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Puccinia graminis chitin deacetylase as well as preparation method and application thereof

A deacetylase, chitin technology, applied in the directions of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve problems such as limited development and lack of chitin deacetylase preparations

Pending Publication Date: 2020-11-03
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 preparations for industrial use, which limits the development of this field

Method used

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  • Puccinia graminis chitin deacetylase as well as preparation method and application thereof
  • Puccinia graminis chitin deacetylase as well as preparation method and application thereof
  • Puccinia graminis chitin deacetylase as well as 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, the chitin deacetylase of Puccinia graminearum was artificially designed (as shown in the sequence SEQ ID NO.1, GenBank accession number: XP_003323413.1 ) 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_003323365.2) is 78%. The optimized gene sequence was entrusted to Beijing Qingke Xinye Biotechnology Co., Ltd. for the total synthesis, and the synthesized gene sequence was named the chitin deacetylase gene pgtcda.

Embodiment 2

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

[0040]The signal peptide sequence shown in SEQ ID NO.4 in the expression vector pPIC9 was codon optimized to obtain a signal peptide sequence suitable for expression in Pichia pastoris as shown in SEQ ID NO.5; using Nsi I / Xho I double Restriction digestion, replacing 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] The cloning vector containing the chitin deacetylase gene pgtcda was double-digested with restriction endonucleases Xho I and Not I to obtain the target gene fragment, and the same endonuclease was used to double-digest the expression vector pGBG1, Recycle large fragments. The two recovered products were connected to obtain a recombinant vector named pgtcda-pGBG1. In order to confirm that the target chitin deacetylase gene has been constructed into the vecto...

Embodiment 3

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

[0043] After the obtained recombinant plasmid pgtcda-pGBG1 was linearized by restriction endonuclease Sac 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 the...

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Abstract

The invention discloses a puccinia graminis chitin deacetylase as well as 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 puccinia graminis is obtained by utilizing a whole-gene synthesis method. Furthermore, a pichia pastoris expressionsystem is used for outperforming secretory expression on the optimized chitin deacetylase encoding gene to obtain chitin deacetylase, and the amino acid sequence of the chitin deacetylase is shown asSEQ ID NO.1. The obtained chitin deacetylase disclosed by the invention can be used for removing acetyl in chitin, chitosan and chitosan oligosaccharide, so that the chitosan or the chitosan oligosaccharide adopting a specific structure is obtained. The chitosan or the chitosan oligosaccharides adopting specific structures can have new or higher biological activity. Therefore, the puccinia graminis chitin deacetylase and a 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 Puccinia graminis 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 ac...

Claims

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Application Information

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IPC IPC(8): C12N9/80C12N15/55C12N15/81C12P19/26C12P19/00C12P19/04C12P19/12C12R1/84
CPCC12N9/80C12Y305/01041C12N15/815C12P19/26C12P19/00C12P19/04C12P19/12
Inventor 焦思明任立世张毓宸冯翠王颖程功孙明杜昱光
Owner ZHONGKE RUNXIN SUZHOU BIOLOGICAL TECH CO LTD
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