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Protease K heat-resistant mutant

A protease and mutant technology, which is applied in the field of genetic engineering and enzyme engineering, can solve the problems of poor stability and high cost of cross-border low-temperature long-distance transportation, and achieve good thermal stability, stability and specific activity.

Active Publication Date: 2021-09-03
WUHAN HANHAI NEW ENZYMES BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After the outbreak of the new coronavirus in 2020, the world’s demand for proteinase K for molecular diagnosis is increasing. The poor stability of wild-type proteinase K when stored at room temperature leads to high costs for long-distance transnational low-temperature transportation
However, after many years of verification by domestic and foreign scholars, only a few mutation sites improve the specific activity of proteinase K, and have no effect on further improving the thermal stability of proteinase K.

Method used

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  • Protease K heat-resistant mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0164] Embodiment 1: Determination of protease mutation site

[0165] According to the three-dimensional crystal structure of proteinase K, the 350th amino acid in the amino acid sequence shown in SEQ ID NO.2 was determined as the key site through rational design. Specifically, a single-point mutant of proteinase K was designed through the structure-assisted Consensus Concept method, and a proteinase K mutant with significantly improved thermal stability was obtained. Consensus Concept is an emerging theoretical system for improving protein thermal stability in rational protein design. Different from rational design based on information such as precise protein structure-function relationship and catalytic mechanism, this method is based on the sequence information of homologous proteins, and explores factors that may lead to improved thermal stability from an evolutionary perspective, thereby improving thermal stability. Combine the existing proteinase K structural informatio...

Embodiment 2

[0174] Embodiment 2: Construction of the recombinant expression vector carrying the nucleotide sequence encoding proteinase K mutant

[0175] As described in Example 1, it is determined that the 350th amino acid of the amino acid sequence shown in SEQ ID NO.2 is used as the key site, and the mutants shown in SEQ ID NO.3 and SEQ ID NO.4 are designed, that is: single Mutant D350V, combined mutant Y151A / K208H / S273T / G293A / K332R / S337N / D350V. Specific steps are as follows:

[0176] The amino acid sequence of SEQ ID NO.2 was reverse-transcribed and optimized according to the codons of Pichia pastoris to obtain the nucleotide sequence SEQ ID NO.5; the amino acid sequence of SEQ ID NO.3 was reverse-transcribed according to the Pichia The yeast codon was optimized to obtain the nucleotide sequence SEQ ID NO.6; the amino acid sequence of SEQ ID NO.4 was reverse-transcribed and then optimized according to the Pichia pastoris codon to obtain the nucleotide sequence SEQ ID NO.7; respective...

Embodiment 3

[0178] The construction of the recombinant microorganism cell of embodiment 3 expression proteinase K mutants

[0179] After the recombinant expression vector constructed in Example 2 was linearized with BglII, it was transformed into Pichia pastoris X33 competent cells by electroporation to obtain recombinant strains expressing proteinase K wild type and mutant, respectively named PRK-WT (for expression of wild-type proteinase K), PRK-D350V (for expression of mutant D350V), PRK-7M (for expression of combined mutant Y151A / K208H / S273T / G293A / K332R / S337N / D350V). The protein sequences of the expressed proteinase K correspond to the amino acid sequences shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 respectively.

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Abstract

The invention discloses a protease K heat-resistant mutant, and belongs to the field of gene engineering and enzyme engineering. According to the protease K heat-resistant mutant, From the molecular structure of Tritirachium album protease K, a mutant with improved thermal stability is obtained, an encoding protease K wild type and a mutant gene thereof are connected with a pPICZ alpha A plasmid to construct a recombinant expression vector, and the recombinant expression vector is transformed into pichia pastoris to construct a recombinant engineering bacterium for heterologous secretory expression. Compared with wild protease K, the protease K mutant PRK-7M provided by the invention has the advantages that the stability and the specific activity are greatly improved.

Description

technical field [0001] The invention relates to a heat-resistant mutant of proteinase K, belonging to the fields of genetic engineering and enzyme engineering. Background technique [0002] Proteinase K is an important serine protease secreted by Candida albicans (Tritirachium album), which has a broad substrate spectrum and high proteolytic activity. Proteinase K can be used not only in biochemical experiments, for example, in nucleic acid extraction to remove DNase and RNase in nucleic acid, in in situ hybridization to degrade proteins surrounding target DNA, and to process samples before hybridization to improve detection efficiency. Sensitivity; can also be used for IVD biochemical detection reagents and molecular diagnostic reagents. [0003] Early commercial supply of proteinase K was mainly secreted from Candida albicans limberii. Candida albicans limberii grows slowly, it is difficult to culture in high density, and the yield of proteinase K is low. Candida albican...

Claims

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Application Information

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IPC IPC(8): C12N9/58C12N15/57C12N15/81C12N1/19C12R1/84
CPCC12N9/58C12N15/815
Inventor 杨广宇罗漫杰徐灿施婧妮宫安彭晶
Owner WUHAN HANHAI NEW ENZYMES BIOLOGICAL TECH CO LTD
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