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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
  • Description
  • 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 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 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

Embodiment 1

[0027] Example 1: Simulation of MTG crystal structure derived from Streptomyces hygroscopicus

[0028] 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

[0029] Embodiment 2: the acquisition of mutant

[0030] The gene sequence of the short peptide was designed on the primer, and inserted into the C-terminus of the leading peptide by site-directed mutagenesis. Using the S. hygroscopicus pro-TGase expression plasmid pBB1-1011 as a template (in the previous study, our laboratory screened out a new strain of transglutaminase production (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 documents are 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 whole plasmid PCR. The primers are listed in Table 1 and were synthesized by Shanghai Sangon Bioengineering Company. The primer Pro-52-R is the d...

Embodiment 3

[0034] Embodiment 3: Detection of mutant enzymatic properties

[0035] In order to allow the leader peptide to 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). GG, GGG, GGGG, GGGGS and PTPPTTPT were selected as insertion short peptides, and the corresponding mutants were pro-52GG, pro-52GGG, pro-52GGGG, pro-52GS and pro-52-PT, respectively. The above-mentioned mutants were fermented, and intracellular and extracellular pro-TGase were detected by SDS-PAGE, and all mutant enzymes could be secreted extracellularly ( figure 1 b), and no intracellular accumulation ( figure 1 c), showing that the insertion of the above short peptide at the C-terminus of the leader peptide has no obvious effect on its secretion.

[0036] Purification of the above mutant enzymes ( figure 2 ), to detect enzymatic properties. Compared with the wild enzyme, inserting short peptides ...

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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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