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Chitin deacetylase gene, chitin deacetylase and preparation method and application of chitin deacetylase

A deacetylase and chitin technology, applied in the field of genetic engineering, can solve problems such as low fermentation activity, and achieve the effects of high-efficiency expression, good stability and lower production costs

Pending Publication Date: 2021-01-05
深圳润康生态环境股份有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The object of the present invention is to provide chitin deacetylase gene cdaba and corresponding recombinant expression vector, recombinant expression strain, and chitin deacetylase CdaBa and its preparation method and application, aiming to solve the problem of existing chitin deacetylation The technical problem of low fermentation activity of enzymes

Method used

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  • Chitin deacetylase gene, chitin deacetylase and preparation method and application of chitin deacetylase
  • Chitin deacetylase gene, chitin deacetylase and preparation method and application of chitin deacetylase
  • Chitin deacetylase gene, chitin deacetylase and preparation method and application of chitin deacetylase

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preparation example Construction

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

[0045] S1. Provide chitin deacetylase gene cdaba, expression vector and expression strain;

[0046] S2. Amplifying the chitin deacetylase gene cdaba, 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 chitin deacetylase CdaBa;

[0049] Wherein, the nucleotide sequence of the chitin deacetylase gene cdaba is the nucleotide sequence shown in SEQ ID NO: 1, or the nucleotide sequence shown in SEQ ID NO: 1 through deletion, insertion or replacement The resulting nucleotide sequence has the same function; the amino acid sequence of chitin deacetylase CdaBa is th...

Embodiment 1

[0075] This embodiment provides chitin deacetylase gene sequence analysis and codon optimization process, including the following steps:

[0076] (11) Based on the many shortcomings of the natural bacteria Bacillus atrophaeus, it is proposed to improve its expression efficiency through heterologous recombination expression. Among them, the Pichia pastoris expression system was selected as the heterologous expression host. The first step of heterologous recombination expression is to find the Bacillus atrophaeus chitin deacetylase gene. Its whole genome sequence was submitted to the NCBI database in 2017, and its accession number is CP024051.1. By analyzing its genome data, it was found that the nucleotides in the interval 4031023-4031853 of its genome are the coding gene of chitin deacetylase CdaBa. The full length of the gene is 831bp, encoding 276 amino acids. The first 38 amino acids of chitin deacetylase CdaBa were found to be its signal peptide through the prediction a...

Embodiment 2

[0079] This example provides a construction process of a recombinant expression vector containing the chitin deacetylase gene cdaba 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, the signal peptide of cdaba itself needs to be removed during the construction process. details as follows:

[0080] (13) Design a pair of primers cdaba-F and cdaba-R according to the sequence of the synthetic gene cdaba, its nucleotide sequence is shown in SEQ ID NO:3-4 respectively, for amplifying the cdaba gene of removing signal peptide;

[0081] (14) Obtain the gene cdabas that removes the signal peptide by PCR amplification, digest the expression vector pPICZαA and the gene cdabas with restriction endonucleases EcoRI and XbaI respectively overnight, and purify and recover the expression vector pPICZαA and cdabas overnight. ligation reaction;

[0082] (15) Transfer...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a chitin deacetylase gene, chitin deacetylase and a preparation method and application of the chitin deacetylase. A nucleotide sequence of the chitin deacetylase gene cdaba is as shown in SEQ ID NO:1, and is obtained by comprehensively optimizing a sequence of the chitin deacetylase derived from Bacillusatrophaeus. An amino acid sequence of the chitin deacetylase CdaBa is as shown in SEQ ID NO:2, the varieties and source ways of the chitin deacetylase are enriched, the enzyme specific activity can reach 352 U / mg, good stability is shown in a relatively wide temperature range and a relatively wide pH range, and the chitin deacetylase CdaBa 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 chitin deacetylase gene cdaba, a corresponding recombinant expression vector, a recombinant expression strain, a chitin deacetylase CdaBa and a preparation method and application thereof. Background technique [0002] Chitin is a straight-chain polymer composed of N-acetyl-β-D-glucosamine randomly connected by β-1,4 glycosidic bonds. Its structure is similar to that of cellulose, and it is second only to cellulose in nature. regenerated polysaccharides. Due to its dense crystalline structure, chitin has poor solubility and is almost insoluble in water, dilute acid, dilute alkali, concentrated alkali and general organic solvents, which limits the commercial application of chitin. It has been found through research that chitosan, a derivative of chitin (the product of chitin deacetylation), can be well dissolved in dilute nitric acid, hydrochloric acid, ac...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N9/80C12N15/81C12N1/19C12P19/26C12R1/84
CPCC12N9/80C12N15/815C12P19/26C12Y305/01041
Inventor 王建荣王平祝木金余思孔建曹革
Owner 深圳润康生态环境股份有限公司
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