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Recombinant bacillus subtilis and method for producing transglutaminase by utilizing recombinant bacillus substilis

A technology of Bacillus subtilis and transglutaminase, which is applied in the field of transglutaminase genetic engineering, can solve the problems of difficult industrial production, inactivity, cumbersome process, etc., to simplify the fermentation production process and save the cell crushing Effect

Inactive Publication Date: 2012-07-18
SOUTH CHINA UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The second, construct the engineering bacterial strain that highly expresses MTG by the method for genetic engineering, but the used host of genetic engineering production MTG is mainly E. Li Pingzuo. Cloning and expression of transglutaminase gene in Escherichia coli. China Biotechnology Journal. 2004, 11: 56-60) Constructed genetically engineered bacteria to successfully express MTG, and the produced MTG not only exists in the cell , and also exists in the form of inclusion bodies. To obtain active MTG, complex processes such as cell disruption, inclusion body denaturation, and renaturation are required. The process is very cumbersome and it is difficult to achieve industrial production
Luo Ning et al. (Luo Ning, Yang Huilin, Shen Xukai, Zheng Mingying. Expression of transglutaminase zymogen in Bacillus subtilis WB800. Modern Food Science and Technology. 2011, 7: 734-737) reported the production of MTG by Bacillus subtilis method, but it is expressed in the form of zymogen and has no activity. After purification, it needs to be activated with trypsin to have biological activity

Method used

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  • Recombinant bacillus subtilis and method for producing transglutaminase by utilizing recombinant bacillus substilis
  • Recombinant bacillus subtilis and method for producing transglutaminase by utilizing recombinant bacillus substilis
  • Recombinant bacillus subtilis and method for producing transglutaminase by utilizing recombinant bacillus substilis

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Experimental program
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Embodiment 1

[0035] A recombinant Bacillus subtilis is constructed by the following steps:

[0036] Step 1: Construction of Bacillus subtilis B.subtilis WB800S

[0037] (1) PCR amplification of subtilisin-like protease gene:

[0038] The nucleotide sequence of Streptomyces albogriseolus subtilisin-like protease was obtained from NCBI, see SEQ ID No.1 in the sequence listing. Using 1 μL of Streptomyces albus (purchased from Guangdong Microbial Culture Collection Center) genomic DNA as a PCR reaction template, the subtilisin-like protease gene forward primer and reverse primer are SEQ ID No.4 and SEQ ID No.5, respectively As shown, a DNA fragment matching the size of the subtilisin-like protease gene was amplified, such as figure 1 As shown, lane 1 in the figure is a DNA marker; lanes 2 and 3 are PCR products (subtilisin-like protease encoding gene).

[0039] (2) PCR amplification of P43 promoter + samyQ signal peptide coding gene:

[0040] For the nucleotide sequence of the P43 promoter...

Embodiment 2

[0054] A method for producing transglutaminase by Bacillus subtilis B.subtilis WB800S / proMTG obtained by the embodiments, comprising the following steps:

[0055] (1) Pick a single colony of Bacillus subtilis WB800S / proMTG in 10mL LB medium (containing Kan 10μg / mL), activate at 37°C and 200rpm for 12h, and inoculate the activated seeds in 50mL LB medium ( Containing Kan 10μg / mL) the inoculum amount is 1-2% (volume ratio), 37 ℃, 200rpm ferments respectively 24h, 48h, 60h, 72h and centrifuges to take the supernatant, the SDS-PAGE electrophoresis of the supernatant is as follows Figure 5 As shown, it was found that MTG (transglutaminase) yield was the highest when fermenting for 48h.

[0056] (2) Purification of MTG by HisTrap column affinity chromatography

[0057] Purify the protein using GE's AKTApurifier chromatograph and 5mL HisTrap FF crude column, including the following steps:

[0058] 2.1 Wash the column with distilled water until the baseline is stable.

[0059] 2.2...

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Abstract

The invention discloses a recombinant bacillus subtilis and a method for producing transglutaminase by utilizing the recombinant bacillus substilis. The recombinant bacillus subtilis is obtained by the following steps of: (1) by virtue of fusion PCR (polymerase chain reaction), connecting a P43 promoter and a signal peptide gene with a subtilisin-like protease gene, and converting a fusion PCR amplification product into bacillus subtilis WB800 to obtain bacillus subtilis B.subtilis WB800S; and (2) carrying out PCR amplification on streptoverticillium mobaraense transglutaminase protogene, andthen converting amplification product into the bacillus subtilis B.subtilis WB800S to obtain bacillus subtilis B.subtilis WB800S / proMTG. The bacillus subtilis B.subtilis WB800S / proMTG is fermented and purified to obtain soluble transglutaminase. By adopting the method disclosed by the invention, the transglutaminase with biological activity can be directly obtained in supernatant of fermentation liquor, fussy processes of breaking cells and activating proenzyme after purification are eliminated, and a fermentation production process is simplified.

Description

technical field [0001] The invention belongs to the field of transglutaminase genetic engineering, and in particular relates to a recombinant bacillus subtilis and a method for producing transglutaminase therefrom. Background technique [0002] Microbial transglutaminase (MTG) is a transferase that catalyzes the transfer of acyl groups in polypeptide chains to cause covalent cross-linking reactions between proteins. It catalyzes the cross-linking reaction between protein molecules, improves the water solubility, water holding capacity, plasticity and thermal stability of protein, thereby improving the application value of protein. 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. [0003] There have been many reports on the characteristics and application of transglutaminase in foreign countries, mainly using enzyme sa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N9/10C12N15/63C12R1/125
Inventor 潘力王坤杨慧林钟景儒
Owner SOUTH CHINA UNIV OF TECH
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