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Transglutaminase mutant expressed in active form

A glutamine and mutant technology, applied in the field of enzyme engineering, can solve problems such as inability to meet requirements, and achieve the effects of easy separation and purification, easy cultivation, and simple fermentation method

Active Publication Date: 2018-03-02
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And the production of transglutaminase produced by recombinant bacteria is generally 2.0-8.0U / mL, which cannot meet the demand

Method used

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  • Transglutaminase mutant expressed in active form
  • Transglutaminase mutant expressed in active form
  • Transglutaminase mutant expressed in active form

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] The construction of embodiment 1 genetically engineered bacteria po1h / hpro-mTG

[0049] The plasmid pINA1297 / N355Q reserved in the laboratory was used as a template, and P1 and P2 were used as primers to carry out PCR, and the 1297 expression vector containing the hpro zymogen region was amplified by PCR. The PCR amplification system is: template 1 μL, upstream and downstream primers 1 μL, primeSTAR 25 μL, double distilled water 22 μL. The PCR conditions are: 98°C for 3min, 98°C for 10s, 60°C for 5s, 72°C for 5min30s, 72°C for 20min, 30 cycles. The plasmid pET 20b / mpro-mTG reserved in the laboratory was used as a template, and P3 and P4 were used as primers to carry out PCR, and the gene fragment containing mTG was amplified by PCR. The PCR amplification system is the same as above, and the PCR conditions are: 98°C for 3min, 98°C for 10s, 60°C for 5s, 72°C for 1min 20s, 72°C for 10min, 30 cycles. The two PCR products were digested by Dpn I and recovered from the gel. ...

Embodiment 2

[0052] Determination of embodiment 2 mutation sites

[0053] Use Discovery Studio2017 software to carry out virtual amino acid mutations, determine the key amino acids in the active site, and perform targeted mutations on the sites that affect the affinity between the enzyme and the substrate according to the prediction results, namely Y24W, R89W, E300W, and Y302R.

Embodiment 3

[0054] Example 3 Construction of site-directed mutagenesis expression strain

[0055] (1) The transglutaminase gene derived from Streptomyces Maoyuan and the zymogen region hpro gene derived from Streptomyces hygroscopicus were fused and ligated using the One Step Cloning Kit to construct plasmid pINA1297 / hpro-mTG.

[0056] (2) Use the plasmid pET 20b / mpro-mTG reserved in the laboratory as a template, and use P5 and P4, P6 and P4, P3 and P7, P3 and P8 as primers to carry out PCR, and amplify by PCR to obtain a protein containing Y24W, R89W, E300W, The mutant gene fragment of Y302R. The PCR amplification system is: template 1 μL, upstream and downstream primers 1 μL, primeSTAR 25 μL, double distilled water 22 μL. The PCR conditions were: 98°C for 3min, 98°C for 10s, 60°C for 5s, 72°C for 1min, 72°C for 10min, 30 cycles. After the two PCR products were digested by Dpn I, they were gel-recovered separately. The plasmid pINA297 / hpro-MTG was used as a template, and the recovered...

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Abstract

The invention discloses a transglutaminase mutant expressed in an active form, belonging to the fields of enzyme engineering and fermentation engineering. According to the technical scheme disclosed by the invention, site-directed mutation is carried out on a mature region of transglutaminase, so that amino acid residues near the active site of the transglutaminase are changed, the catalytic efficiency of transglutaminase is improved, and recombinant bacterium hpro-E300W with the enzyme activity of the transglutaminase further improved is obtained. The fermentation enzyme activity of a recombinant bacterium hpro-E300W fermentation tank can reach 59.85U / mL, which is the maximal value of the currently reported fermentation level. In addition, by inserting yarrowia lipolytica endogenous proteinase Kex2 recognition sites in transglutaminase, the active expression of the transglutaminase in the yarrowia lipolytica is embodied. During the fermentation production, little impure protein is generated, the purification is simple, and the cost of industrial production of transglutaminase can be reduced.

Description

technical field [0001] The invention relates to a transglutaminase mutant expressed in an active form and belongs to the field of enzyme engineering. Background technique [0002] Transglutaminase (Transglutaminase, EC 2.3.2.13, TGase) can catalyze the transacylation reaction between the γ-carboxamide group in the glutamine residue in the peptide chain and the acyl receptor, so that the protein or polypeptide covalently cross-linked. TGase is widely used in the field of food processing. For example, TGase can cross-link proteins with essential amino acids (such as lysine) to improve the nutritional value of some foods. TGase can bind minced meat into pieces, increase the utilization rate of meat products, and improve the elasticity of meat products. In addition, TGase has a huge market demand in the fields of medicine, cosmetics, biotechnology research, textile industry and leather processing. [0003] Microorganisms have the advantages of low production cost, easy cultiv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N1/19C12N15/81C12R1/645
CPCC12N9/1044C12N15/815C12Y203/02013
Inventor 刘松任蕊蕊李江华陈坚堵国成冯岳
Owner JIANGNAN UNIV
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