Method for improving specific activity of microbial transglutaminase

A technology of transglutaminase specific enzyme activity and transglutaminase, applied in the direction of microorganism-based methods, transferases, botany equipment and methods, etc., can solve the problem that TGase cannot be secreted, affects TGase secretion and Catalytic activity and other issues, to achieve the effect of being suitable for industrial applications, improving enzyme activity, and improving specific enzyme activity

Inactive Publication Date: 2013-12-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although the C-terminal of the leader peptide is not an essential region of TGase, it can affect the secretion and catalytic activity of TGase
At present, the transformation of TGase is limited to the interior of the mature enzyme molecule, ignoring the influe

Method used

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  • Method for improving specific activity of microbial transglutaminase
  • Method for improving specific activity of microbial transglutaminase
  • Method for improving specific activity of microbial transglutaminase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Simulation of MTG crystal structure from Streptomyces hygroscopicus

[0040] Using the reported Streptomyces mobaraensis pro-TGase (PDB code: 3IU0) as a template (the similarity between the two amino acids is 73.1%), the online simulation software SWISS-MODEL was used to simulate the crystal structure of Streptomyces hygroscopicus TGase.

Embodiment 2

[0041] Example 2: Obtaining mutants

[0042] (1) Acquisition of mutant genes

[0043] The gene sequence of the short peptide (the underlined part in Table 1 is the base sequence of the short peptide) was designed on the primers, and the S.hygroscopicus pro-TGase expression plasmid pBB1-1011 was used as the template to perform full-plasmid PCR using site-directed mutagenesis technology Insert a short peptide into the C-terminus of the leader peptide. The plasmid pBB1-1011 was constructed based on the genome of Streptomyces hygroscopicus (obtained from the China Type Culture Collection, with an accession number of CCTCC NO: M203062) as a template. For the construction method, see the article 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 in the expression plasmid pBB1- Construction method of 1010 / pBB1-1020. The prime...

Embodiment 3

[0052] Example 3: Detection of mutant enzyme properties

[0053] In order to make the leader peptide mutated and still be cleaved normally, in the present invention, the short peptide insertion site is selected before the leader peptide cleavage site (L53-F54), that is, before L53, and the short peptide is added to the N-terminus or C-terminus of the protein. The peptides are different. Through the formation of new interactions between the short peptide and its surrounding amino acids, TGase modification is realized, so short peptide selection is the key to modification. The present invention screens important short peptides from pro-TGase for TGase molecular modification, and the short peptides are limited to 9 amino acids.

[0054] (1) Choose 4 loops from the mature enzyme as insert short peptides

[0055] Since the C-terminus of the TGase leader peptide is a loop structure, firstly select 4 loops that are important for the catalytic activity of TGase from the mature enzyme as th...

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Abstract

The invention discloses a method for improving specific activity of microbial transglutaminase and belongs to the field of enzyme engineering. Mutant enzyme with specific activity improved by 40 percent is obtained by using a site-specific mutagenesis technology to insert a loop, coming from the interior of the transglutaminase maturase and playing an importance effect to TGase catalytic activity, or an amino acid sequence, coming from the vicinity of an catalytic activity center, between streptomyces hygroscopicus transglutaminase leading peptide and maturase, and the method is more suitable for industrial production and application.

Description

Technical field [0001] The invention relates to a method for improving the specific enzyme activity of transglutaminase, and belongs to the technical field of enzyme engineering. Background technique [0002] Microbial transglutaminase (protein-glutamate-transglutaminase, 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 epsilon-acyl or other acyl groups react to form epsilon-(γ-glutamyl)lysine covalent bonds. The special catalytic ability makes TGase widely used in food engineering, textile and leather processing, material engineering, biomedicine and other fields. However, due to the low secretion of MTG heterologous expression and other defects, the application scope of MTG is limited. [0003] The special catalytic ability of TGase makes it widely used in industrial production, but its low catalytic activity and poor thermal stability severely restrict the appli...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/10C12N15/70C12N1/21C12R1/55
Inventor 陈坚王广圣陈康康刘松堵国成
Owner JIANGNAN UNIV
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