sample with 0.5μL of blue loading buffer;

[0060] ③Spotting: Spot the solidified agarose gel and the sample mixed with the loading buffer into the sample hole of the gel.

[0061] ④Recovery: Use a blue light gel cutter to cut off the target band, and use Gel Extraction Kit (OMEGA) according to the instructions to recover DNA through a DNA adsorption c...

Embodiment 2

[0093] Example 2 The enzyme activity and protein of the main cellulase genes of the transformants of Trichoderma reesei were tested under glucose conditions. 2% glucose was used as the carbon source, and the seed culture medium was used for inoculation for 2-3 days → transfer 10% to 2% glucose As a carbon source enzyme production medium, filter paper enzyme activity (FPA), pNPC, protein amount and biomass were measured on 5 days.

[0094] Experiment parallel and repetition: 3 technical parallels and 2 biological repetitions.

[0095] (1) Filter paper enzyme activity (FPA):

[0096] Use a 1*6.4cm Whatman rapid qualitative filter paper strip for the substrate. Fold the filter paper strip and put it into a 25mL colorimetric tube to find the one. Add 1.5mL citric acid buffer (pH 4.8), and add 0.5mL cellulose to the experimental group. Enzyme enzyme solution, and mix well (no control group), 50℃ water bath reaction for 1h, control group add 0.5mL enzyme solution, experimental group and c...

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Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a method for increasing the yield of cellulase produced by Trichoderma reesei. According to the invention, the method is based on gene modification of the transcription factor Cre1 derived from Trichoderma reesei, CCTCC NO: M2015804; by mutating the tail end of the transcription factor, inhibition of the CCR effect on cellulase expression under the glucose condition can be relieved; and finally, the enzyme activity and the protein content of main cellulase are improved, and cellulase production under the condition of the presence of glucose is facilitated. The research result has an important guiding significance for inducer development in the industrial production process of cellulase.

Description

Technical field [0001] The present disclosure belongs to the field of microbial technology, and specifically relates to a carboncatabolite repression (CCR) effect caused by quick-acting simple carbon sources such as glucose during microbial culture by modifying transcription factors in Trichoderma reesei , Improve the method of strains to produce cellulase. Background technique [0002] Disclosure of the background information is only intended to increase the understanding of the overall background of the present disclosure, and is not necessarily regarded as an acknowledgement or in any form suggesting that the information constitutes the prior art known to those of ordinary skill in the art. [0003] Filamentous fungi are the main microorganisms in nature that degrade lignocellulosic biomass. The cellulase produced by them mainly includes three components (endonuclease, exonuclease and β-glucosidase). These three enzymes cooperate with each other to effectively degrade cellulose...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/80C12N9/42C12R1/885
CPCC07K14/37C12N9/2437C12N15/80C12N2800/22
Inventor 方诩韩丽娟蒋艺谭银霜
Owner SHANDONG UNIV
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