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Alkaline protease mutant with improved specific activity and coding gene of alkaline protease mutant

A mutant and protease technology, applied in genetic engineering, plant gene improvement, hydrolytic enzymes, etc., can solve the problems of high production cost, low specific activity of BmP, and limited industrial application, and achieve the goal of reducing production cost and improving specific activity Effect

Active Publication Date: 2018-08-10
珠海市双指环投资有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the specific activity of BmP is relatively low and the production cost is high, which limits its industrial application

Method used

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  • Alkaline protease mutant with improved specific activity and coding gene of alkaline protease mutant
  • Alkaline protease mutant with improved specific activity and coding gene of alkaline protease mutant
  • Alkaline protease mutant with improved specific activity and coding gene of alkaline protease mutant

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Effect test

Embodiment 1

[0026] Embodiment 1, the cloning of bacillus mojavensis (Bacillus mojavensis) alkaline protease BmP gene

[0027] The target gene was directly synthesized according to the reported alkaline protease gene sequence of Bacillus mohaiwei (Genebank: AY665611.1). Two primers (R: 5'-ATCGGGATCCGCTCAACCGGCGAAAAATGTT-3' and F: 5'-TCTAGCGGCCGC TTATTGAGCG GCAGCTTCGAC-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

[0028] Embodiment 2, Rational Design Fixed-point Saturation Mutation

[0029] The alkaline protease BmP was modeled by homology modeling software, and the three-dimensional conformation map of BmP was obtained. The built BmP model was docked and analyzed by bioinformatics software, and the key amino acid position 178 was found. The effect of position 178 on the specific activity of alkaline protease BmP was studied by saturation mutation.

[0030] The process of site-specific saturation mutation is as follows: To construct a good phyP 43 L-BmP was used as a template, and PCR amplification was carried out with corresponding mutant primers; the amplified PCR product was subjected to agarose electrophoresis, and the PCR product was purified and recovered. The original plasmid was decomposed with the restriction endonuclease DpnI, and the decomposed product was transformed into E. coli Top10 by the heat shock method. The recombinant transformants were verified by bacterial liqui...

Embodiment 3

[0032] Embodiment 3, original alkaline protease BmP and mutant specific activity analysis

[0033] Alkaline protease BmP and mutants were purified by nickel column purification. The purified alkaline protease BmP and mutants were measured for their specific activity. The experimental results are shown in Table 1. It can be seen from Table 1 that the 178th position is a key amino acid site that affects the hydrolysis activity of alkaline protease BmP. When it is mutated to D , M, G, Q and T, the relative specific activity increased by 35%, 21%, 28%, 19% and 25%, respectively.

[0034] Table 1 Original alkaline protease BmP and mutant specific activity analysis

[0035] Numbering

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Abstract

The invention relates to the field of gene engineering, and in particular relates to an alkaline protease BmP mutant with the improved specific activity and a coding gene of the alkaline protease BmPmutant. The mutation site of the alkaline protease BmP mutant refers to that the 178th site of the alkaline protease BmP as shown in amino acid SEQ ID NO.7 mutates into D,M,G,Q or T from S. The obtained mutant has higher specific activity than the original enzyme.

Description

technical field [0001] The invention relates to the field of protein molecular transformation, in particular to an alkaline protease BmP mutant with improved specific activity, its coding gene and application. Background technique [0002] Protease is a class of enzymes that catalyze protein hydrolysis. Protease can be divided into acid protease, neutral protease and alkaline protease according to its optimum reaction pH environment. Alkaline protease is a class of enzymes that hydrolyze proteins under alkaline conditions. Alkaline protease is widely used in food, washing, feed and other industrial fields because of its strong hydrolysis ability. At present, alkaline proteases mainly come from Bacillus licheniformis, Bacillus subtilis and Bacillus circulans, and there are relatively few reports on alkaline proteases from other bacillus. [0003] In previous experiments, our laboratory cloned, expressed and analyzed the alkaline protease BmP from Bacillus mojavensis. BmP has...

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 珠海市双指环投资有限公司
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