A kind of trypsin mutant with improved enzyme activity and its construction method

A trypsin and mutant technology, applied in the field of trypsin mutants and their construction, can solve the problems of low trypsin enzyme activity and low protein expression, and achieve the effects of improving extracellular secretion, high enzyme activity and simple fermentation process
CN104328102BActive Publication Date: 2017-01-25JIANGNAN UNIV
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
JIANGNAN UNIV
Publication Date
2017-01-25

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Abstract

The invention discloses a trypsin mutant capable of improving enzyme activity and a construction method thereof, belonging to the technical field of genetic engineering. According to the mutant disclosed by the invention, on the basis of an amino acid with a sequence as shown in SEQ ID NO. 2, tyrosine on the 1st site is mutated into phenylalanine, meanwhile arginine on the 123rd site is mutated into isoleucine. The mutant disclosed by the invention is expressed in pichia pastoris; the enzyme activity of amidase (BAPNA) fermented in a 3L tank is 85.3U / mL and enzyme activity of esterase (BAEE) is 5954U / mL, which are respectively improved by 3.3 times and 2.7 times in comparison with those of a strain before mutation; therefore, a problem of low extracellular enzyme activity of trypsin is solved. When applied to producing the trypsin, the mutant disclosed by the invention is high in yield, simple in process and convenient for industrial application.
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Description

technical field

[0001] The invention relates to a trypsin mutant with improved enzyme activity and a construction method thereof, belonging to the technical field of genetic engineering. Background technique

[0002] Trypsin, as an important type of serine protease that was discovered earlier, was first found in the intestinal digestive juice of mammals. Commercial trypsin is mostly extracted from mammalian pancreas. Because the pancreas is rich in protease, it is difficult to separate and purify trypsin. In addition, trypsin from mammals is mostly a mixture of trypsin, which has immunogenic hazards to the human body in medical applications.

[0003] Streptomyces griseus trypsin research, Koo-Bon-Joon et al. used Streptomyces lividans1326, a common host of Streptomyces, and the pWHM3 plasmid (Ermp, erythromycin promoter) commonly used by Streptomyces to obtain recombinant trypsin secreted and expressed in the same genus, Chi-Won -Jae et al. transformed this plasmid into St...

Claims

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