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Referred to a few butadase CHI304 mutant and its preparation methods and applications

A technology of chitinase and mutants, applied in botany equipment and methods, biochemical equipment and methods, applications, etc., can solve the problem of low enzyme activity and expression, poor thermal stability of enzymes, and failure to reach industrial applications, etc. problem, to achieve the effect of improving conversion ability

Active Publication Date: 2021-03-19
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Most of the currently known chitinases are isolated from cultivable microorganisms by traditional methods. Due to the limitation of microbial growth conditions, the isolated enzymes have poor thermal stability, low enzyme activity and expression, and cannot reach industrialization. application requirements

Method used

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  • Referred to a few butadase CHI304 mutant and its preparation methods and applications
  • Referred to a few butadase CHI304 mutant and its preparation methods and applications
  • Referred to a few butadase CHI304 mutant and its preparation methods and applications

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Embodiment 1

[0045] Example 1 Design of Chi304 single point mutant, construction and induced expression of single point mutant

[0046] (1) Design of Chi304 single point mutant

[0047] Chi304 is the thermophilic chitinase (the amino acid sequence of which is shown in SEQ ID No.1) screened from the marine metagenomics in the inventor's laboratory. In order to further improve its comprehensive properties, the gene consensus method was used to identify Among them, the key amino acid sites are mutated, and the specific method is as follows:

[0048] Firstly, the Chi304 amino acid sequence was used to perform Blast comparison on the National Center for Biotechnology Information (NCBI), and the download e value was less than 10 -4 sequence, and then use the multiple sequence alignment software ClustualW to perform homologous alignment on these sequences, and use the inventor's laboratory PIRD website (http: / / www.elabcaas.cn / pird / premuse.html) to calculate each amino acid position Point the oc...

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Abstract

The invention discloses a thermophilic chitinase Chi304 mutant, a preparation method and application thereof. The present invention designs a series of single-point mutants whose amino acid sequence is chitinase Chi304 shown in SEQ ID No.1 through gene consensus, and finally successfully screens and obtains the single-point mutant Y166F with improved enzyme activity and thermal stability, as well as its expression, Single point mutant D421E with improved temperature optimum, thermostability and substrate binding ability. In addition, the substrate conversion ability of the two single point mutants, Y166F and D421E, was significantly improved compared with the wild type. The invention lays a theoretical foundation for the industrial high-temperature enzymatic degradation of chitin, and creates conditions for the industrial application of chitinase.

Description

technical field [0001] The present invention relates to chitinase mutants, in particular to a single-point mutant of thermophilic chitinase Chi304, and the invention also relates to a preparation method of thermophilic chitinase Chi304 mutant and the ability of the mutant to degrade chitin The application in the invention belongs to the field of chitinase mutants and applications. Background technique [0002] Chitin, also known as chitin and chitin, is a natural polymer polysaccharide polymerized by N-acetylglucosamine through β-1,4 glycosidic bonds. It is a white or off-white amorphous solid under natural conditions. Usually in the range of 30-3000kDa, the acetylation degree is over 90%. It is widely distributed in the shells of shrimp and crabs, fungi and algae cell walls of seafood. It is the most important renewable nitrogen and carbon source in the growth and reproduction of marine microorganisms. The content in nature It is second only to cellulose. [0003] With th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56
CPCC12N9/2442C12Y302/01014C12N15/70
Inventor 关菲菲伍宁丰田健刘晓青张岩田晓倩
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI