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Mutant-type Sus scrofa swine trypsin and encoding gene thereof as well as acquisition method and application

A trypsin gene and trypsin technology, which is applied in the fields of molecular enzymology and biology, and can solve the problems of complicated analysis steps and the like

Active Publication Date: 2016-02-03
TIANJIN UNIV
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  • Claims
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Problems solved by technology

Denatured proteins are usually added with chemical reagents, adding additional steps, which complicates the analysis steps

Method used

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  • Mutant-type Sus scrofa swine trypsin and encoding gene thereof as well as acquisition method and application
  • Mutant-type Sus scrofa swine trypsin and encoding gene thereof as well as acquisition method and application
  • Mutant-type Sus scrofa swine trypsin and encoding gene thereof as well as acquisition method and application

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Experimental program
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Effect test

Embodiment Construction

[0063] (1) Construction of wild-type Susscrofa pig-derived trypsinogen expression vector

[0064] The constructed wild-type recombinant porcine trypsinogen expression vector pPIC9K-rpTry vector was synthesized from the whole gene of Beijing Jinweizhi Co., Ltd., as shown in figure 1 shown. Among them, the first 20 amino acids of the first 25 amino acids of the N-terminal zymogen activating peptide of the original gene were removed, and the remaining sequence of the N-terminal activating peptide was the enterokinase recognition site (DDDDK). The shortened Susscrofa porcine zymogen sequence was double digested with EcoRI and NotI and inserted into the corresponding single cloning site of pPIC9K.

[0065] (2) Quick-ChangePCR technology to obtain mutant vector

[0066] According to the primer design principles of the TransGenFastMutagenesisSystem kit, we designed mutation primers for the mutation N84S, as shown in Table 1-1. The PCR reaction polymerase was Phusion High-fidelity ...

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Abstract

The invention relates to a mutant-type Sus scrofa swine trypsin and an encoding gene thereof as well as an acquisition method and an application. According to the invention, the original wild type Sus scrofa swine trypsin is subjected to mutation to obtain the mutant enzyme with heat stability obviously improved; the invention further provides the encoding gene for encoding the trypsin mutant, the amino acid sequence and a construction method of a trypsin yeast engineering bacteria, wherein the amino acid sequence is SEQ ID NO.1, and the protein DNA sequence in encoding is SEQ ID NO.2. The invention lays a foundation for the research, development and production of the protease through a bioanalysis method, and is beneficial for the early realization of the clean and low-energy biological tanning, and the bio-safe production of leather. With the strain for producing swine trypsin, the yield is relatively high, the process is relatively simple, and the industrial application is facilitated.

Description

technical field [0001] The invention relates to molecular enzymology and biotechnology, more specifically to a coding gene of Susscrofa pig-derived trypsin with high thermal stability and high activity, as well as a method for obtaining the enzyme and an application thereof. Background technique [0002] Trypsin (EC.3.4.21.4) is a single-chain serine protease consisting of 223 amino acid residues, and its precursor trypsinogen contains a signal peptide of 15 amino acids, followed by a signal peptide of 8 amino acids Leader peptide, zymogen activation peptide. Trypsinogen is produced in the ribosomes of the rough endoplasmic reticulum in the pancreatic cells of vertebrate animals, transported by the Golgi apparatus, enters the pancreatic transport duct, and is finally released into the intestinal lumen, where it utilizes the duodenal intestine Enterokinase secreted by mucosal cells specifically recognizes the N-terminal short peptide chain DDDDK of the trypsinogen sequence, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/76C12N15/57C12N15/10C12N15/81C12N1/19C12R1/84
CPCC12N9/6427C12Y304/21004
Inventor 黄鹤杜坤郭超刘冶
Owner TIANJIN UNIV
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