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

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...

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Problems solved by technology

At present, the production of MTG is mainly improved through the following methods: first, through mutagenesis breeding to screen high-efficiency enzyme-producing strains, including traditional mutagenesis breeding and space mutagenesis breeding. The variable Streptomyces mobara fermented to produce MTG (AndoH, Adachi M, Umeda K, et al. Purification and characteristics of a novel transglutaminase derived from microorganisms [J]. Agric Biol Chem. 1989, 53: 2613-2617.), but this The high-yield bacteria obtained by this method are easy to degenerate, and the production is unstable; domestic Wang Zhang et al. .Food and Fermentation Industry, 2003, 29(1): 6-12.) Carrying MTG-producing bacteria on the "Shenzhou IV" spacecraft for breeding research, but the effect is not satisfactory

[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

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