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Endoglucanase mutant, gene, engineering bacterium and application thereof

A glucanosidase and mutant technology, applied in the field of genetic engineering, can solve the problem of low transformation efficiency

Active Publication Date: 2020-09-22
ZHEJIANG UNIV OF TECH +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, due to the special structure of lignocellulose, the resulting anti-biodegradation barrier makes its conversion efficiency not high

Method used

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  • Endoglucanase mutant, gene, engineering bacterium and application thereof
  • Endoglucanase mutant, gene, engineering bacterium and application thereof
  • Endoglucanase mutant, gene, engineering bacterium and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0043] Example 2 Expression and Enzyme Activity Measurement of Endo-Glucanidase Mutant Gene Engineering Bacteria

[0044] 1. Expression of genetically engineered bacteria

[0045] Pick the sequenced single colony and inoculate it on the Congo red screening solid medium, culture it upside down at 30°C for 3 days, measure the diameter of the colony (D) and the diameter of the surrounding hydrolysis circle (H). According to the size of D / H, the ability of the strain to degrade cellulose was initially judged, and the colony with a large ratio was screened to induce expression in the next step.

[0046] The endoglucosidase mutant genetically engineered bacteria screened in Example 1 was inoculated into 5 mL of YPD medium, cultivated at 30° C. for 24 h, and then transferred to 30 mL of BMGY containing 1% glycerol with a volume concentration of 1% inoculum. Medium, cultivate at 30°C for 16-20h, centrifuge at 4000rpm for 10min, discard the supernatant, resuspend the bacterial pellet ...

Embodiment 3

[0055] Example 3 Endogluconsidase Mutant Degradation Application Research on Cellulose

[0056] Substrate: sodium carboxymethylcellulose (CMC-Na), bacterial microcrystalline cellulose (BMCC), corncob (dried corncob waste residue soaked in 12% KOH aqueous solution at 70°C for 12h, washed with water to pH9 hereinafter, freeze-dried).

[0057] The reaction system is: add different substrates with a final concentration of 10 mg / mL respectively, add 0.05 mL of the endoglucosidase pure enzyme prepared in Example 2 (wild enzyme Endo-WT or mutant enzyme Endo-K202R, wherein the mutant enzyme Endo- The enzyme activity of K202R is 16.80U / mL) as a catalyst, 0.4mL of reaction system was formed in 50mM pH6.5 phosphate buffer, reacted at 65°C and 500rpm for 48h, after the reaction, each reaction system was added with a final concentration of 1mg / mL Cellulase (mix 100mg / mL exoglucosidase sterile aqueous solution and 100mg / mL β-glucosidase sterile aqueous solution at a volume ratio of 3:1, wh...

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Abstract

The invention discloses an endoglucanase mutant, a genetically engineered bacterium and application of the endoglucanase mutant in degradation of cellulose. The endoglucanase mutant is obtained by mutating lysine at the 202 <nd> position of an amino acid sequence shown in SEQ ID No. 3 into arginine. The specific activity of the endoglucanase mutant disclosed is obviously improved by about 1.4 times compared with that of a wild enzyme. When microcrystalline cellulose is used as a substrate; through the common reaction of cellulase and mutant enzyme, the reaction catalytic efficiency is remarkably improved by 23.5% compared with that of single degradation of a substrate with cellulase, it can be seen that the mutant enzyme remarkably improves the enzyme activity, the economic cost of industrial application can be reduced, and an application basis is provided for the mutant enzyme in the field of industrial production of food bioethanol and the like.

Description

[0001] (1) Technical field [0002] The invention belongs to the field of genetic engineering, and in particular relates to the preparation of an endoglucosidase mutant by a site-directed mutagenesis method and its application. [0003] (2) Background technology [0004] Facing the dual pressures brought about by the scarcity of petrochemical energy and environmental pollution, the search for renewable clean energy has become the focus of researchers from all over the world. Among them, lignocellulose has the advantages of wide sources, low price, and strong regeneration. It is the most abundant renewable resource on the earth. The world produces about 100 million tons of plants through photosynthesis every year. In order to avoid competing with people for food, lignocellulose will be Become the most potential raw material for bioethanol production. [0005] The use of cellulase to convert lignocellulosic biomass has important strategic significance for solving energy crisis a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/81C12N1/19C12P19/02C12P19/14C12R1/84
CPCC12N9/2437C12Y302/01004C12N15/815C12P19/02C12P19/14
Inventor 柳志强周海岩易晓男周建宝薛亚平郑裕国陈德水程新平李勉王红艳陈凯茜
Owner ZHEJIANG UNIV OF TECH
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