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Chitinase gene, chitinase and preparation method and application of chitinase

A chitinase gene and a chitinase technology, applied in the field of genetic engineering, can solve problems such as low fermentation activity, and achieve the effects of simple steps, good stability and good industrial value

Inactive Publication Date: 2020-11-27
深圳润康生态环境股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide chitinase gene chiam and corresponding recombinant expression vector, recombinant expression strain, and chitinase ChiAm and its preparation method and application, aiming to solve the low fermentation activity of existing chitinase technical issues

Method used

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  • Chitinase gene, chitinase and preparation method and application of chitinase

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preparation example Construction

[0044] Correspondingly, the embodiment of the present invention also provides a preparation method of chitinase ChiAm, which comprises the following steps:

[0045] S1. Provide chitinase gene chiam, expression vector and expression strain;

[0046] S2, amplifying the chitinase gene chiam, and connecting the obtained amplified product with an expression vector to obtain a recombinant expression vector;

[0047] S3. Transforming the recombinant expression vector into an expression strain to obtain a recombinant expression strain;

[0048] S4. Cultivate the recombinant expression strain to obtain chitinase ChiAm;

[0049] Wherein, the nucleotide sequence of chitinase gene chiam is shown in SEQ ID NO:1, and the amino acid sequence of chitinase ChiAm is shown in SEQ ID NO:2.

[0050] In the preparation method of chitinase ChiAm provided in the embodiment of the present invention, the method of heterologous recombination is adopted to amplify the chitinase gene chiam to obtain the...

Embodiment 1

[0074] This embodiment provides chitinase gene sequence analysis and codon optimization process, including the following steps:

[0075] (11) By analyzing the genome data of Amycolatopsis mediterranei in NCBI (GenBank: CP003729.1), it was found that the nucleotides in the interval 7823925-7825130 of its genome are the coding gene of chitinase ChiAm. The full length of the gene is 1206bp, encoding 401 amino acids (the amino acid sequence is shown in SEQ ID NO.2). The first 32 amino acids of chitinase ChiAm were found to be its signal peptide by the signal peptide prediction software SignalP-5.0Server prediction analysis.

[0076] (12) Due to the difference in gene coding between Amycolatopsis mediterranei and Pichia pastoris, chitinase ChiAm needs to be optimized according to the codon preference of Pichia pastoris. The optimized chitinase ChiAm coding gene is named chiam (the nucleotide sequence is shown in SEQ ID NO.1). Compared with the original gene, the GC content of chi...

Embodiment 2

[0078] This embodiment provides a construction process of a recombinant expression vector containing the chitinase gene chiam obtained in Example 1. The recombinant expression vector uses pPICZαA as the expression vector, and the signal peptide used is the α signal peptide carried by pPICZαA. Therefore, in The signal peptide of chiam itself needs to be removed during the construction process. details as follows:

[0079] (13) Design a pair of primers chiam-F and chiam-R according to the sequence of the synthetic gene chiam, the nucleotide sequences of which are shown in SEQ ID NO:3-4 respectively, for amplifying the chiam gene for removing the signal peptide;

[0080] (14) Obtain the gene chiams that removes the signal peptide by PCR amplification, digest the expression vector pPICZαA and gene chiams with restriction endonucleases EcoRI and XbaI respectively overnight, purify and recover the expression vector pPICZαA and chiams digested overnight ligation reaction;

[0081] ...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a chitinase gene, chitinase and a preparation method and application of the chitinase. The nucleotide sequence of the chitinase gene chiam is shown as SEQ ID NO: 1, and the chitinase gene chiam is obtained by comprehensively optimizing the sequence of the chitinase derived from the Amycolatopsis mediterranei. The amino acid sequence of the chitinase ChiAm is shown as SEQ ID NO: 2. The types and source ways of the chitinase are enriched, the enzyme specific activity is 48U / mg, the chitinase has good stability in a wide temperature range and a wide pH range, and the chitinase has good application prospects and industrial value.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a chitinase gene chiam, a corresponding recombinant expression vector, a recombinant expression strain, a chitinase ChiAm and a preparation method and application thereof. Background technique [0002] Chitin is a straight-chain polymer composed of N-acetyl-β-D-glucosamine randomly linked by β-1,4 glycosidic bonds, and its structure is similar to cellulose. Chitin is a renewable polysaccharide second only to cellulose in nature, and it is also one of the most abundant natural polymer compounds. Chitin oligosaccharide, the hydrolyzed product of chitin, has good physiological functions as a biologically active substance, and can be used in biomedicine, food processing, agricultural planting, animal husbandry and other industries. At present, the preparation of chitooligosaccharides is mainly divided into physical method, chemical method and enzymatic method....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/42C12N15/81C12N1/19C12P19/26C12P19/14C12R1/84
CPCC12N9/2442C12N15/815C12P19/26C12P19/14C12Y302/01014C12N2800/22
Inventor 王建荣祝木金王平余思曹革
Owner 深圳润康生态环境股份有限公司
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