Method for improving specific activity and activation efficiency of transglutaminase

A technology of transglutaminase and glutamine is applied in the field of improving the specific enzyme activity and activation efficiency of transglutaminase, which can solve the problem of low heterologous expression and secretion, inability to secrete TGase, and affecting the secretion and catalytic activity of TGase. and other problems to achieve the effect of improving cutting efficiency, shortening reaction time, and improving specific enzyme activity

Active Publication Date: 2014-01-29
TAIXING YIMING BIOLOGICAL PRODS
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  • Summary
  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to defects such as heterologous expression and low secretion of MTG, the scope of application of MTG is limited.
[0003] 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 t

Method used

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  • Method for improving specific activity and activation efficiency of transglutaminase
  • Method for improving specific activity and activation efficiency of transglutaminase
  • Method for improving specific activity and activation efficiency of transglutaminase

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0027] Example 1: MTG crystal structure simulation from Streptomyces hygroscopicus

[0028] Using the reported S. 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 S. hygroscopicus TGase.

Example Embodiment

[0029] Example 2: Obtaining mutants

[0030] The gene sequence of the short peptide is designed on the primer and inserted into the C-terminus of the leader peptide using site-directed mutagenesis. S.hygroscopicus pro-TGase expression plasmid pBB1-1011 was used as the template (in the previous study, this laboratory screened a new transglutaminase-producing strain (Streptomyces hygroscopicus CCTCC M203062), through gene cloning method, Obtained the MTG gene sequence and its upstream and downstream sequences, including MTG's own promoter and terminator (Genbank: EU477523), the specific literature is 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), and the whole plasmid PCR was performed. The primers are shown in Table 1, synthesized by Shanghai Shenggong Biological Engineering Company. The primer Pro-52-R is the dow...

Example Embodiment

[0034] Example 3: Detection of mutant enzyme properties

[0035] In order to ensure that the leader peptide can still be cleaved normally after mutation, the short peptide insertion site is selected before the leader peptide cleavage site (L53-F54), that is, before L53 ( figure 1 a). Choose GG, GGG, GGGG, GGGGS and PTPTPPT as insert short peptides, and the corresponding mutants are pro-52GG, pro-52GGG, pro-52GGGG, pro-52GS and pro-52-PT, respectively. Ferment the above mutants, use SDS-PAGE to detect intracellular and extracellular pro-TGase, all mutant enzymes can be secreted outside the cell ( figure 1 b), and no accumulation in the cell ( figure 1 c), indicating that the C-terminus of the leader peptide inserted into the above short peptide has no significant effect on its secretion.

[0036] Purify the above mutant enzyme ( figure 2 ), to detect the properties of enzymes. Compared with the wild enzyme, inserting a short peptide of different length into the leader peptide can ...

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Abstract

The invention discloses a method for improving specific activity and activation efficiency of transglutaminase. A connecting peptide frequently used in fusion protein is inserted into the C end of transglutaminase, the gene sequence of the connecting peptide is designed on a primer, and by the site-specific mutagenesis technology, the connecting peptide is inserted into the C end of leading peptide, and is preferably GS or PT. With the method disclosed by the invention, under the premise of keeping secretion efficiency of transglutaminase, the specific activity of transglutaminase is improved remarkably, which has important significance. Moreover, the invention also discovers that TGase leading peptide cutting efficiency can be improved remarkably due to application of the oligopeptide provided by the invention, so that the reaction time is decreased by 2/3.

Description

technical field [0001] The invention relates to a method for improving the specific enzyme activity and activation efficiency of glutamine transaminase, in particular to a method for improving the specific enzyme activity and activation efficiency of glutamine transaminase by using short peptides. 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] Although the C-terminal of the leader peptide is not an essenti...

Claims

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Application Information

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IPC IPC(8): C12N9/10
CPCC12N9/1044C12Y203/02013
Inventor 陈坚王广圣陈康康刘松堵国成
Owner TAIXING YIMING BIOLOGICAL PRODS
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