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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: 2015-01-07
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 influence of the leader peptide on the catalytic performance of TGase
Preliminary research in the laboratory found that co-expression of TGase leader peptide and mature enzyme can realize the active expression of TGase in E.coli, but TGase cannot be secreted extracellularly

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 derived from Streptomyces hygroscopicus

[0040] Using the reported Streptomyces 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 Streptomyces hygroscopicus TGase.

Embodiment 2

[0041] Embodiment 2: the acquisition of mutant

[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 primer, and the expression plasmid pBB1-1011 of S. hygroscopicus pro-TGase was used as a template to carry out PCR of the whole plasmid, using the site-directed mutagenesis technique A short peptide is inserted into the C-terminus of the leader peptide. The construction of plasmid pBB1-1011 is based on the genome of Streptomyces hygroscopicus (obtained from China Center for Type Culture Collection, deposit number: CCTCC NO: M203062) as a template. For the construction method, please refer to 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 Expression plasmid pBB1- Construct...

Embodiment 3

[0052] Embodiment 3: Detection of mutant enzymatic properties

[0053] In order to allow the leader peptide to be cleaved normally after mutation, in the present invention, the short peptide insertion site is selected before the leader peptide cleavage site (L53-F54), that is, before L53, which is different from adding a short peptide at the N-terminal or C-terminal of the protein. Peptides are different. The transformation of TGase is achieved by forming new interactions between short peptides and their surrounding amino acids, so the selection of short peptides is the key to transformation. The invention screens important short peptides from pro-TGase for molecular transformation of TGase, and the short peptides are limited to 9 amino acids.

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

[0055] Since the C-terminus of the TGase leader peptide has a loop structure, four loops that play an important role in the catalytic activity ...

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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, which belongs to the technical 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] The special catalytic ability of TGase makes it widely used in industrial production, but its low catalytic activity and poor thermal stability seriously restrict the application of...

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
Inventor 陈坚王广圣陈康康刘松堵国成
Owner JIANGNAN UNIV