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A recombinant strain of Streptomyces mogenes and its application in the production of transglutaminase

A technology of glutamine and prostreptomyces, applied in the biological field, can solve the problems of low yield and hinder the large-scale industrial production of microbial-derived transglutaminase, and achieve high application prospects

Active Publication Date: 2022-07-05
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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  • A recombinant strain of Streptomyces mogenes and its application in the production of transglutaminase
  • A recombinant strain of Streptomyces mogenes and its application in the production of transglutaminase
  • A recombinant strain of Streptomyces mogenes and its application in the production of transglutaminase

Examples

Experimental program
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Effect test

Embodiment 1

[0058] Example 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] Take the genome of Streptomyces mobaraensis smY2019 as a template, and use primer-F and primer-R as primers to carry out PCR amplification to obtain a nucleotide sequence such as the transglutaminase gene expression cassette shown in SEQ ID No.1 ; The transglutaminase gene expression frame and the pSET152 plasmid are connected after being cut by restriction enzymes Xba I and BamH I to obtain a recombinant vector pSET152-1TG carrying a transglutaminase gene expression frame; the recombinant vector pSET152-1TG was digested with restriction enzymes Xba I and Bgl II to obtain a linearized recombinant vector; the linearized recombinant vector and the transglutaminase gene expression cassette were ligated to obtain two ...

Embodiment 2

[0072] Example 2: Production of transglutaminase

[0073] Specific steps are as follows:

[0074] Streptomyces mobaraensis smY2019 and the recombinant Streptomyces mobaraensis smY2019-nC (n=2, 3, 4, 5) obtained in Example 1 were coated on GYM solid medium, and cultured at 30°C for 5 days to GYM Spores grow on the solid medium; scrape the spores on the GYM solid medium into sterile water, disperse them with glass beads, and filter them with sterile filter paper to obtain a spore suspension; use sterile water to separate the spore suspensions. The density is adjusted to 1×10 7 After spores / mL, 50 μL of spore suspension was inoculated into the seed medium, and cultured at 30°C for 24 h to obtain seed liquid; the seed liquid was inoculated into the fermentation medium at Fermentation at 30°C for 72h to obtain fermentation broth.

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Abstract

The invention discloses a strain of recombinant Streptomyces mogenes and its application in the production of glutamine transaminase, and belongs to the field of biotechnology. The invention provides a recombinant Streptomyces mobara smY2019-nC capable of high glutaminase production, the recombinant Streptomyces mobara smY2019-nC is obtained by integrating several nucleotide sequences such as SEQ ID NO. The transglutaminase gene expression frame shown in 1 is obtained; this recombinant Streptomyces mogenes smY2019-nC is inoculated into the fermentation medium for fermentation for 72h, so that the enzymatic activity of transglutaminase in the fermentation broth can be as high as 40U / mL, which is relatively high. Wild-type Streptomyces mobara smY2019 increased by 100%, therefore, this recombinant Streptomyces mobara smY2019‑nC has a very high application prospect in the production of transglutaminase.

Description

technical field [0001] The invention relates to a strain of recombinant Streptomyces mogenes and its application in the production of glutamine transaminase, and belongs to the field of biotechnology. Background technique [0002] Transglutaminase (TGase) is a class of enzymes that can introduce covalent cross-links between glutamine residues and various primary amines through acyl transfer reactions. Due to this unique catalytic ability, transglutaminase has been widely used in the fields of food, feed, biomedical engineering, materials science, textile and leather processing. [0003] Transglutaminase is widely distributed in vertebrates, invertebrates, mollusks, plants and microorganisms. Different sources of transglutaminase have different properties. Among them, transglutaminase derived from microorganisms does not depend on Ca. 2+ The existence of , easy extraction and isolation, therefore, the production of microbially derived transglutaminase has been widely studied...

Claims

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Application Information

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Patent Type & Authority Patents(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