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Glutamine transaminase mutant with improved enzymatic activity

A technology of glutamine and transaminase, applied in the field of enzyme engineering, can solve problems such as low production of transglutaminase and increase of production of transglutaminase, and achieve a large amount of expression of transglutaminase, easy separation and purification, and pH adaptation range wide effect

Active Publication Date: 2018-02-27
TAIXING DONGSHENG FOOD TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In recent years, it has become a new trend to use genetic engineering technology to clone transglutaminase gene into heterologous hosts such as E. coli to express MTG. However, on the one hand, heterologous hosts such as E. On the one hand, the yield of transglutaminase produced by recombinant bacteria is relatively low. For example, in Liu Song's "Overproduction of pro-transglutaminase from Streptomyces hygroscopicus in Yarrowia lipolytica and its biochemical characterization", the mutant N355Q of pro-TGase derived from Streptomyces hygroscopicus was heterologously expressed The yield of recombinant bacteria can reach 35U / mL, and the yield of recombinant bacteria producing transglutaminase has a small improvement

Method used

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  • Glutamine transaminase mutant with improved enzymatic activity
  • Glutamine transaminase mutant with improved enzymatic activity
  • Glutamine transaminase mutant with improved enzymatic activity

Examples

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Embodiment 1

[0034] Example 1 Determination of Mutation Sites

[0035] The Discovery Studio2017 software was used to perform virtual amino acid mutation to determine the key amino acids in the active site, and according to the predicted results, targeted mutations were made to the sites that affect the affinity of the enzyme and the substrate, namely Y24W, R89W, E300W, Y302R.

Embodiment 2

[0036] Example 2: Construction of site-directed mutagenesis expression strain

[0037] (1) The transglutaminase gene derived from Streptomyces maoyuan and the pro-glutamine transaminase hpro gene derived from Streptomyces hygroscopicus were fused and ligated using One Step Cloning Kit to construct plasmid pINA1297 / hpro-mTG.

[0038](2) Use the plasmid pET 20b / mpro-mTG reserved in the laboratory as a template, and use P1 and P2, P3 and P4, P5 and P4, P1 and P6 as primers to perform PCR, and amplify by PCR 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 are: 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 product was us...

Embodiment 3

[0041] Embodiment 3 genetically engineered bacteria hpro-Y24W, hpro-R89W, hpro-E300W, hpro-Y302R shake flask fermentation

[0042] The genetically engineered bacteria hpro-Y24W, hpro-R89W, hpro-E300W, hpro-Y302R constructed in Example 1 and the starting strain hpro-MTG constructed earlier were inoculated in YPD liquid medium, cultured at 28°C and 200rpm for 24h, On the next day, 10% of the inoculum was transferred to Yarrowia lipolytica fermentation medium, and cultured in a shaker flask (specification: 250 mL) at 28° C. and 200 rpm for 120 h. The fermentation broth was centrifuged at 4°C and 4000rpm for 10 minutes, and the supernatant was the crude extracellular enzyme solution. After being activated by dispase, the enzyme activity was measured. The detected enzyme activities were 0.916U / mL, 9.87U / mL, 16.995U / mL and 0.635U / mL, and the hpro-MTG enzyme activity was 11.7U / mL, that is, the mutation of the 300th amino acid produced glutamine more than the original strain Amide tr...

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Abstract

The invention discloses a glutamine transaminase mutant with improved enzymatic activity, which belongs to the field of enzyme engineering. The site-specific mutagenesis is performed on glutamine transaminase, an amino acid residue close to an active site of the glutamine transaminase is changed, so that the catalytic efficiency of the glutamine transaminase is improved, and the enzymatic activityof the glutamine transaminase is further improved. Recombinant yarrowia lipolytica with improved enzymatic activity is established by the glutamine transaminase mutant, the enzymatic activity of theglutamine transaminase is improved by 1.45 times compared with an original strain, the enzymatic activity of the shake flask fermentation can reach 16.995U / mL, and the enzymatic activity of the fermenting tank fermentation can reach 59.85U / mL which is the highest fermentation value reported at present.

Description

technical field [0001] The invention relates to a glutamine transaminase mutant with improved enzyme activity, and belongs to the field of enzyme engineering. Background technique [0002] Glutamine transaminase (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 acceptor, so that the protein or polypeptide can interact with each other. Covalent cross-linking. 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 bond minced meat into pieces, improve the utilization rate of meat products, and improve the elasticity of meat products. In addition, TGase has huge market demand in the fields of medicine, cosmetics, biotechnology research, textile industry and leather processing. [0003] Originally, TGase was derived...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N1/19C12N15/81
CPCC12N9/1044C12N15/815C12Y203/02013
Inventor 刘松任蕊蕊李江华陈坚堵国成
Owner TAIXING DONGSHENG FOOD TECH
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