Chitosanase gene, chitosanase as well as preparation method and application of chitosanase

A technology of chitosanase and gene, applied in the field of genetic engineering, can solve the problems of insufficient research on fungal chitosanase, achieve good stability and codon adaptability, good application prospects and industrial value, and improve sequence stability Effect

Pending Publication Date: 2020-11-06
深圳润康生态环境股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide polycanase gene csn and corresponding recombinant expression vector, recombinant expression bacterial strain,

Method used

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  • Chitosanase gene, chitosanase as well as preparation method and application of chitosanase
  • Chitosanase gene, chitosanase as well as preparation method and application of chitosanase
  • Chitosanase gene, chitosanase as well as preparation method and application of chitosanase

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Example Embodiment

[0045] Correspondingly, the embodiment of the present invention also provides a preparation method of chitosanase Csn, which includes the following steps:

[0046] S1. Provide chitosanase gene csn, expression vector and expression strain;

[0047] S2. Amplify the chitosanase gene csn, and connect the amplified product to the expression vector to obtain a recombinant expression vector;

[0048] S3. Transform the recombinant expression vector into an expression strain to obtain a recombinant expression strain;

[0049] S4. Culturing the recombinant expression strain to obtain chitosanase Csn;

[0050] Among them, the nucleotide sequence of the chitosanase gene csn is shown in SEQ ID NO:1.

[0051] In the method for preparing chitosanase Csn provided by the embodiment of the present invention, the chitosanase gene csn is amplified to obtain the corresponding recombinant expression vector and recombinant expression strain, and then the chitosanase Csn is obtained by culturing. The method ha...

Example Embodiment

[0075] Example 1

[0076] This embodiment provides the chitosanase CsnLr gene sequence analysis and codon optimization process, including the following steps:

[0077] By consulting the glycoside hydrolase bioinformatics database (http: / / www.cazy.org / ), it is found that the chitosanase GH46 family has the most sequence and related information. There are currently 852 chitosanases recorded in the GH46 family. Among them, 6 species are derived from archaea, 784 species are derived from bacteria, 60 species are derived from viruses and 2 species are derived from fungi. According to the results of literature review, the current recombinant expression and characterization of GH46 family chitosanase are of bacterial origin, and fungal chitosanase has not been reported. In view of the above analysis, the embodiment of the present invention hopes to express fungal-derived chitosanase through recombinant expression to lay the foundation for the excavation of GH46 family fungal-derived chit...

Example Embodiment

[0079] Example 2

[0080] This embodiment provides a construction process of a recombinant expression vector containing the chitosanase gene csn, which is specifically as follows:

[0081] (1) Design a pair of primers CSN-F and CSN-R according to the sequence of the chitosanase gene csn, the nucleotide sequence of which is shown in SEQ ID NO: 3-4, used to amplify the chitosanase gene csn ;

[0082] (2) The chitosanase gene csn was obtained by PCR amplification, and the expression vector pPICZαA and chitosanase gene csn were digested overnight with restriction enzymes EcoRI and XbaI, and the expression vector pPICZαA and chitosan were digested overnight. Carbohydrase gene csn is purified and recovered and then ligated;

[0083] (3) Use heat shock method to transfer the ligation reaction product into E. coli Top10, and use bacterial liquid PCR to verify the recombinant transformant;

[0084] (4) The successfully verified transformants are connected to the LBZ liquid medium, the plasmid ...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a chitosanase gene, chitosanase as well as a preparation method and application of the chitosanase. A nucleotide sequence of the chitosanase gene csn is as shown in SEQ ID NO: 1, and the chitosanase gene csn is obtained by comprehensively optimizing the sequence of the chitosanase from lichtheimia ramosaaccording to the preference of pichia pastoris codons. An amino acid sequence of the chitosanase Csn is as shown in SEQ ID NO: 2. The chitosanase Csn is fungal chitosanase, not only enriches the variety and source ways of the chitosanase, but also has relatively high enzymatic activity, shows good stability in relatively wide temperature and pH ranges, and has good application prospects and industrial values.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to chitosanase gene csn, corresponding recombinant expression vector, recombinant expression strain, chitosanase Csn and its preparation method and application. Background technique [0002] As a biologically active substance, oligochitosan is composed of glucosamine and N-acetylglucosamine connected randomly through β-1,4 glycosidic bonds. Studies have shown that chitosan has antibacterial, anti-tumor, and immune-enhancing effects, so it is widely used in agriculture, animal husbandry, food, and medicine. At present, the preparation of oligochitosan is mainly divided into physical method, chemical method and enzymatic method. Compared with physical and chemical methods, enzymatic preparation of chitooligosaccharides has many advantages such as mild reaction conditions, complete product structure, easy control of the process, and no pollution to the environm...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/24C12N15/81C12N1/19C12P19/26C12R1/84
CPCC12N9/2402C12N15/815C12P19/26C12Y302/01132C12N2800/22
Inventor 王建荣祝木金余思王平孟建军刘小慧曹革
Owner 深圳润康生态环境股份有限公司
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