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Chitosanase gene csnbaa, chitosanase and preparation method and application of chitosanase

A chitosanase and gene technology, applied in the field of genetic engineering, can solve the problems of little research on chitosanase, achieve good application prospects and industrial value, reduce production costs, and improve stability.

Pending Publication Date: 2021-03-02
深圳润康生态环境股份有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the research on Bacillus chitosanase mainly focuses on Bacillus circulans and Bacillus subtilis, and there is little research on other Bacillus-derived chitosanases.

Method used

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  • Chitosanase gene csnbaa, chitosanase and preparation method and application of chitosanase
  • Chitosanase gene csnbaa, chitosanase and preparation method and application of chitosanase
  • Chitosanase gene csnbaa, chitosanase and preparation method and application of chitosanase

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preparation example Construction

[0046] Correspondingly, the embodiment of the present invention also provides a kind of preparation method of chitosanase CsnBaa, it comprises the steps:

[0047] S1. Provide chitosanase gene csnbaa, expression vector and expression strain;

[0048] S2, the chitosanase gene csnbaa is amplified, and the resulting amplified product is connected to the expression vector to obtain a recombinant expression vector;

[0049] S3. Transforming the recombinant expression vector into an expression strain to obtain a recombinant expression strain;

[0050] S4. Cultivate the recombinant expression strain to obtain chitosanase CsnBaa;

[0051] Wherein, the nucleotide sequence of chitosanase gene csnbaa is the nucleotide sequence shown in SEQ ID NO: 1, or the nucleotide sequence shown in SEQ ID NO: 1 is obtained through deletion, insertion or replacement with Nucleotide sequence of identical function; The amino acid sequence of chitosanase CsnBaa is the amino acid sequence shown in SEQ ID ...

Embodiment 1

[0079] The present embodiment provides chitosanase gene sequence analysis and codon optimization process, comprising the steps:

[0080] (11) The research on chitosanase derived from Bacillus is currently mainly focused on chitosanase from Bacillus circulus and chitosanase from Bacillus subtilis, while chitosanase from other Bacillus is less reported. In order to further excavate chitosanase derived from spores, it is necessary to analyze the genome sequences of different Bacillus species. In this example, by analyzing the genome sequence of Bacillus atrophaeus, it is found that there is a chitosanase gene in its genome, and the accession number of this gene in the NCBI database is CP024051.1. The gene is 825bp in full length and encodes 274 amino acids. The chitosanase gene belongs to the GH46 family and is a typical Bacillus chitosanase gene. The first 32 amino acids were found to be signal peptides through the prediction analysis of the signal peptide prediction software S...

Embodiment 2

[0083] This embodiment provides a construction process of a recombinant expression vector containing the chitosanase gene csnbaa obtained in Example 1. The recombinant expression vector uses pPICZαA as the expression vector. details as follows:

[0084] (13) A pair of primers csnbaa-F and csnbaa-R are designed according to the sequence of the synthetic gene csnbaa, and its nucleotide sequence is shown in SEQ ID NO:3-4 respectively, for amplifying the csnbaa gene;

[0085] (14) The gene csnbaa was obtained by PCR amplification, and the expression vector pPICZαA and the gene csnbaa were digested overnight with restriction enzymes EcoRI and XbaI, respectively, and the expression vectors pPICZαA and csnbaa digested overnight were purified and recovered, followed by a ligation reaction;

[0086] (15) Transfer the ligation reaction product into Escherichia coli Top10 by the heat shock method, and verify the recombinant transformant by bacterial liquid PCR;

[0087] (16) Insert the ...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a chitosanase gene csnbaa, chitosanase and a preparation method and application thereof. The nucleotide sequence of the chitosanase gene csnbaa is shown as SEQ ID NO: 1, and the chitosanase gene csnbaa is obtained by comprehensively optimizing the sequence of chitosanase derived from Bacillus atrophaeus.The amino acid sequence of the chitosanase CsnBaa is as shown in SEQ ID NO: 2, so that not only are the types and source ways of chitosanase enriched, but also the fermentation enzyme activity can reach 4630 U / mL, and the chitosanase CsnBaa shows good stability in a relatively wide temperature range and a relatively wide pH range and has good application prospect and industrial value.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to chitosanase gene csnbaa, a corresponding recombinant expression vector, a recombinant expression strain, chitosanase CsnBaa, a preparation method and application thereof. 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 chitosan oligosaccharides has many advantages, such as mild reaction conditions, complete product structure, easy control of the process, and no pollutio...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/42C12N15/81C12N1/19C12P19/26C12P19/14C12R1/84
CPCC12N9/2434C12N15/815C12P19/26C12P19/14C12Y302/01132
Inventor 王建荣祝木金王平孔建王永华余思曹革
Owner 深圳润康生态环境股份有限公司
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