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Chitin deacetylase and application thereof

A technology of vegetable deacetylase and vegetable deacetylase, which is applied in the field of chitin deacetylase, can solve the problems of optimal temperature difference, inadaptability to industrial material reaction conditions, etc., achieve low production conditions, reduce production costs, and reduce environmental pollution Effect

Active Publication Date: 2019-04-12
JILIN COFCO BIOCHEM +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The CDA derived from fungi found so far are basically single-peptide chain glycoproteins, showing good thermal stability, but the position, optimum pH value, optimum temperature, molecular weight, isoelectricity, etc. point and the reaction to metal ions and acetic acid are quite different
In addition, the optimum pH of the CDA (hereinafter also referred to as CliCDA) derived from the reported plant pathogenic bacterium Bean anthracnose is a slightly alkaline environment, which is not suitable for the reaction conditions of industrial materials (slightly acidic)
[0006] In summary, the activity of CDA found so far (especially the activity under acidic conditions) still cannot fully meet the actual needs of industrialized production of chitosan, therefore, screening highly active CDA is still a need in industrialized applications. important issues to solve

Method used

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  • Chitin deacetylase and application thereof
  • Chitin deacetylase and application thereof
  • Chitin deacetylase and application thereof

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Embodiment approach

[0043] According to a preferred embodiment of the present invention, Escherichia coli transformed cells or Bacillus subtilis transformed cells can be obtained by the following method: using chemical transformation method, the expression vector of the present invention is transformed into Escherichia coli competent cells or Bacillus subtilis competent cells cells; then the bacterial suspension was spread on the plate and cultured until a single colony appeared.

[0044] Five. The test kit of the present invention

[0045] The kit of the present invention is a kit comprising one or more of the chitin deacetylase of the present invention, the DNA molecule encoding the chitin deacetylase of the present invention, the expression vector of the present invention and the transformed cell of the present invention .

[0046] The kit can comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syri...

Embodiment 1

[0058] Design and screening of embodiment 1 chitin deacetylase

[0059] Searching for potential thermostable sugar isomerases in the NCBI database, and after sequence alignment analysis and strain source investigation, an unknown protein from Verticillium dahliae Vlo was found, whose amino acid sequence is shown in SEQ ID NO: 1 As shown, it is speculated that it has deacetylase activity.

[0060] Furthermore, codon optimization is performed on the coding sequence of the amino acid sequence to obtain the nucleotide sequence shown in SEQ ID NO:2.

Embodiment 2

[0061] Example 2 Expression of chitin deacetylase in Escherichia coli

[0062] The nucleotide sequence shown in SEQ ID NO: 2 was synthesized by General Biosystems (Anhui) Co., Ltd., a BamH I restriction site was added at its 5' end, and a hexahistidine tag was added before the stop codon. coding sequence, and add Pst I and Not I restriction sites at the 3' end. The synthesized fragment was double-digested by BamH I and Not I (New England Biolabs, NEB), and the double-digested fragment was ligated with T4 DNA ligase (Takara) to a DNA that was also double-digested by BamH I and NotI pET24a(+) vector (General Biosystems (Anhui) Co., Ltd.). The ligation product was transformed into Escherichia coli DH5α competent cells (Beijing Quanshijin Biotechnology Co., Ltd.), cultured on LB solid plates with kanamycin, and positive clones were screened. Pick a single colony for colony PCR verification; and perform sequencing verification for clones that are positive for colony PCR.

[0063...

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Abstract

By analyzing and studying different genomic sequences of different species, several unknown proteins are screened out, and chitin deacetylase with excellent deacetylase activity is further identified.More specifically, the invention relates to a chitin deacetylase derived from verticillium longisporum Vlo. Compared with the reported chitin deacetylase, the chitin deacetylase has lower requirements for production conditions, has higher biosafety, and has characteristics more suitable for industrial applications, and shows wide application potential.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a chitin deacetylase derived from Verticillium longisporum VL1 and application thereof. Background technique [0002] Chitin, also known as chitin, is a straight-chain polymer aminopolysaccharide composed of N-acetylamino-D-glucose monomers (D-GlcNAc) linked by β-1,4 glycosidic bonds, mainly found in invertebrates (especially the shells of crustaceans such as shrimps, crabs, and insects), seaweeds (such as green algae), fungi (such as molds), etc., are another important class of polysaccharides besides cellulose. However, chitin is insoluble in water, dilute acid, alkali, ethanol or other organic solvents, so its utility value is greatly limited, and the product chitosan after deacetylation depends on the degree of deacetylation and can be dissolved in acid and neutral aqueous solution. Because chitosan has excellent characteristics such as biological fu...

Claims

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Application Information

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IPC IPC(8): C12N9/80C12N15/55C12N15/70C12N15/75C12N1/21C12P19/26C12R1/125C12R1/19
CPCC12N9/80C12N15/70C12N15/75C12P19/26C12Y305/01041
Inventor 佟毅沈雪梅王靖张媛刘颖慰王小艳陈博彭超李义周勇卢宗梅满云
Owner JILIN COFCO BIOCHEM
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