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Engineering bacterium for producing heterologous alkaline protease and construction method thereof

A technology of protease and genetically engineered bacteria, applied in the field of microbial genetic engineering, can solve problems such as low expression level and unstable expression system, and achieve the effect of increasing expression level

Active Publication Date: 2021-03-19
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Bacillus amyloliquefaciens still has the problem of unstable expression system and low expression level in terms of heterologous protein expression, because Bacillus amyloliquefaciens can secrete a variety of extracellular proteases, and these enzymes can have a strong effect on heterologous proteins. Therefore, reducing the degradation of the bacteria's own proteases is the key to increasing the expression of exogenous proteases

Method used

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  • Engineering bacterium for producing heterologous alkaline protease and construction method thereof
  • Engineering bacterium for producing heterologous alkaline protease and construction method thereof
  • Engineering bacterium for producing heterologous alkaline protease and construction method thereof

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Experimental program
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Effect test

Embodiment 1

[0058] A strain of genetically engineered bacteria producing heterologous alkaline protease and its construction method.

[0059] 1. Obtaining overlapping fragments of the homology arms of the knockout gene (taking epr as an example).

[0060] According to the whole genome sequence information of Bacillus amyloliquefaciens, taking the epr gene as an example, the primers for the upstream and downstream homology arms of epr were designed by SnapGene, and the target fragment was amplified by PCR, and the amplified upstream and downstream homology arm fragments were purified and recovered. Overlap PCR was performed using the recovered product as a template. The primer sequences and restriction sites used are shown in Table 1.

[0061] The reaction system used to amplify the Epr homology arm is 50 μL, as follows:

[0062]

[0063] The annealing temperature of Epr is 56°C, and the extension time corresponds to the length of the gene. The reaction procedure is as follows:

[006...

Embodiment 2

[0111] Expression and analysis of heterologous alkaline protease genetically engineered bacteria.

[0112] Inoculate single colonies of recombinant strains J1, J2, and J3 on fresh LB plates into 50 mL of kanamycin sulfate-resistant seed medium, culture with shaking at 37°C and 220 r / min for 12 hours, and transfer to 2% inoculum Inoculated in the fermentation medium containing kanamycin resistance, fermented and cultured at 37°C and 220r / min for 48h.

[0113] According to the national standard GB / T 23527-2009 Appendix B, the activity of alkaline protease in the fermentation supernatant of the recombinant strains was determined by the Folin phenol method, and the activity of alkaline protease in the fermentation supernatants of the three recombinant strains reached the highest at 48 hours , the recombinant alkaline protease activity expressed by the recombinant strain J3 reached 19524U / mL, which was 1.40 times that of the recombinant strain J1 (enzyme activity was 13985U / mL) and...

Embodiment 3

[0115] Scale-up experiment of recombinant alkaline protease genetically engineered bacteria.

[0116] Pick a single colony of recombinant bacteria on the plate and inoculate it into 100 mL of LB liquid medium containing 50 μg / mL kanamycin at 37°C and 220 r / min. After culturing for 4 hours, transfer to 100mL fermentation medium with 2% inoculum amount, 37°C, 220r / min, and after 8 hours of cultivation, transfer to 5L with a total volume of 3L with 2% inoculum amount Scale-up experiments were carried out in fermenters. Wherein, the dissolved oxygen content in the fermenter is controlled to be more than 40% during the fermentation process, the stirring speed is 600-700rpm, and the temperature is 37°C. When pH=7, feed (feed medium: cottonseed protein 50g / L, dextrin 300g / L), the initial feeding amount was 100g / h, and the DE value was controlled by feeding during the entire fermentation period to 15mg / mL-18mg / mL. In the early stage of fermentation, antifoaming agent is added approp...

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Abstract

The invention aims to provide a gene engineering bacterium capable of efficiently and stably producing heterologous alkaline protease through directional transformation of a gene engineering host. Bacillus amyloliquefaciens is used as the host, and firstly, eight extracellular proteases aprE, epr, mpr, vpr, nprE, bpr, wprA and aprX of the host are deleted to obtain an extracellular protease defecttype strain; alpha-amylase and an extracellular polysaccharide gene cluster eps of the host are also deleted; and high-copy plasmids expressing the heterologous alkaline protease are also introducedinto the host to obtain the gene engineering bacterium capable of efficiently producing the heterologous alkaline protease. In addition, the viscosity of the engineering bacterium in the fermentationprocess is greatly reduced, and the separation and purification of the product protease are facilitated.

Description

technical field [0001] The invention belongs to the technical field of microbial genetic engineering, and in particular relates to an engineering bacterium for producing heterologous alkaline protease and a construction method thereof. Background technique [0002] Alkaline protease, a class of enzymes that can catalyze the hydrolysis of peptide bonds. Its active center contains serine, also known as serine protease, is an enzyme that hydrolyzes protein peptide bonds in the alkaline pH range. It can not only hydrolyze peptides bond, and also has the function of hydrolyzing amide bond, ester bond, transesterification and transpeptide. This type of enzyme widely exists in animal pancreas, bacteria, and mold, and its enzyme activity can be specifically controlled by diisopropylphosphoryl fluoride (DFP), phenylmethylsulfonyl fluoride (PMSF) and potato inhibitor (PI). Sexual inhibition. Alkaline protease is widely used in industries such as food, washing and tanning. Since the...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N9/54C12N15/57C12N15/75C12R1/07
CPCC12N9/54C12N9/2417C12N15/75C07K14/32C12Y302/01001
Inventor 路福平李玉刘逸寒陈雪佳王茂军王兴吉李庆刚王克芬刘文龙刘夫锋张杰佟新伟
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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