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Transglutaminase mutant based on leader peptide displacement mutation

A technology of live transglutaminase and mutant, applied in the field of enzyme engineering, can solve the problem of no catalytic activity of pro-TGase

Inactive Publication Date: 2015-07-08
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Its N-terminal 25 amino acids (9-33) are located in the active cleft of the mature enzyme, in which Y12 and Y16 are located above the catalytic site Cys64-Asp255-His274, which prevents the entry of substrate acyl acceptors and donors, thus making pro -TGase has no catalytic activity

Method used

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  • Transglutaminase mutant based on leader peptide displacement mutation
  • Transglutaminase mutant based on leader peptide displacement mutation
  • Transglutaminase mutant based on leader peptide displacement mutation

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1 Simulation of crystal structure of MTG derived from Streptomyces hygroscopicus

[0030] Using the reported S. mobaraensis pro-TGase (PDB code: 3IU0) as a template (the amino acid similarity between the two is 73.1%), the online simulation software SWISS-MODEL was used to simulate the crystal structure of S. hygroscopicus TGase.

Embodiment 2

[0031] Example 2 Functional Analysis of Leading Peptides

[0032] To analyze the function of the leader peptide, use the Discovery studio software to analyze the structure of S. hygroscopicus pro-TGase obtained by homology modeling. Seven hydrogen bonds were found between TGase leader peptide and mature enzyme, distributed between Y12 and N334, D362, N27 and W309, N311, N30 and N311, R32 and D295. Hydrogen bonds play an important role in protein structure and its physiological function.

Embodiment 3

[0033] Example 3 Obtaining of mutant strains

[0034] (1) Site-directed mutagenesis of the leader peptide

[0035] The S. hygroscopicus pro-TGase expression plasmid pBB1-1011 was constructed using the genome of Streptomyces hygroscopicus (obtained from the China Center for Type Culture Collection, collection number CCTCC NO: M203062) as a template, and the site-specific mutagenesis technique, the leader peptide C-terminal α-helix 37-42 6 amino acids (QPGNSL) were replaced with 3 alanines (AAA) or glycines (GGG) respectively to obtain the gene of the TG mutant. For the construction of plasmid pBB1-1011, see Liu S, Zhang D, Wang M, Cui W, Chen K, Liu Y, Du G, Chen J, Zhou Z, (2011). The pro-region of Streptomyces hygroscopicus transglutaminase affects its secretion by Escherichia coli. FEMS Microbiol Lett324(2):98-105. The PCR reaction conditions were: 95°C for 5min, 24 cycles (95°C for 5min, 65°C for 30s, 72°C for 1min40s), 72°C for 10min.

[0036] leader peptide α-helix 37...

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Abstract

The invention discloses a transglutaminase mutant with high enzyme activity based on leader peptide displacement mutation, and belongs to the field of enzyme engineering. Alpha-helix 37-42 (QPGNSL) at pro-TGase leader peptide C terminal of Streptomyces hygroscopicus is respectively replaced into three lactamic acids or three glycines by using site-directed mutagenesis. Compared with S.hygroscopicus wild enzyme, the enzyme activities of the hygroscopicus TGase mutants pro-37-42AAA and pro-37-42GGG provided by the invention are respectively improved by 22.2% and 24.8%, Km value is reduced, combination of a substrate and a catalytic activity center is facilitated, the transglutaminase mutant is applicable to industrial application, and a new idea is provided for transformation of similar protein molecules.

Description

technical field [0001] The invention relates to the transformation of glutamine transaminase, in particular to a mutant of transglutaminase with improved specific enzyme activity by substituting a leader peptide and belonging to the field of enzyme engineering. Background technique [0002] Microbial transglutaminase (protein-glutamic acid-glutaminase, Microbial Transglutaminase, EC2.3.2.13 referred to as MTG) can catalyze the γ-carboxamide group of glutamine residues in protein peptide chains and lysine Amino acid ε-acyl or other acyl react to form ε-(γ-glutamyl) lysine covalent bond. The special catalytic ability makes TGase widely used in food engineering, textile and leather processing, material engineering, biomedicine and other fields. However, due to defects such as heterologous expression and low secretion of MTG, the scope of application of MTG is limited. [0003] Streptomyces TGase is synthesized and secreted by an inactive zymogen (pro-TGase), and its N-termina...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/10C12N15/70C12N1/21C12R1/55
CPCC12N9/1044C12Y203/02013
Inventor 陈坚王广圣陈康康刘松堵国成
Owner JIANGNAN UNIV
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