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Gene engineering bacteria highly expressing transglutaminase, and applications thereof

A technology of transglutaminase and transglutaminase enzyme, which is applied in the field of genetically engineered bacteria expressing high-efficiency transglutaminase, to reduce production costs and simplify production steps

Active Publication Date: 2013-09-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the process of studying the active TGase mature enzyme, various difficulties were encountered

Method used

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  • Gene engineering bacteria highly expressing transglutaminase, and applications thereof
  • Gene engineering bacteria highly expressing transglutaminase, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The acquisition of embodiment 1 proTG zymogen

[0038] proTG can be amplified from the previously constructed vector pET22b(+) / proTG (published articles) in our laboratory or artificially synthesized according to the relevant information of Genebank: EU477523.

[0039] Specifically, the recombinant strain construction method is as follows:

[0040] (1) proTG primer design

[0041] The PCR primers P1 and P2 of proTG gene were designed according to pET22b(+) / proTG gene sequence.

[0042] P1: 5'-TTG GGCCGTTCTGGCC GCCAGCGGCGGCGACG-3'(SfiI)

[0043] P2:5'-CGC GGATCC TTACGACCAGCCCTGCTTCACCTC-3' (BamHI)

[0044] (2) Construction of recombinant expression vector pINA1297-proTG

[0045] Using primers P1 and 2, using the pET22b(+) / proTG plasmid as a template, the PCR product of proTG was amplified, and the PCR product was recovered according to the instructions of the gel recovery kit of Fermentas, and the recovered fragment and plasmid pINA1297 were subjected to SfiI sing...

Embodiment 2

[0046] Embodiment 2: The primer design of introducing the cleavage site Lys-Arg of Kex2 protease

[0047] According to the gene sequence of plasmid pINA1297-proTG, PCR mutation primers Kex1 and Kex2 introducing the cleavage site Lys-Arg (the corresponding nucleotide sequence is AAGCGA) were designed.

[0048] Kex1: 5'- CGA GACGCTGCCGACGAGAGGG-3' (the underlined part is the base sequence introduced)

[0049] Kex2: 5'- CTT GGGGGCCCGGAAGAGCG-3' (the underlined part is the base sequence introduced)

Embodiment 3

[0050] Example 3: Amplification of mutant genes

[0051] Using primers Kex1 and Kex2, using the plasmid pINA1297-proTG as a template, the amplification conditions are: 95°C pre-denaturation, 5min, one cycle; 95°C denaturation, 30s, 65°C annealing, 30s, 72°C extension, 6min35s, 34 cycles ; 72°C, 10min, one cycle; 12°C, 10min, one cycle. PCR amplification system: 5×PCR buffer (Mg 2+ Plus) 10 μL, dNTP Mix 4 μL, template 1 μL, upstream and downstream primers 0.1 μL, sterilized double distilled water 35 μL, Prime Star DNA polymerase 0.5 μL. The PCR product was purified and recovered using a gel recovery kit, and the concentration of the recovered product was checked by electrophoresis. The recovered product was stored in a 1.5mL centrifuge tube and stored in a -20°C refrigerator for future use.

[0052] Example 3: Construction of recombinant expression vector pINA1297-proKexTG

[0053] Phosphorylate and ligate the recovered fragments with the Takara Mutation Kit. The specific s...

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Abstract

The present invention discloses gene engineering bacteria highly expressing transglutaminase, and applications thereof, wherein a cleavage site Lys-Arg of yeast endogenous Kex2 protease is introduced between a proenzyme region gene and a maturase region gene of the expressed streptomyces hygroscopicus CCTCC M203062 transglutaminase proenzyme, and the proenzyme is expressed in Yarrowia lipolytica by using the expression vector. According to the present invention, the gene engineering bacteria is adopted to carry out fermentation to produce transglutaminase so as to achieve independent activation of the transglutaminase without an exogenous protease effect, effectively reduce production cost, and simplify production steps.

Description

technical field [0001] The invention relates to a genetically engineered bacterium for highly expressing glutamine transaminase and its application, belonging to the technical field of enzyme engineering. technical background [0002] Microbial glutamine transaminase (TransglutaminaseEC2.3.2.13 full name R-glutaminyl-peptide: amine-γ-glutayle-transferase referred to as TGase), can catalyze the γ-carboxamide group of glutamine residue in the protein peptide chain and each An acyl receptor undergoes an amido transfer reaction to form a covalent bond of ε-(γ-glutamyl) lysine, which causes intramolecular and intermolecular cross-linking of proteins, connections between proteins and amino acids, and intramolecular valleys in proteins. Hydrolysis of the aminophthalamide group. At present, Streptomyces TGase has been widely used in the food industry. Adding an appropriate amount of TGase can significantly improve the functional properties and nutritional value of various food prot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N9/10C12N15/63C12N15/54C12R1/645
Inventor 陈坚堵国成王淼刘松万丹
Owner JIANGNAN UNIV
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