Alkaline protease BmP mutants with improved thermal stability and genes and application thereof

A technology of thermal stability and mutant, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of poor thermal stability of BmP, easy decomposition and inactivation, and limited industrial application, and achieve the effect of improving stability.

Active Publication Date: 2018-05-08
刘丹妮
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, BmP has poor thermal stability and is easily decomposed and inactivated at high temperatures above 70 °C, which limits its industrial application.

Method used

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  • Alkaline protease BmP mutants with improved thermal stability and genes and application thereof
  • Alkaline protease BmP mutants with improved thermal stability and genes and application thereof
  • Alkaline protease BmP mutants with improved thermal stability and genes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1, the cloning of bacillus mojavensis (Bacillus mojavensis) alkaline protease BmP gene

[0048] The target gene was directly synthesized according to the sequence of the alkaline protease gene of Bacillus mohaiwei (Genebank: AY665611.1). Two primers (R: 5'-ATCGGGATCCGCTCAACCGGCGAAAAATGTT-3' and F: 5'-TCTAGCGGCCGC TTATTGAGCGGCAGCTTCGAC-3') were designed according to the synthesized target gene to amplify the BmP gene of Bacillus mohaiwei alkaline protease. The amplified PCR product was purified and recovered, and connected to the expression vector phyP 43 L, get the expression vector phyP 43 L-BmP.

Embodiment 2

[0049] Embodiment 2, site-directed saturation mutation

[0050] The effect of position 193 and position 288 on the thermostability of alkaline protease BmP was studied by saturation mutagenesis. The process of site-specific saturation mutation is as follows: To construct a good phyP 43 L-BmP was used as a template, and the corresponding mutant primers were used for PCR amplification; the amplified PCR product was subjected to agarose electrophoresis, and the PCR product was purified and recovered. Decompose the original plasmid with the restriction endonuclease DpnI, transfer the decomposed product into E. coli Top10 by heat shock method, verify the recombinant transformant by bacterial liquid PCR, extract and verify the correct transformant plasmid for sequencing, so as to determine the corresponding mutants. The mutants with correct sequencing were transformed into Bacillus subtilis WB600 by electrotransformation.

[0051] The screening of recombinant transformants is as ...

Embodiment 3

[0054] Embodiment 3, combinatorial mutation

[0055] Combined mutations were carried out on the basis of single point mutations, and four combined mutations were finally obtained through experiments, named BmP1, BmP2, BmP3, and BmP4, respectively. The mutation sites contained in BmP1 are S193K and S288M; the mutation sites contained in BmP2 are S193L and S288M; the mutation sites contained in BmP3 are S193L and S288K; the mutation sites contained in BmP3 are S193K and S288K.

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Abstract

The present invention relates to the field of bioengineering, in particular to alkaline protease BmP mutants with improved thermal stability and genes and application thereof. The mutants are obtainedfrom a mutant of an alkaline protease BmP by mutating amino acid S at the 193 site into L or K or mutating amino acid S at the 288 site into K or M, wherein the amino acid sequence of the alkaline protease BmP is shown as SEQ ID NO.6. The thermal stability of the alkaline protease BmP of bacillus mojavensis is improved. Under the premise of not destroying other excellent enzymatic properties of the alkaline protease, the thermal stability of the alkaline protease is significantly improved, and a foundation is laid for industrialized application.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to an alkaline protease BmP mutant with improved thermostability, its gene and application. Background technique [0002] Protease is a kind of enzyme that catalyzes the hydrolysis of protein. As an important industrial enzyme preparation, protease is widely used in feed processing, brewing, washing, food processing and other fields. Proteases are widely distributed. Currently, the proteases on the market are mainly derived from microorganisms. Compared with animals and plants, microbial proteases have a wider pH range and lower production costs. As the most important type of microbial protease, Bacillus protease is mainly divided into neutral protease and alkaline protease. At present, Bacillus protease has been widely used in feed, washing, food and other fields. [0003] Bacillus mojavensis alkaline protease is called BmP for short. It has been found through experiments in the earl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/54C12N15/57
CPCC12N9/54
Inventor 刘丹妮
Owner 刘丹妮
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