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A self-cleavage resistant and high specific activity trypsin mutant

A technology of trypsin and trypsinogen, applied in the directions of enzymes, peptidases, hydrolase, etc., can solve the problems of affecting the application efficiency and level, and the decline of enzyme activity, and achieves the improvement of easy self-cleavage and reduced activity, high specific activity, good The effect of anti-self-cutting properties

Active Publication Date: 2022-07-19
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Trypsin can not only hydrolyze other protein molecules, but also attack the lysine and arginine sites on its surface, resulting in a rapid decline in its enzyme activity during storage or use, thereby affecting its application performance and level

Method used

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  • A self-cleavage resistant and high specific activity trypsin mutant
  • A self-cleavage resistant and high specific activity trypsin mutant
  • A self-cleavage resistant and high specific activity trypsin mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1. Cloning of porcine trypsinogen gene.

[0058] According to the amino acid sequence of wild porcine trypsinogen, Pichia pastoris was selected as the expression host for codon optimization to obtain the optimized gene of Pichia trypsinogen (SEQ ID NO. 1), and the wild-type trypsinogen gene (SEQ ID NO. 1) was synthesized. .1), design primers and amplify the gene by PCR technology, use T4 ligase to connect the amplified gene fragment to the vector pMD19T to obtain the recombinant cloning vector pMD19T-try. Transform into E. coli JM109 competent cells.

Embodiment 2

[0059] Example 2. Site-directed mutagenesis

[0060] The process of site-directed mutagenesis is as follows: using the constructed PMD19T-try as a template, design corresponding primers for inverse PCR, at the corresponding amino acid positions 107, 115, 133, 147, 157, 208 and 210. Single point or combined mutation was introduced, and the primer sequences are shown in Table 2. Use Dpn I restriction endonuclease to remove the original template, carry out agarose electrophoresis to verify the amplified product and purify the recovered product, transform the recovered plasmid into E. coli, extract the plasmid from the constructed strain, and perform single enzyme digestion and double-enzyme digestion to verify and sequence, and extract the recombinant plasmid pMD19T-M-try (M represents the corresponding mutant); the constructed recombinant plasmid pMD19T-M-try (M represents the corresponding mutant) and the expression vector pPIC9K were respectively used Ecol I and Not I were do...

Embodiment 3

[0064] Embodiment 3, recombinant Pichia pastoris shake flask fermentation

[0065] The strains were inoculated into YPD solid medium for three-district streaking, and cultured at 28°C for 2 days; a single colony was picked from the YPD solid plate to YPD liquid medium, 28°C, 200rpm for 24h; YPD liquid culture was inoculated at 2% of the inoculum The well-cultivated seed liquid was cultured to BMGY medium, 28°C, 200rpm to the bacterial liquid OD 600 For 2-6, then transfer it to a sterile centrifuge tube and centrifuge at 5000rpm for 10min to retain the bacteria; add an appropriate amount of BMMY and repeat the centrifugation to remove residual glycerol; transfer to BMMY medium, add methanol to a final concentration of 0.5%; then every 12h Add methanol with a final concentration of 0.5% to induce culture for 96 h.

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Abstract

The invention relates to a trypsin mutant with resistance to self-cleavage and high specific activity, in particular to a mutant of trypsin with resistance to self-cleavage and high specific activity obtained by molecular modification and screening, and its encoding gene and application, belonging to protein and genetic engineering technology field. The trypsin mutant provided by the present invention is obtained by mutating at least one of the following sites on the basis of the amino acid sequence shown in SEQ ID NO. 2: R107H, R107L, R115S, R115T, K133A, K133A, K147D, K157E, K208P , K210A. Compared with the original enzyme, it has better anti-self-cleavage performance and higher specific activity. Compared with wild-type trypsin, the anti-self-cleavage performance is increased by 0.67-2.88 times, and the specific enzyme activity is increased by 0.07-2.98 times. The protease is easy to cut itself and reduce its activity during storage and use.

Description

Technical field: [0001] The invention relates to a trypsin-resistant mutant with high specific activity obtained by molecular modification and screening, and its encoding gene and application, and belongs to the technical field of protein and genetic engineering. Background technique: [0002] Trypsin (EC 3.4.21.4) is an alkaline serine proteolytic enzyme widely used in food processing, medicine and scientific research. As a digestive enzyme, trypsin can supplement the lack of endogenous enzymes in animals and promote the absorption of nutrients in animals; trypsin can dilute blood clots and pus without damaging other tissues, accelerate wound healing, and degrade intercellular matrix and adhesion. Protein has important medical value; trypsin can clarify alcohol and beverages, and can improve the elasticity and softness of leather; because of its strong amino acid site cleavage specificity, it is used as an important tool enzyme in the field of scientific research. [0003]...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/76C12N15/57C12N15/81C12N1/19C12R1/84
CPCC12N9/6427C12Y304/21004C12N15/815
Inventor 王洪彬路福平曾芳冯永蕊
Owner TIANJIN UNIV OF SCI & TECH
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