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Bacillus subtilis not producing cellulase as well as construction method and application thereof

A bacillus and construction method technology, applied in the field of genetic engineering, can solve the problems of ramie fiber damage, the impact of unassessed strains on the ecological environment, etc., and achieve the effect of not causing environmental pollution

Active Publication Date: 2018-04-20
INST OF BAST FIBER CROPS CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The ramie biological degumming strains in the existing published patents all belong to wild-type microbial strains, and it has not been confirmed from the perspective of molecular biology whether the strains used produce exocrine cellulase, whether the ramie fibers are damaged during the degumming process, and The impact of the strain on the ecological environment has not been evaluated

Method used

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  • Bacillus subtilis not producing cellulase as well as construction method and application thereof
  • Bacillus subtilis not producing cellulase as well as construction method and application thereof
  • Bacillus subtilis not producing cellulase as well as construction method and application thereof

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Effect test

preparation example Construction

[0027] Preferably, the preparation method of the competent Bacillus subtilis strain 168 cells comprises:

[0028] Bacillus subtilis strain 168 is cultured in GM I medium and GM II medium in sequence;

[0029] The composition of the GMI medium includes: dipotassium hydrogen phosphate 13g / L~13.8g / L, potassium dihydrogenphosphate 5.3g / L~6.1g / L, ammonium sulfate 1.7g / L~2.1g / L, lemon Sodium sulfate 0.8g / L~1.2g / L, magnesium sulfate heptahydrate 0.15g / L~0.25g / L, glucose 0.4g / L~0.6g / L, casein 0.15g / L~0.25g / L, yeast Extract 0.8g / L~1.2g / L;

[0030] More preferably, the composition of described GMI medium comprises: dipotassium hydrogen phosphate 13.4g / L, potassium dihydrogen phosphate 5.7g / L, ammonium sulfate 1.9g / L, sodium citrate 1g / L, magnesium sulfate heptahydrate 0.2g / L, glucose 0.5g / L, casein 0.2g / L, yeast extract 1g / L;

[0031] The components of the GM II medium include: dipotassium hydrogen phosphate 13g / L~13.8g / L, potassium dihydrogenphosphate 5.3g / L~6.1g / L, ammonium sulfate...

Embodiment 1

[0043] Embodiment 1 is used for the construction of the homology arm of homologous recombination double exchange

[0044] The engineering strain ΔeglS is to knock out the cellulase Endoglucanase coding gene (eglS) in B. subtilis strain 168 to construct a recombinant Bacillus subtilis that cannot degrade ramie fibers. The principle of genome modification is as follows figure 1 As shown, it mainly includes the following steps:

[0045] (1) Clarify the metabolic pathway of cellulose degradation in B. subtilis strain 168

[0046] Bioinformatics analysis shows that the cellulose metabolism pathway shown on the KEGG website (http: / / www.kegg.jp / kegg-bin / show_pathway?bsu00500) of the B.subtilis strain 168 strain is Starch and Sucrose Metabolism, wherein the fiber Endoglucanase (EC: 3.2.1.4) is an enzyme required for the first step of cellulose degradation.

[0047] (2) Obtain the information of the cellulase Endoglucanase coding gene (eglS)

[0048] The registration information of...

Embodiment 2

[0053] This example relates to a method for knocking out the gene encoding cellulase Endoglucanase (eglS) in B. subtilis strain 168.

[0054] B. subtilis strain 168 was inoculated on LB plate (Tryptone 10g / L, NaCl 10g / L, yeast extract 5g / L) and cultured at 37°C for 1 day, and the culture was inserted into 6mL GMI medium, at 30 Cultivate in a slow shaker at 130 rpm for 16 hours to obtain the first bacterial solution; transfer 1.5 mL of the first bacterial solution to 16 mL of GMI medium, and culture in a fast shaker (230 rpm) at 37°C for 4 hours to obtain the second bacterial solution; Then take 4 mL of the second bacterial solution and transfer it to 40 mL of GM II medium, culture it on a slow shaker (120 rpm) at 37° C. for 80 min, and then centrifuge at 5000 g for 10 min to collect the bacterial cells. Use 5mL of the original culture supernatant to gently suspend the bacterial cells, and the suspended bacterial cells are competent cells.

[0055] Wherein, the formula of the ...

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Abstract

The invention relates to the technical field of genetic engineering, in particular to bacillus subtilis not producing cellulase as well as a construction method and application thereof. The construction method comprises the following steps: knocking out a cellulase Endoglucanase encoding gene eglS of the bacillus subtilis strain 168 to obtain the bacillus subtilis not producing the cellulase. Themutant strain obtained by the construction method can generate various kinds of degummase such as pectinase and hemicellulase, can directly decompose the colloid component in ramie bast, can be applied to microbial degumming of the ramie, has the advantages of no damage to ramie fiber and safety for people and livestock, and is favorable for protecting the ecological environment.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a non-cellulase-producing Bacillus subtilis and its construction method and application. Background technique [0002] Ramie (Boehmeria nivea) is a crop of the genus Ramie in the family Urticaceae. Its bast has long single fibers, high strength, rapid moisture absorption and dissipation, and excellent thermal conductivity. It is an important raw material for the textile industry. my country is the main producing area of ​​ramie, accounting for more than 90% of the world's output, and has great economic benefits and international influence. However, ramie raw hemp contains about 30% bonded non-cellulose substances (commonly known as "colloids") composed of pectin and hemicellulose. The existence of these colloids will affect the quality of ramie fibers and cannot be used directly. textile. Degumming is a key step before ramie fiber is used for textile. Currently, ram...

Claims

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Application Information

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IPC IPC(8): C12N15/75C12N1/21D01C1/04C12R1/125
CPCC12N9/2437C12N15/75C12Y302/01004D01C1/04
Inventor 杨琦彭源德段盛文成莉凤冯湘沅刘志远郑科
Owner INST OF BAST FIBER CROPS CHINESE ACADEMY OF AGRI SCI
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