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A kind of transglutaminase mutant and its preparation method and application

A technology of glutamine and transaminase, applied in the field of bioengineering, can solve problems such as difficult to obtain satisfactory results

Active Publication Date: 2021-08-03
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is generally difficult to obtain satisfactory results for a gene mutated once, so a sequential error-prone PCR (Sequential Error-prone PCR) strategy has been developed.

Method used

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  • A kind of transglutaminase mutant and its preparation method and application
  • A kind of transglutaminase mutant and its preparation method and application
  • A kind of transglutaminase mutant and its preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The acquisition of embodiment 1 wild-type transglutaminase gene btg

[0045] 1. Grinding Bacillus subtilis (Bacillus subtilis) thallus ATCC 23857 in liquid nitrogen, and extracting the Bacillus subtilis genome according to the instructions of the genome extraction kit.

[0046] 2. Using the extracted Bacillus subtilis genome as a template, design a pair of primers on the upstream and downstream of the ORF frame according to the transglutaminase sequence registered in Genbank sequence number CP032315.1, and introduce restriction enzyme sites BamH I and Hind respectively III, the amplification primers of the transglutaminase gene of the present invention are as follows:

[0047] Upstream primer P1 (SEQ ID NO.1):

[0048] 5'-CGCGGATCCGATGATTATTGTATCAGGACAATTGC-3'

[0049] Downstream primer P2 (SEQ ID NO.2):

[0050] 5'-CCCAAGCTTTTAGCGGACGATGCGGA-3'

[0051] Using P1 and P2 as upstream and downstream primers, and using the Bacillus subtilis transglutaminase genome as a ...

Embodiment 2

[0055] Obtaining of embodiment 2 transglutaminase mutant gene

[0056] 1. Random mutation based on error-prone PCR technology to construct a new type of transglutaminase, and design primers as follows:

[0057] Upstream primer P1 (SEQ ID NO.1):

[0058] 5'-CGCGGATCCGATGATTATTGTATCAGGACAATTGC-3'

[0059] Downstream primer P2 (SEQ ID NO.2):

[0060] 5'-CCCAAGCTTTTAGCGGACGATGCGGA-3'

[0061] In the error-prone PCR reaction system, P1 and P2 were used as upstream and downstream primers, and the wild-type transglutaminase gene btg was used as a template to perform error-prone PCR.

[0062] The reaction conditions for its amplification are:

[0063] 10×PCR buffer (Mg-free 2+ )

10 μL dATP 0.2 μL dGTP 0.2 μL dCTP 1.0 μL dTTP 1.0 μL Upstream primer P1 1.5μL downstream primer P2 1.5μL wild-type transglutaminase gene 1.0 μL Taq DNA polymerase 1.0 μL Mg 2+ (7mM)

28 μL mn 2 +(0.15mM)

2μL ...

Embodiment 3

[0080] Embodiment 3 Construction of bacillus mutant transglutaminase recombinant bacteria

[0081] 1. Construction of expression vector pBSA43

[0082] pBSA43 is obtained by using the Escherichia coli-Bacillus shuttle cloning vector pBE2 as the backbone, cloning into a strong Bacillus constitutive promoter P43, and the fructan sucrase signal sequence sacB that can directly secrete the recombinant protein into the medium. it comes with amp r Gene that can use ampicillin resistance as a selectable marker in E. coli; also has Km r Gene, kanamycin resistance can be used as a selection marker in Bacillus subtilis and Bacillus licheniformis. For the construction of the pBSA43 plasmid, refer to the Chinese invention patent "Production Process of Bacillus amyloliquefaciens Adding Precursor Fermentation to Produce Antiviral Drug Ribavirin" CN 103146785B.

[0083] 2. Construction of transglutaminase expression vector pBSA43-btgm

[0084] The transglutaminase mutant gene btgm amplifi...

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Abstract

The invention belongs to the technical field of bioengineering, and in particular relates to a transglutaminase and a preparation method and application thereof. In the present invention, the genomic DNA of Bacillus subtilis ATCC 23857 is extracted, and the sequence of the wild-type transglutaminase btg gene with the zymogen region is obtained by PCR amplification, and the amplified wild-type btg gene is randomly mutated by error-prone PCR to obtain a mutant gene btgm. The mutant gene was constructed as a recombinant vector and successfully expressed in Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus licheniformis to obtain a recombinant strain with improved enzyme production activity, and further obtain a new type of transglutaminase through optimization of the fermentation process.

Description

Technical field: [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a transglutaminase and a preparation method and application thereof. [0002] technical background: [0003] Transglutaminase (TG for short) is an enzyme that can catalyze the acyl transfer reaction, which can make the ε-amino group on the lysine residue and the γ-hydroxyamide on the glutamine residue in the protein molecule The group interacts to form an ε-(γ-glutamyl) lysine bond, allowing covalent cross-linking between proteins (or polypeptides). Thereby improving the structure and function of protein, affecting the properties of protein, such as foaming, emulsifying, emulsifying stability, thermal stability, water retention and gel ability, etc., and then improving the flavor, taste, texture and appearance of food, etc. At present, it has been widely used in food, textile, pharmaceutical and other fields. This enzyme has been popularized and applied in th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/75C12N1/21C12R1/125C12R1/07C12R1/10
Inventor 路福平刘逸寒单孟颖张元夫
Owner TIANJIN UNIV OF SCI & TECH