Chitin deacetylase derived from saccharomyces cerevisiae, encoding gene and applications

A technology of deacetylase and Saccharomyces cerevisiae, applied in the directions of application, genetic engineering, plant genetic improvement, etc., can solve the problems such as chitin deacetylase that have not been reported yet, and achieve the effect of a wide range of pH adaptation

Inactive Publication Date: 2019-05-07
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

At present, the research on thermophilic enzymes is still in its infancy, and no one has reported chitin deacetylases with hyperthermophilic and high temperature resistance properties.

Method used

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  • Chitin deacetylase derived from saccharomyces cerevisiae, encoding gene and applications
  • Chitin deacetylase derived from saccharomyces cerevisiae, encoding gene and applications
  • Chitin deacetylase derived from saccharomyces cerevisiae, encoding gene and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Full-length cloning of chitin deacetylase gene.

[0036] The target gene was upregulated from NCBI, and the target gene was synthesized in Zhongmei Taihe Biotechnology (Beijing) Co., Ltd. After performing multiple sequence alignment analysis on chitin deacetylases of the carbohydrate esterase family 4 (CE4), primers CDA-F: 5'-CCATGG GAAGCTAATAGGGAAGATTTA-3'; CDA-R5' were designed -CCGCTCGAGGGACAAGAATTCTTTTATGT AATC-3'. PCR amplification was performed using the synthesized DNA as a template. The PCR reaction conditions were: 94°C for 5 min, 1 cycle; 94°C for 30 s, 45°C for 30 s, 72°C for 1 min 30 s, 30 cycles; 72°C for 10 min, 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.

Embodiment 2

[0037] Embodiment 2 Chitin deacetylase gene complete sequence analysis

[0038] The sequencing results were analyzed using the Basic Local Alignment Search Tool (BLAST) in the GenBank database, and the Vector NTI Suite 8.0 software was used for multiple sequence alignments to analyze their homology. The domains of the sequences were analyzed using the SimpleModular Architecture Research Tool (SMART) online tool.

[0039] The obtained chitin deacetylase (named ScCDA 2 ) coding region is 861bp long, and its nucleotide sequence is shown in SEQ ID NO 1. The nucleotide sequence of ScCDA2 has the highest identity (100%) with the deacetylated protein gene (accession number CP020202.1) derived from Saccharomyces cerevisiae strainY12chromosome XII sequence. cCDA2 encodes 287 amino acids and a stop codon, its amino acid sequence is shown in SEQ ID NO 2, the protein molecular weight is 34kDa, and the predicted isoelectric point is 7.0. SMART analysis showed that the protein domain cha...

Embodiment 3

[0040] Example 3 ScCDA 2 The recombinant expression of the gene in Pichia pastoris uses the synthesized DNA as a template, and uses the designed primers CDA-F: 5'-CCATGGGAAGCTAATAGGGAAGATTTA-3'; CDA-R5'-CCGCTCGAGGGACAAGAATTCTTTTATGTAATC-3' to amplify the target gene sequence. Construct the target gene on the PMD-19T cloning vector, spread it on the solid plate of Luria-Bertani medium containing 100 μg / mL ampicillin, culture at 37°C for 14 hours, and pick a single clone; Ampicillin was cultured in liquid Luria-Bertani medium, and the plasmid was extracted; the plasmid was verified by colony PCR with the forward primer ScCDA-F and the reverse primer ScCDA-R, and the result was an amplified product of the correct size, which preliminarily proved that the constructed recombinant The plasmid is correct; then the recombinant plasmid is sent to Yingwei Jieji Company for sequencing, and the results show that the ScCDA shown in SEQ ID NO 1 is inserted between the Nco I and Xho I sites ...

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Abstract

The invention relates to clone expression and applications of chitin deacetylase derived from saccharomyces cerevisiae. The amino acid sequence of the saccharomyces cerevisiae chitin deacetylase is asshown in SEQ ID NO.2; and the nucleotide sequence of the saccharomyces cerevisiae chitin deacetylase is as shown in SEQ ID NO.1. The invention also provides a method for preparing the chitin deacetylase, namely cloning the gene of the chitin deacetylase onto a pichia pastoris expression vector pPICZ alpha by utilizing a genetic engineering technical method so as to obtain a pichia pastoris recombinant strain capable of expressing the chitin deacetylase in a heterologous way. The chitin deacetylase ScCDA2 prepared by the heterologous expression of the strain has wide pH adaptation range and has the characteristic of a single-minded deacetylation mode, and can be used for treating chitin and chitosan oligosaccharide of different sources such as shrimp and crab shells. The chitin deacetylaseprovided by the invention can be widely applied to the aspects of food science, biological medicines and chemical engineering materials.

Description

technical field [0001] The invention relates to a chitin deacetylase coding gene in Saccharomyces cerevisiae, which is hyperthermophilic, high temperature resistant, and has a wide range of pH adaptability, as well as its preparation and application. The invention also provides the recombinant plasmid and recombinant genetic engineering strain of the chitin deacetylase. Chitin deacetylase ScCDA of the present invention 2 Can be widely used in food science, biomedicine, chemical materials and other aspects. 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 biocompa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/80C12N15/55C12P19/26
Inventor 尹恒朱先玉王文霞赵勇
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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