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Aspergillus nidulans chitin deacetylase, and preparation method and application thereof

A technology of deacetylase and Aspergillus nidulans, applied in the field of Aspergillus nidulans chitin deacetylase and its preparation, can solve the problems of limited development, lack of chitin deacetylase preparations, etc., and achieve the effect of high biological activity

Inactive Publication Date: 2018-12-18
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is still a lack of chitin deacetylase preparations for industrial use, which limits the development of this field

Method used

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  • Aspergillus nidulans chitin deacetylase, and preparation method and application thereof
  • Aspergillus nidulans chitin deacetylase, and preparation method and application thereof
  • Aspergillus nidulans chitin deacetylase, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Codon optimization and total gene synthesis of chitin deacetylase gene

[0032]On the premise of not changing the amino acid sequence, the chitin deacetylase of Aspergillus nidulans was artificially designed using the preferred codon of Pichia pastoris (as shown in the sequence SEQ ID NO.1, GenBank accession number: XP_682649.1) The coding gene, the specific nucleotide sequence is shown in SEQ ID NO.2. The homology between the optimized nucleotide sequence and the original coding gene sequence (as shown in SEQ ID NO.3, GenBank accession number: XM_677557.1) is 72%. The optimized gene sequence was entrusted to Beijing Qingke Xinye Biotechnology Co., Ltd. for the total synthesis, and the synthesized gene sequence was named chitin deacetylase gene ancdal.

Embodiment 2

[0033] Example 2 Construction of expression vector of chitin deacetylase gene ANCDA1

[0034] The signal peptide sequence shown in SEQ ID NO.4 in the expression vector pPIC9 was replaced by the signal peptide sequence shown in SEQ ID NO.5 by using Nsi I / Xho I double enzyme digestion to obtain the expression vector pGBG1. Use restriction endonucleases Xho I and Not I to double-enzyme digest the cloning vector containing chitin deacetylase gene ancda1 to obtain the target gene fragment, and use the same endonuclease to double-enzyme digest the expression vector pGBG1, Recycle large fragments. The two recovered products were connected to obtain a recombinant vector named ancda1-pGBG1. In order to confirm that the target chitin deacetylase gene has been constructed into the vector, we use Xho I / Not I and Bgl II to perform double digestion and single digestion of the recombinant vector, and perform agarose gel electrophoresis on the product. The result is as figure 1 Shown: afte...

Embodiment 3

[0035] Example 3 Screening of chitin deacetylase Pichia pastoris engineered bacteria and preparation of chitin deacetylase

[0036] After linearizing the obtained recombinant plasmid ancda1-pGBG1 with restriction endonuclease Bgl II, gel electrophoresis was used to separate and excise the nucleotide fragment containing the target gene (such as figure 1 The larger fragment shown) was introduced into Pichia pastoris GS115 by electroporation, and the obtained recombinants were screened on the histidine auxotrophic MD plate. Pick 6 single colonies and inoculate them in 200mL BMGY medium, culture at 30°C and 250rpm for 48 hours, centrifuge to discard the supernatant, and add an equal amount of BMMY to induce expression. After 24 hours, methanol was added to a final concentration of 1%, and added every 24 hours thereafter. After a total of 120 hours of induction, centrifugation was performed, and SDS-PAGE was used to detect the expression of the target protein in the protein superna...

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Abstract

The invention discloses an Aspergillus nidulans chitin deacetylase, and a preparation method and an application thereof. The sequence of a chitin deacetylase coding gene in Aspergillus nidulans is obtained by a whole gene synthesis technology according to the codon preference of Pichia yeast, and the optimized nucleic acid sequence is represented by SEQ ID NO.2. The optimized chitin deacetylase encoding gene is secreted and expressed by a Pichia pastoris expression system to obtain the Aspergillus nidulans chitin deacetylase, and the amino acid sequence of the Aspergillus nidulans chitin deacetylase is represented by SEQ ID NO.1. The Aspergillus nidulans chitin deacetylase obtained in the invention can remove acetyl groups in chitosan and chitosan oligosaccharides in order to obtain the chitosan or chitosan oligosaccharides with a specific structure. The modified chitosan or chitosan oligosaccharides have new or higher biological activities than unmodified chitosan or chitosan oligosaccharides. So the above enzyme and its deacelation product have good industrial application prospects.

Description

technical field [0001] The invention belongs to the technical field of chitosan or chitosan oligosaccharide preparation, and in particular relates to a chitin deacetylase from Bradyrhizobium diazoefficiens and a preparation method and application thereof. Background technique [0002] Chitosan has a series of biological activities such as antibacterial and stimulating animal and plant immunity. At present, chitin is mainly obtained by chemical deacetylation with strong alkali, which is easy to cause environmental pollution. It is possible to obtain chitosan with controllable acetyl site distribution by directly using chitin deacetylase to deacetylate chitin, which is environmentally friendly. Oligochitosan is an oligomer produced by degrading chitin or its deacetylated product chitosan by physical, chemical or enzymatic methods. ) or a combination of the two through β-1,4 glycosidic bonds, usually with a degree of polymerization (DP)<20. Oligochitosan has a series of b...

Claims

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Application Information

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IPC IPC(8): C12N9/80C12N15/55C12N15/81C12P19/26
CPCC12N9/80C12N15/815C12P19/26C12Y305/01041
Inventor 杜昱光焦思明程功张毓宸冯翠任立世李建军未金花
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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