Pestalotiopsis fungus chitin deacetylase and preparation method and application thereof

A technology of polychaetesis and deacetylase, which is applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of limited development, lack of chitin deacetylase preparations, etc., and achieve wide application Effect

Active Publication Date: 2020-10-30
ZHONGKE RUNXIN SUZHOU BIOLOGICAL TECH CO LTD
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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 prepar...

Method used

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  • Pestalotiopsis fungus chitin deacetylase and preparation method and application thereof
  • Pestalotiopsis fungus chitin deacetylase and preparation method and application thereof
  • Pestalotiopsis fungus chitin deacetylase and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Codon optimization and total gene synthesis of chitin deacetylase gene

[0038] On the premise of not changing the amino acid sequence, using the preferred codon of Pichia pastoris, artificially designed the chitin deacetylase of the fungus of the genus Discochlein sp. (as shown in the sequence SEQ ID NO.1, GenBank accession number: APH81274 .1) the coding gene, see SEQ ID NO.2 for the specific nucleotide sequence. 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 82%. 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 pescda.

Embodiment 2

[0039] The expression vector construction of embodiment 2 chitin deacetylase gene pescda

[0040]The signal peptide sequence shown in SEQ ID NO.4 in the expression vector pPIC9 is codon-optimized to obtain a signal peptide sequence suitable for expression in Pichia pastoris as shown in SEQ ID NO.5; using Nsi I / XhoI double enzyme Cut, and replace the sequence shown in SEQ ID NO.4 in the expression vector pPIC9 with the signal peptide sequence shown in SEQ ID NO.5 to obtain the expression vector pGBG1.

[0041] Use restriction endonucleases Xho I and Not I to double-enzyme digest the cloning vector containing chitin deacetylase gene pescda 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 pescda-pGBG1. In order to confirm that the target chitin deacetylase gene has been constructed into the vector, the reco...

Embodiment 3

[0042] Example 3 Screening of chitin deacetylase Pichia pastoris engineered bacteria and preparation of chitin deacetylase

[0043] After the obtained recombinant plasmid pescda-pGBG1 was linearized by the restriction endonuclease Bgl I, the nucleic acid fragment containing the target gene was recovered by gel electrophoresis, introduced into Pichia pastoris GS115 by electroporation, and obtained by screening on the histidine auxotrophic MD plate Recombinant. A single colony was picked and inoculated in 25 mL of BMGY medium, cultured at 30°C and 250 rpm for 48 hours, the supernatant was discarded by centrifugation, and an equal amount of BMMY was added to induce expression. Methanol was added after 24 hours to a final concentration of 1%, and added once every 24 hours thereafter for a total of 72 hours of induction, followed by centrifugation, and SDS-PAGE was used to detect the expression of the target protein in the supernatant of the fermentation broth. see figure 2 , is...

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Abstract

The invention discloses a pestalotiopsis fungus chitin deacetylase and a preparation method and application thereof. According to codon preference of pichia pastoris, an optimized nucleic acid sequence is shown as SEQ ID NO.2, and a chitin deacetylase encoding gene sequence of the pestalotiopsis fungus is obtained by using a whole-gene synthesis method. Furthermore, a pichia pastoris expression system is used for carrying out secretory expression on the optimized chitin deacetylase encoding gene to obtain chitin deacetylase, and the amino acid sequence of the chitin deacetylase is shown as SEQID NO.1. The chitin deacetylase obtained by the invention can be used for removing acetyl in chitin, chitosan and chitosan oligosaccharide, so that chitosan or chitosan oligosaccharide with a specific structure is obtained. These chitosan or chitosan oligosaccharides of specific structures may have a new or higher biological activity. Therefore, the pestalotiopsis fungus chitin deacetylase and the deacetylation product thereof 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 Pestalotiopsis sp. 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 biological a...

Claims

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Application Information

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IPC IPC(8): C12N9/80C12N15/55C12N15/81C12P19/26C12R1/84
CPCC12N9/80C12N15/815C12P19/26C12Y305/01041C12N2800/22
Inventor 任立世冯翠张毓宸焦思明王颖程功孙明杜昱光
Owner ZHONGKE RUNXIN SUZHOU BIOLOGICAL TECH CO LTD
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