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Chitin deacetylase, encoding gene and application

A deacetylase, encoding gene technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problems of difficult separation, harsh reaction conditions, serious environmental pollution and other problems

Active Publication Date: 2020-05-15
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method not only requires harsh reaction conditions, but also produces a large number of by-products, serious environmental pollution, and technical defects such as separation difficulties.

Method used

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  • Chitin deacetylase, encoding gene and application
  • Chitin deacetylase, encoding gene and application
  • Chitin deacetylase, encoding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1: Phylogenetic tree construction

[0024] Penicillium (American Strain Classification Number Penicillium oxalicum 114-2, Taxonomy ID: 933388) Penicillium sp.1805 Isolation and Purification Fungal Genome Extraction Kit (Manufacturer: Sangon, Model: B518229-0050), Genome Extraction, and ITS1 and ITS4 The primers were cloned, the fungi were identified, and the sequencing results were compared on NCBI, and the sequences with close relatives were downloaded to construct the phylogenetic tree through MEGA7.

Embodiment 2

[0025] Example 2: Full-length cloning of chitin deacetylase gene.

[0026] RNA was extracted from Penicillium oxalicum (Penicillium oxalicum 114-2, Taxonomy ID: 933388), reverse-transcribed to form cDNA, and primers CDA-F: 5'-CTAGTCTAGAATGGCCCCCAAGAGAGTATTG-3'; CDA-R5'-CCGCTCGAGCTACTTCTTGAGCATCTGACC- 3'. The reverse transcribed cDNA was used as a template for PCR amplification. The PCR reaction conditions were: 94°C for 5min, 1 cycle; 94°C for 30s, 45°C for 30s, 72°C for 1min30s, 30 cycles; 72°C for 10min, 1 cycle. After the PCR product was analyzed by agarose gel electrophoresis, the target fragment was recovered by cutting the gel, connected to the pMD19-T vector and then sequenced.

[0027] Sequence listing:

[0028]Information on SEQ ID No.1

[0029] (a) Sequential features

[0030] Length: 927 nucleotides

[0031] Type: Nucleotide

[0032] Chain type: single chain

[0033] (b) Molecule type: DNA

[0034] Sequence description: The gene encoding chitin deacetylase ...

Embodiment 3

[0046] Embodiment 3: the heterologous expression of target gene

[0047] The PCR amplified product and the pET-22b vector plasmid were double-digested with BamHI and HindIII restriction endonucleases, digested at 37°C for 12h, and the fragment was ligated to the pET-22b vector plasmid, and ligated at 16°C for 18h. Take 5 μL of the ligation product and transfer it into E.coliDH5α, select a single clone, and perform bacterial liquid PCR detection and sequencing verification to obtain the recombinant plasmid psccda-PET-22b.

[0048] The recombinant plasmid extracted and sequenced correctly was transferred into Escherichia coli BL21 (DE3) expression-competent cells, and monoclonal PCR was selected for verification. The correct single clone was cultured in 10 mL LB medium containing ampicillin with a final concentration of 100 μg / ml for 12 hours as the seed solution. The seed solution was added to 1L fermentation medium at a volume ratio of 1:100, induced for 12 hours at 16°C, cen...

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Abstract

The invention relates to chitin deacetylase and an encoding gene, as well as preparation and application thereof. The invention further provides a method for preapring the chitin deacetylase. The method comprises the following steps: cloning a gene of the chitin deacetylase onto an escherichia coli expression vector PET-22b by utilizing a genetic engineering technical method to obtain an escherichia coli recombinant strain capable of heterogeneously expressing the enzyme. By using the chitin deacetylase PsCDA prepared by heterogeneous expression of the strain, chitin tetrasccharide (A4) can perform specific deacetylation to generate chitin oligose (A3D1) having the deacetylation degree of 75 percent and chitosan oligosaccharide (A2D2) having the deacetylation degree of 50 percent. The chitin deacetylase can act on chitin and chitin oligose from different sources, such as shrimp and crab shells and the like. The chitin deacetylase can be widely applied to the aspects of food science, biomedicine, chemical materials and the like.

Description

technical field [0001] The invention relates to a gene encoding chitin deacetylase in Penicillium oxalicum (American strain classification number Penicillium oxalicum 114-2, TaxonomyID: 933388) and a patent specification for its preparation and application. The invention also provides the recombinant plasmid and recombinant genetic engineering strain of the chitin deacetylase. The chitin deacetylase PsCDA of the invention can be widely used in food science, biomedicine, chemical materials and the like. Background technique [0002] Chitin is the second largest renewable natural resource next to cellulose in nature and widely exists in the ocean. (Chen Shaobo, Wu Genfu. Science and Technology Bulletin, 2004, (03): 258-262.) Chitosan is the deacetylation product of chitin, and is the only alkaline polysaccharide that exists in nature. Its molecular formula is (C6H11NO4)n, and it has Broad-spectrum antibacterial properties, water retention, reproducibility, good biocompatibil...

Claims

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Application Information

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IPC IPC(8): C12N9/80C12N15/55C12P19/04C12P19/00
CPCC12N9/80C12Y305/01041C12P19/04C12P19/00
Inventor 尹恒朱先玉
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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