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A method for increasing the expression level of pyrethroid-degrading enzymes

A technology for degrading enzymes and expressing them, which is applied in the field of prokaryotic expression systems, can solve the problems of low yield of enzyme powder, and achieve the effect of high enzyme powder production

Active Publication Date: 2019-11-15
浙江杭海新城控股集团有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Huang Hao et al used lactose fermentation in CN 201210412214.0 to increase the yield of degrading enzymes, but the yield of the above-mentioned enzyme powder is still low

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] 1. Use recombinant E. coli BLR(DE3) / pET-28a-est825 as the production strain, and store the production strain at -86°C in a glycerol tube.

[0017] 2. Seed cultivation: insert the production bacteria in the glycerol tube into the shake flask seed medium at an inoculum size of 1.5%, and cultivate it in a constant temperature shaker for 10 hours with a rotation speed of 220r / min, a temperature of 37°C, and a culture condition; The pH of the shake flask seed culture medium is 7.0, and the shake flask seed culture medium is an LB medium containing kanamycin 50mg / L.

[0018] 3. Fermentation culture: insert the cultured seed liquid into a fermenter equipped with 5.5L fermentation medium with 4% inoculum amount, and carry out fermentation culture. The time of fermentation culture is 26h, and the dissolved oxygen value is maintained at 20%. The ventilation volume is 3vvm, the stirring speed is 1000r / min, the fermentation temperature is 37°C, the pH of the fermentation broth is c...

Embodiment 2

[0023] 1. Use recombinant E. coli BLR(DE3) / pET-28a-est825 as the production strain, and store the production strain at -86°C in a glycerol tube.

[0024] 2. Seed cultivation: insert the production bacteria in the glycerol tube into the shake flask seed medium at an inoculum size of 1.5%, and cultivate it in a constant temperature shaker for 10 hours with a rotation speed of 220r / min, a temperature of 37°C, and a culture condition; The pH of the shake flask seed culture medium is 7.0, and the shake flask seed culture medium is an LB medium containing kanamycin 50mg / L.

[0025] 3. Fermentation culture: insert the cultured seed liquid into a fermenter equipped with 5.5L fermentation medium with 4% inoculum amount, and carry out fermentation culture. The time of fermentation culture is 26h, and the dissolved oxygen value is maintained at 20%. The ventilation volume is 3vvm, the stirring speed is 1000r / min, the fermentation temperature is 37°C, the pH of the fermentation broth is c...

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Abstract

The invention provides a method for improving the expression quantity of a pyrethroid degeneration enzyme. The method comprises the following steps: preparing and producing strain escherichia coli (E.coli) BLR (DE3) / pET-28a-est825; culturing seeds; carrying out fermentation culture: cooling a mixed induction solution of lactose and isopropylthio-galactoside (IPTG) (at the molar concentration ratio of 7 to 3) to 30 DEG C to 35 DEG C and starting to induce, wherein a mixed material supplementing solution of glycerin and yeast powder is continually added in a flowing way in an induction process, the concentration of the glycerin in a fermentation solution is kept to be 1g / L to 2g / L, and induction time lasts for 6h to 7h; finally, preparing the strain into a fungal suspension solution with the mass concentration of 15 percent to 20 percent by utilizing a phosphate buffering solution containing 0.005mol / L of phenylmethylsulfonyl fluoride and having the pH (Potential of Hydrogen) of 6.5 to 7.0, and carrying out ultrasonic crushing, centrifuging and freeze-drying to obtain high-enzyme-activity degeneration enzyme powder.

Description

【Technical field】 [0001] The invention relates to the field of microbial fermentation, in particular to a prokaryotic expression system and a method capable of increasing the expression of pyrethroid-degrading enzymes. 【Background technique】 [0002] Prokaryotic expression system is one of the most widely used exogenous gene expression systems at present. The commonly used expression systems are E. coli expression system, Bacillus subtilis expression system, Streptomyces expression system, etc. Among them, E. coli expression system is the most common. The advantage of the Escherichia coli expression system is that it can obtain gene expression products in a short period of time and the required cost is relatively low, and it has been applied to the expression of pyrethroid-degrading enzymes. BLR(DE3) is a type B protease-deficient strain, a BL21recA derivative constructed by A. Roca (University of Wisconsin), which can stabilize some target genes with repetitive sequences. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/18C12N1/21C12R1/19
CPCC12N9/18C12Y301/01
Inventor 龙燕妮
Owner 浙江杭海新城控股集团有限公司
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