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Method for using transgenic Coprinus cinereus to efficiently express recombinant enzyme

The invention is a kind of gray-capped ghost and transgenic technology, which is applied in the field of high-efficiency expression of recombinant multifunctional cellulase, can solve the problems of single component, high production cost, complex cellulase production process, etc., and achieves the effects of low activity and difficult purification.

Inactive Publication Date: 2013-08-28
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

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Prepare the medium:

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

[0034] Crayfish 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 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, dilute to 1000 mL with distilled water, add a few drops of chloroform and store at room temperature.

[0036] Stock B buffer: 0.04 g thiamine, dilute to 1000 mL with distilled water, and store at 4°C after filter sterilization.

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

[0038] Save under.

[0039...

Embodiment 2

[0049] (1) The TCles11 strain prepared in Example 1 was inoculated on a solid medium plate of Pseudomonas serrata, cultivated at 37°C for 7 days, inoculated with 1-5% in 100 mL of seed medium, and cultured with shaking at 37°C, 210r / min for 96h;

[0050] (2) Preparation of bagasse and soybean meal powder mixed medium, bagasse powder 30g / L, soybean meal powder 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 triangular bottles, each bottle contains 100mL liquid, and sterilize by autoclaving;

[0051] (3) Inoculate each bottle with 1 mL of the bacteria cultured in step (1) during the ultra-clean work, and place it in a shaker at 37°C for culture at a speed of 210r / min.

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

[0053] Among them, the solid medium formula of Pseudomonas serrata is composed o...

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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. 3.2.1.4...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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