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Production method of recombinant glutamine transaminase

A glutamine and production method technology, applied in the field of separation and purification of biological and recombinant proteins, can solve the problems of difficulty in screening high-yield TGase strains, high production costs, and restrictions on industrial production, and achieve stable protein structure and good fragmentation rate , the effect of improving the success rate of renaturation

Inactive Publication Date: 2016-11-23
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although many microorganisms can produce TGase in the natural state, the yield of most enzymes is extremely low, and it is time-consuming and laborious to screen high-yielding strains of TGase through conventional breeding techniques, which results in high production costs and limits industrial production

Method used

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  • Production method of recombinant glutamine transaminase
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  • Production method of recombinant glutamine transaminase

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1 This example illustrates the construction of transglutaminase recombinant Escherichia coli

[0035] 1. Design synthesis with EcoR I Upstream primers with restriction sites and Bam H I The downstream primer of restriction site (the underlined part is the corresponding restriction site),

[0036] Primer1 upstream primer: 5'- GGTCGTCAACAACTACATAC-3';

[0037] Primer2 downstream primer: 5'-GTTCGCCAGTTCCTTCTT-3'.

[0038] 2. According to the MTG gene reported by Fu, R.Y. (GenBank: DQ132977), to Streptomyces mobaraensis Genomic DNA was used as a template, and the target gene fragment was amplified by PCR. The reaction conditions were: 95°C for 5 min; (95°C for 30 s, 60°C for 60 s, 72°C for 45 s, 30 cycles); 72°C for 10 min. Purified and amplified mtg gene (refer to Takara DNA Fragment Purification Kit instructions) and expression plasmid pET30a (purchased from Takara Company) were digested with EcoR I and bamH I respectively, ligated and transformed into ...

Embodiment 2

[0040] Example 2 This example illustrates the fermentation control process of recombinant glutamine transaminase Escherichia coli

[0041] Pick a single clone on the flat plate of the recombinant transglutaminase Escherichia coli BL21(DE3) / mtg obtained in Example 1 and inoculate it in a test tube containing 4 ml of LB culture solution at 37°C and cultivate it at 200 rpm for 16h, then culture a well The grade seed solution was inoculated at 2% inoculum amount in an Erlenmeyer flask containing 200ml LB culture solution and cultivated at 37°C for 16h as the seed solution for fermentation. Kanamycin, peptone 12g / L, yeast extract 24g / L, glycerol 5g / L, K 2 HPO 4 ·3H 2 O 15.2g / l, KH 2 PO 4 2.33g / l) in a 10L fermenter at 37°C, the rotation speed is set to 300rpm, the tank pressure is maintained at 0.03-0.05Mpa, and the initial ventilation volume is 1.5m 3 / h, maintain DO greater than 25% by adjusting the ventilation, cultivate until the OD600 is between 2-3, add a final concentr...

Embodiment 3

[0042] Example 3 This example illustrates the purification of recombinant transglutaminase protein

[0043] Take 26.7g of the collected cells and add 10 times the volume (260ml) of 50mmol / L Tris-HCl (pH 8.0), 0.15mol / L NaCl, 0.5% Triton X-100, 1mM DTT, 0.2mM PMSF, 1mM EDTA and stir well Finally, it was slowly fed into the high-pressure homogenizer, and the pressure was controlled at 800Mpa. After two cycles, the homogenate was centrifuged at 6000rpm at 4°C, and the precipitate was added to 10 times the volume of 50mmol / L Tris-HCl (pH 8.0), 0.15 mol / L NaCl, 0.5% Triton X-100, 1mM DTT, 2mol / L urea, after stirring well, slowly poured into the high-pressure homogenizer, the pressure was controlled at 800Mpa, after two cycles, the homogenate was mixed at 6000rpm, 4℃ Centrifuge, then add 10 times the volume of 50mmol / L Tris-HCl (pH 8.0), 0.15mol / L NaCl, 0.5%Triton X-100, 1mM DTT, 4 mol / L urea to the obtained precipitate, stir well, and flow slowly Add it to a high-pressure homogeni...

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Abstract

The invention discloses a production method of recombinant glutamine transaminase. The production method comprises the following steps of 1 fermentation of recombinant glutamine transaminase, 2 cell collection and lysis after fermentation, 3 obtaining and purification of an inclusion body and 4 renaturation of the glutamine transaminase. According to the method, target protein exists in the inclusion body form by adopting high temperature culturing and inducing in the fermentation process, therefore, the problem that the target protein generates a crosslinking reaction in cells to influence normal growth of thalli is prevented, the expression quantity of the target protein is increased, and the inhibition effect of metabolic by-products is reduced; the raw material utilization rate is increased, and therefore the production cost is reduced; in addition, the processes are simple, the obtained glutamine transaminase is high in quality and low in cost, and the production method is suitable for being applied to industrialized production.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a production method of recombinant glutamine transaminase, which belongs to the technical field of recombinant protein separation and purification. Background technique [0002] Transglutaminase (Transglutaminase, TG.2.3.2.13) through acyl transfer reaction causes internal protein cross-linking between proteins, connection between proteins and amino acids, and transfer and hydrolysis of glutamine groups in protein molecules function, forming ε-(γ-glutamyl) lysine covalent isopeptide bonds, thereby improving the structural and functional properties of proteins, due to its excellent cross-linking properties, TG is considered to be the best choice for the production of various new protein processing products As an important enzyme source, it has important potential application value in the fields of food processing, storage, and fresh-keeping, cosmetics, textiles, and biomedicine. [0...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12R1/19
CPCC12N9/1044C12Y203/02013
Inventor 虞龙吴奎李玉燕
Owner NANJING UNIV OF TECH
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