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Autotomy-resistant high-specific-activity trypsin mutant

A technology of trypsin and mutants, which is applied in the field of protein and genetic engineering, can solve the problems of affecting the application efficiency and level, the decrease of enzyme activity, etc., and achieve the characteristics of easy self-cutting and low activity, high specific activity, and good anti-self-cutting performance Effect

Active Publication Date: 2020-09-18
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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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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  • Autotomy-resistant high-specific-activity trypsin mutant
  • Autotomy-resistant high-specific-activity trypsin mutant
  • Autotomy-resistant high-specific-activity trypsin mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1. Cloning of pig-derived 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 trypsinogen Pichia optimized gene (SEQ ID NO.1), and the wild-type trypsinogen gene (SEQ ID NO.1) was synthesized. .1), designing primers and amplifying the gene by PCR technology, using T4 ligase to connect the amplified gene fragment to the vector pMD19T to obtain the recombinant cloning vector pMD19T-try. Transformation into Escherichia coli JM109 competent cells.

Embodiment 2

[0059] Embodiment 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 107th, 115th, 133rd, 147th, 157th, 208th and 210th amino acid positions Point-introduced single-point or combined mutations, the primer sequences are shown in Table 2. Use Dpn I restriction endonuclease to remove the original template, verify the amplified product by agarose electrophoresis and purify the recovered product, transform the recovered plasmid into Escherichia coli, extract the plasmid from the constructed strain, and perform single enzyme digestion and double enzyme digestion verification and sequencing, 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 ...

Embodiment 3

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

[0065] Inoculate the strains into YPD solid medium for three-section line, culture at 28°C for 2 days; pick a single colony from the YPD solid plate to YPD liquid medium, culture at 28°C, 200rpm for 24h; inoculate YPD liquid culture with 2% inoculum The cultured seed solution was transferred to BMGY medium, and cultivated at 28°C and 200rpm to the OD of the bacterial solution 600 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 centrifugation to remove residual glycerol; transfer to BMMY medium, add methanol to a final concentration of 0.5%; then every 12h Add methanol at a final concentration of 0.5% to induce culture for 96 hours.

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Abstract

The invention relates to an autotomy-resistant high-specific-activity trypsin mutant, particularly relates to the autotomy-resistant high-specific-activity trypsin mutant obtained through molecular modification and screening, a coding gene thereof and application of the autotomy-resistant high-specific-activity trypsin mutant and belongs to the technical fields of proteins and genetic engineering.The trypsin mutant provided by the invention is obtained through mutating at least one of the following loci: R107H, R107L, R115S, R115T, K133A, K133A, K147D, K157E, K208P and K210A on the basis of an amino acid sequence shown in SEQ ID No. 2. Compared with a protoenzyme, the trypsin mutant has better autotomy resistance and higher specific activity; and compared with wild type trypsin, the autotomy resistance is improved by 0.67 to 2.88 times, the specific enzyme activity is improved by 0.07 to 2.98 times, and thus, the problem that the trypsin is prone to activity lowering due to autotomy during storage and use is solved.

Description

Technical field: [0001] The invention relates to the anti-self-cutting and high specific activity mutant of trypsin obtained through molecular transformation and screening, its coding 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 deficiency of endogenous enzymes in animals and promote the absorption of nutrients; 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 cutting specificity, it is used as an important tool enzyme in the field of scientific research. ...

Claims

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Application Information

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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 UNIVERSITY OF SCIENCE AND TECHNOLOGY
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