Colibacillus and method for performing soluble expression of transglutaminase proenzyme thereof

A technology of transglutaminase enzyme and glutaminase enzyme, which is applied in the field of transglutaminase genetic engineering, can solve the problems of unstable production, cumbersome process, and easy degradation of high-yield bacteria, and achieve simplified fermentation production process, The process is simple, and the effect of eliminating inclusion body denaturation and renaturation process

Inactive Publication Date: 2010-04-07
SOUTH CHINA UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

One is to increase the yield of production bacteria through mutation breeding and medium optimization. For example, Japan Ajinomoto Company uses Streptomyces Mobara to ferment and produce MTG. However, the high-yielding bacteria obtained by this method are easy to degrade and the production is unstable; the second is By constructing genetically engineered bacteria to express transglutaminase, as published in "Journal of Biological Engineering" in the fifth issue of volume 21 in September 2005, "Streptomyces mobara transglutaminase gene is highly expressed in Escherichia coli" ( Authors: Xu Bin, Han Zhibo, Yang Ping, Liu Yongjun, Li Yanhan, Han Zh...

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  • Colibacillus and method for performing soluble expression of transglutaminase proenzyme thereof
  • Colibacillus and method for performing soluble expression of transglutaminase proenzyme thereof
  • Colibacillus and method for performing soluble expression of transglutaminase proenzyme thereof

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Experimental program
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Effect test

Embodiment 1

[0022] The construction process of engineering bacteria E.coli BL21 / proMTG and the method for soluble expression of transglutaminase zymogen under shake flask scale comprise the following steps:

[0023] (1) PCR amplification of transglutaminase zymogen gene: the nucleotide sequence of Maoyuan Streptomyces mobaraensis transglutaminase zymogen is from the document "Molecular Cloning of the Gene for Microbial Transglutaminase from Streptomyces mobaraensis and Its Obtained in "Expression in Streptomyces lividans" (Biosci.Biotech.Biochem.58(1), 82-87, 1994), the sequence of the transglutaminase zymogen gene is shown in the sequence table SEQ ID No1, with 1 μg of Streptomyces mobara genomic DNA It is a PCR reaction template, and the forward primer and reverse primer are shown in SEQ ID No3 and SEQ ID No4 respectively. The PCR reaction conditions are 95°C for 10 minutes, 94°C for 1 minute, 65°C for 30 seconds, and 72°C for 2.5 minutes. A total of 30 cycles, and finally extended at 7...

Embodiment 2

[0027] Optimization of temperature for induction and expression of E. coli BL21 / proMTG:

[0028]See Example 1 for the construction of Escherichia coli E. coli BL21 / proMTG. The single colony of the above-mentioned engineered bacteria was inoculated in 50mL LB medium, activated at 200rpm for 12h at 37°C, and then inoculated with 2% of the inoculum size in 100mL fermentation medium for 2h, then added IPTG to a final concentration of 1mM, and then inoculated at 20-37°C Induced expression was carried out under the conditions, centrifuged to collect cells after 6-8 hours, supernatant was obtained by ultrasonic crushing and centrifugation, the supernatant was activated with trypsin, and the activity of transglutaminase was measured by colorimetry, and it was found that under the condition of 20-28 ℃, all Soluble expression is formed in different degrees, and the expression level is the highest under the induction condition of 24°C. The SDS-PAGE electrophoresis images of the centrifu...

Embodiment 3

[0030] Optimization of E. coli BL21 / proMTG inoculum volume:

[0031] See Example 1 for the construction of Escherichia coli E. coli BL21 / proMTG. A single colony of Escherichia coli E.coli BL21 / proMTG was inoculated in 50mL LB medium, activated at 37°C and 200rpm for 12h, and 1, 2, 3, 4mL of activated seeds were inoculated in 100mL fermentation medium and fermented to OD 600 After =0.5 or so, add IPTG to a final concentration of 1 mM, and then place at 24°C to induce expression. After 4 hours, the cells were collected by centrifugation, ultrasonically broken and centrifuged to get the supernatant. After the supernatant was activated with trypsin, the activity of transglutaminase was measured by colorimetry. high.

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Abstract

The invention discloses colibacillus and a method for performing soluble expression of transglutaminase proenzyme thereof. The method comprises the following steps: (1) designing PCR primer to perform PCR amplification according to the nucleotide sequence of transglutaminase proenzyme of streptomyces mobaraensis, cloning the related gene to prokaryotic expression vector pET22b (+),constructing expression vector pET22b-proMTG to conform to E.coli BL21/proMTG of which strain preservation number is CCTCC M 208240; (2) performing soluble expression of transglutaminase proenzyme; (3) activating transglutaminase proenzyme; (4) performing high density fermentation; and (5) performing protein purification technology. In the method of the invention, the yield and specific activity of transglutaminase proenzyme can reach the level of that of transglutaminase proenzyme which is produced by firstly performing direct expression in colibacillus to form inclusion body and then performing denaturation and renaturation and the fermentation process is easier.

Description

technical field [0001] The present invention relates to transglutaminase genetic engineering, in particular to a method for soluble expression of transglutaminase zymogen in Escherichia coli to realize soluble expression of transglutaminase zymogen for large-scale industrialized production of transglutaminase enzyme. Background technique [0002] Transglutaminase is a transferase that catalyzes the acyl transfer reaction. It mainly catalyzes the amido transfer reaction between the γ-amide group of glutamine residue and the ε-amino group of lysine in proteins and polypeptides. Carry out covalent cross-linking polymerization between molecules, thereby changing the structure and function of the protein itself and the attached cells and tissues. The enzyme has been widely used in the pharmaceutical industry, food industry and textile industry. It has the reputation of "21st century super adhesive". advantage, which has attracted people's attention. In Japan, the food sales re...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/54C12N15/70C12N9/10C12R1/19
Inventor 潘力杨慧林苗小康崔翠
Owner SOUTH CHINA UNIV OF TECH
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