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Recombinant streptomyces mobaraensis and application thereof in production of transglutaminase

A technology of glutamine and streptomyces, applied in the biological field, can solve the problems of low yield, hinder the large-scale industrial production of microbial source glutamine transaminase, etc., and achieve the effect of high application prospect.

Active Publication Date: 2020-12-25
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the yield of existing Streptomyces mobaraensis producing transglutaminase is not high. For example, people such as Zhang Lili inoculated S. mobaraensis DSM40587 into the fermentation medium and fermented for 96 hours, which could only make the enzyme activity of transglutaminase in the fermentation broth Up to 4.3U / mL (see reference for details: Enhancement oftransglutaminase production in Streptomyces mobaraensis as achieved bytreatment with excessive MgCl 2 ); people such as Tian Shucui inoculate the mutagenic bacteria M-8 into the fermentation medium and ferment for 40h, only the enzyme activity of transglutaminase in the fermentation broth can be made to reach 5.1U / mL (see references for details: Atmospheric pressure room temperature plasma ( ARTP) mutagenizes Streptomyces spp. Maoyuan), which greatly hinders the large-scale industrial production of microbial sources of transglutaminase

Method used

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  • Recombinant streptomyces mobaraensis and application thereof in production of transglutaminase
  • Recombinant streptomyces mobaraensis and application thereof in production of transglutaminase
  • Recombinant streptomyces mobaraensis and application thereof in production of transglutaminase

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Experimental program
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Embodiment 1

[0058] Embodiment 1: Construction of recombinant plasmid pSET152-nTG (n=1,2,3,4) and recombinant Streptomyces mobara smY2019-nC (n=2,3,4,5)

[0059] Specific steps are as follows:

[0060] 1. Construction of recombinant plasmid pSET152-nTG (n=1, 2, 3, 4)

[0061] Using the genome of Streptomyces mobaraensis smY2019 as a template and using primer-F and primer-R as primers for PCR amplification, the nucleotide sequence of the transglutaminase gene expression cassette shown in SEQ ID No.1 was obtained ; The transglutaminase gene expression cassette and the pSET152 plasmid were digested by restriction enzymes Xba I and BamH I and connected to obtain a recombinant vector pSET152-1TG carrying a transglutaminase gene expression cassette; the recombinant vector pSET152-1TG was digested with restriction endonucleases Xba I and Bgl II to obtain a linearized recombinant vector; the linearized recombinant vector was connected to the expression cassette of the transglutaminase gene to obt...

Embodiment 2

[0072] Embodiment 2: the production of transglutaminase

[0073] Specific steps are as follows:

[0074] Spread Streptomyces mobaraensis smY2019 and the recombinant Streptomyces mobaraensis smY2019-nC (n=2, 3, 4, 5) obtained in Example 1 on the GYM solid medium respectively, and culture them at 30°C for 5 days until GYM Spores grow on the solid medium; scrape the spores on the GYM solid medium into sterile water, break up with glass beads and filter with sterile filter paper to obtain a spore suspension; use sterile water to separate the spore suspension Density adjusted to 1×10 7 After 1 / mL, 50 μL of spore suspension was inoculated into the seed medium respectively, and cultured at 30° C. for 24 h to obtain the seed liquid; the seed liquid was inoculated into the fermentation medium according to the inoculation amount of 8% (v / v), and the Ferment at 30°C for 72 hours to obtain a fermentation broth.

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Abstract

The invention discloses a recombinant streptomyces mobaraensis and application thereof in production of transglutaminase, and belongs to the technical field of biology. The invention provides a recombinant streptomyces mobaraensis smY2019-nC capable of producing transglutaminase at high yield. The recombinant streptomyces mobaraensis smY2019-nC is obtained by integrating a plurality of nucleotidesequences such as transglutaminase gene expression cassettes shown in SEQ ID NO.1 on a genome of streptomyces mobaraensis smY2019. The recombinant streptomyces mobaraensis smY2019-nC is inoculated into a fermentation medium to be fermented for 72 hours, so that the enzyme activity of transglutaminase in the fermentation liquor can be up to 40 U / mL and is increased by 100% compared with that of wild streptomyces mobaraensis smY2019, and therefore, the recombinant streptomyces mobaraensis smY2019-nC has an extremely high application prospect in production of transglutaminase.

Description

technical field [0001] The invention relates to a strain of recombinant Streptomyces mobara and its application in the production of transglutaminase, belonging to the field of biotechnology. Background technique [0002] Transglutaminase (TGase) is a class of enzymes that can introduce covalent crosslinks between glutamine residues and various primary amines through acyl transfer reactions. Because of this unique catalytic ability, transglutaminase has been widely used in food, feed, biomedical engineering, material science, textile and leather processing and other fields. [0003] Transglutaminase is widely distributed in vertebrates, invertebrates, molluscs, plants and microorganisms. Different sources of transglutaminase have different characteristics. Among them, transglutaminase from microorganisms does not depend on Ca 2+ Existence, easy extraction and isolation, therefore, the production of microbial sources of transglutaminase has been widely studied. [0004] At ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/76C12N9/10C12R1/465
CPCC12N9/1044C12Y203/02013C12N15/76
Inventor 刘松尹小强周景文陈坚
Owner JIANGNAN UNIV
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