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Alkaline protease BmP mutant for improving specific activity and coding gene thereof

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

Active Publication Date: 2018-09-25
横琴仲泰生物医药有限公司
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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 BmP mutant for improving specific activity and coding gene thereof
  • Alkaline protease BmP mutant for improving specific activity and coding gene thereof
  • Alkaline protease BmP mutant for improving specific activity and coding gene thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1, the cloning of bacillus mojavensis (Bacillus mojavensis) alkaline protease BmP gene

[0029] 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'-TCTAGCGGCCGCTTATTGAGCGGCAGCTTCGAC-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

[0030] Embodiment 2, study the impact of the 236th and 293rd positions on the specific activity of alkaline protease BmP by saturation mutation

[0031] 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.

[0032] The screening of recombinant transformants is as follows: First, insert the recombinant bact...

Embodiment 3

[0033] Embodiment 3, original alkaline protease BmP and single mutant specific activity analysis

[0034]Alkaline protease BmP and single mutants were purified by nickel column purification. The alkaline protease BmP after purification and the mutant are carried out specific activity determination, and experimental result is as shown in table 1, as can be seen from table 1, the 236th and 293 are the key amino acid sites that affect the hydrolysis activity of alkaline protease BmP, when 236 When the mutation is G, C and D, the relative activity is increased by 12%, 15% and 18% respectively; when the 293 mutation is S, K and M, the relative activity is increased by 21%, 28% and 31% respectively .

[0035] Table 1 Original alkaline protease BmP and mutant specific activity analysis

[0036]

[0037]

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Abstract

The invention relates to the field of protein molecular modification, in particular to an alkaline protease BmP mutant for improving specific activity and a coding gene thereof. The mutation site of the alkaline protease BmP mutant is as follows: the 236th site of the amino acid sequence shown as SEQ ID NO.11 of alkaline protease BmP is mutated from N to G or C or D, and the 293th site is mutatedfrom N to S or K or M. Experiments confirm that the mutant has higher specific activity than original enzyme, and the production cost of alkaline protease is reduced, so as to lay a foundation for theindustrial application of BmP.

Description

technical field [0001] The invention relates to the field of protein molecular modification, in particular to an alkaline protease BmP mutant with improved specific activity and its coding gene. 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. According to the optimal reaction pH environment of microbial protease, protease can be divided into acid protease, neutral protease and alkaline protease. Alkaline protease is a class of enzymes that hydrolyze proteins under alkaline conditions. At present, alkaline proteases are mainly derived from Bacillus licheniformis, Bacill...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/50C12N15/57C12N15/75C12N1/21
CPCC12N9/50C12N15/75
Inventor 刘丹妮
Owner 横琴仲泰生物医药有限公司
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