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Heterologous expression of glutamine transaminase and application thereof

A glutamine and transaminase technology, applied in the direction of acyltransferase, application, transferase, etc., can solve the problems of low expression, difficulty in high-efficiency expression of TGase, and no catalytic activity of inclusion bodies

Pending Publication Date: 2022-02-01
SICHUAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the low expression level, the lack of catalytic activity of inclusion bodies, and the need for dilution and renaturation in the later stage make the high-efficiency expression of TGase encounter many difficulties

Method used

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  • Heterologous expression of glutamine transaminase and application thereof
  • Heterologous expression of glutamine transaminase and application thereof
  • Heterologous expression of glutamine transaminase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Embodiment one: the acquisition of TGase gene

[0015] extract Streptomyces Genomic DNA of sp. TYQ1024 was sequenced for whole genome sequence. The obtained genome data is analyzed to obtain the transglutaminase gene TGase.

Embodiment 2

[0016] Embodiment two: the heterologous expression of TGase gene

[0017] Design specific primers to amplify TGase gene, upstream primer (5'-CGC GGATCC TCGCCACCGGCAGTGGCAGTGGCA-3'), downstream primer (5'-CTA GCTAGC TCACGGCCAGCCCTGTGTCA-3'), the TGase gene was obtained by PCR. Perform TGase gene and plasmid pMA LipA Bam H and Nhe Digest and ligate. Then, the ligation product was transformed into E. coli DH5α, the recombinant plasmid pMA LipA-TG was extracted and transformed into B. subtilis SCK6.

Embodiment 3

[0018] Embodiment three: Recombinant Bacillus subtilis engineered bacterium culture condition

[0019] recombinant bacteria B. subtilis SCK6 / pMA LipA-TG was inoculated into 100 mL TB basal medium containing 50 µg / mL Kanamycin, cultured on a shaker at 37°C for 16 hours, and inoculated at a 3% inoculum size into the fermentation culture containing 50 µg / mL Kan Medium (medium composition: glucose 15 g / L, soybean peptone 20 g / L, urea 20 g / L, potassium dihydrogen phosphate 2.31 g / L, dipotassium hydrogen phosphate 0.54 g / L, pH 6.5), 37° C, cultured on a shaker at 220 rpm for 64 hours; centrifuged, collected the fermentation supernatant, and measured the TGase activity in the supernatant.

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Abstract

The invention discloses heterologous expression and characterization of a streptomyces glutamine transaminase gene and an application of the streptomyces glutamine transaminase gene in gelatin crosslinking, and belongs to the technical field of biology. The full length of the glutamine transaminase gene is obtained by mining Streptomyces sp.TYQ1024 whole-genome data, the full length of the glutamine transaminase gene is 1233 bp, 410 amino acids are encoded, and the gene is amplified in vitro through PCR. According to the invention, heterologous expression, purification and characterization of streptomyces glutamine transaminase genes in bacillus subtilis SCK6 are realized for the first time, and recombinant glutamine transaminase is applied to gelatin crosslinking. The enzyme has good enzymatic characteristics and excellent cross-linking characteristics, and has wide application prospects in protein and gelatin cross-linking.

Description

technical field [0001] The invention relates to the technical field of recombinant expression, molecular optimization and application of glutamine transaminase. Background technique [0002] Transglutaminase (TGase) is a kind of enzyme that catalyzes the cross-linking reaction of proteins. Its substrates have a wide range of applications, such as whey protein, soybean protein, casein, bovine myosin, etc., and are used in biomedicine, textiles, etc. , food, cosmetics, leather, materials and other fields have broad application prospects. [0003] TGase obtained from guinea pigs and livers has complex separation and purification methods, which makes the price of the enzyme very expensive and limits large-scale production and application. Coagulation factor XIII is extracted from the blood of pigs and cattle to produce commercial TGase. However, blood TGase needs thrombin to activate its in vitro activity and affects the appearance of the product, so it is rarely used in food....

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/10C12N15/75
CPCC12N9/1044C12N15/75C12Y203/02013
Inventor 田永强王诗婷李晓广
Owner SICHUAN UNIV
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