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Method for using transgenic Coprinus cinereus to efficiently express recombinant enzyme

A high-efficiency expression, gray-covered ghost technology, applied in the direction of recombinant DNA technology, biochemical equipment and methods, enzymes, etc., can solve the problems of high production cost, single component, inability to effectively degrade cellulose, etc., to achieve difficult purification, active low effect

Inactive Publication Date: 2011-07-20
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the main production strains producing cellulase are Trichoderma, Penicillium and Aspergillus, and the production method is to obtain cellulase through solid fermentation or liquid fermentation of these filamentous fungi, because the cellulase produced by these filamentous fungi The composition is single, the activity of the enzyme is low in practical application, and it cannot effectively degrade cellulose (as mentioned above, the degradation of cellulose requires the synergy of multiple enzymes to complete), and the current production process of cellulase is complex and costly. high

Method used

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  • Method for using transgenic Coprinus cinereus to efficiently express recombinant enzyme
  • Method for using transgenic Coprinus cinereus to efficiently express recombinant enzyme
  • Method for using transgenic Coprinus cinereus to efficiently express recombinant enzyme

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Effect test

Embodiment 1

[0032] Prepare medium:

[0033] Seed medium: microcrystalline cellulose 20g, yeast extract 4g, dipotassium hydrogen phosphate 1g, potassium dihydrogen phosphate 0.46g, magnesium sulfate 1g, distilled water to 1000mL, autoclaved for later use.

[0034] Coprinus cinereus solid medium: glucose 10 g, asparagine 2 g, adenine sulfate 0.1 g, Stock A buffer 25 mL, Stock B buffer 1 mL, Stock C buffer 10 mL, agar 14 g, Distilled water to make up to 1000mL, autoclaved for later use.

[0035] Stock A buffer: ammonium tartrate 20 g, KH 2 PO 4 40 g, anhydrous NaSO 4 11.6 g, anhydrous Na 2 HPO 4 90 g, distilled water to 1000mL, add a few drops of chloroform and store at room temperature.

[0036] Stock B buffer solution: 0.04 g of thiamine, distilled water to 1000 mL, stored at 4°C after filter sterilization.

[0037] Stock C buffer: MgCl 2 ·H 2 O 25 g, distilled water to 1000 mL, autoclave, room temperature

[0038] Save it.

[0039] Basic enzyme production medium: carbon source ...

Embodiment 2

[0049] (1) Inoculate the TCles11 strain prepared in Example 1 on the Coprinus cinereus solid medium plate, culture at 37°C for 7 days, inoculate 1-5% into 100mL seed medium, and culture at 37°C for 96 hours with shaking at 210r / min;

[0050] (2) Prepare the intermixing medium of bagasse and soybean meal, bagasse powder 30g / L, soybean meal 6 g / L, dipotassium hydrogen phosphate 1g / L, potassium dihydrogen phosphate 0.46g / L, magnesium sulfate 1g / L , adjust the pH value to 6.25; divide into 250mL Erlenmeyer flasks, each bottle contains 100mL, and sterilize under high pressure;

[0051] (3) In the ultra-clean work, inoculate each bottle with 1mL of the bacteria cultured in step (1), and place it on a shaking table at 37°C after inoculation, and the shaking table speed is 210r / min.

[0052] (4) The culture was terminated after 7 days of cultivation, and the fermentation broth was collected by centrifugation.

[0053] The formula of the Coprinus cinereus solid medium is composed of t...

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Abstract

The invention belongs to the field of microbial fermentation engineering, and relates to a preparation method of a recombinant multi-function cellulase. The method is characterized in that multi-function cellulase genes are used to carry out genetic transformation on Coprinus cinereus to obtain a transgenic Coprinus cinereus strain which is used as a producing strain, a mixture of bagasse powder and bean pulp powder is the optimal culture medium, and the recombinant multi-function cellulase is prepared by shake fermentation under the optimal culture conditions. The formula of the bagasse powder and bean pulp powder mixed culture medium comprises 27.9-30.0g / L bagasse powder, 4.3-6g / L bean pulp powder, 0.5-1g / L dipotassium hydrogen phosphate, 0.23-0.69g / L potassium dihydrogen phosphate and 0.5-1g / L magnesium sulfate, and the pH value is 6.25-7.82. The optimal culture conditions are as follows: the culture temperature is 36-37 DEG C, the culture time is 7 days, and the rotation speed of a shaking table is 210r / min. The optimal enzyme yields are as follows: the enzyme activity of Xylanase is 1.66*10<5>U / L, and the enzyme activity of CMCase is 1.7*10<4>U / L. The method provided by the invention can effectively enable the transgenic Coprinus cinereus strain to produce the recombinant multi-function cellulase from low-cost agricultural waste bagasse and bean pulp powder.

Description

technical field [0001] The invention belongs to the field of microbial fermentation engineering, and in particular relates to a method for efficiently expressing recombinant multifunctional cellulase by utilizing a transgenic Coprinus cinerea strain. A method for efficiently expressing recombinant multifunctional cellulase by using bagasse and soybean meal. Background technique [0002] Cellulose and hemicellulose are one of the most abundant renewable resources in nature and are the main components of plant cell walls. Every year, the earth produces hundreds of millions of tons of plant dry matter (polysaccharides) through photosynthesis, the main components of which are cellulose and hemicellulose. The enzymatic degradation of cellulose and hemicellulose is currently recognized as the most effective and clean conversion method. Its biodegradation requires the action of cellulase. Cellulase is a complex enzyme system, mainly including endo-β-1,4-D-glucanase (E.C. 3.2.1...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N1/21C12N9/42
Inventor 林俊芳娄囡囡郭丽琼杨培周
Owner SOUTH CHINA AGRI UNIV
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