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Glucoside hydrolase CmNAGase and cloning expression and applications thereof

A technology of glycoside hydrolase and expression vector, which is applied in the field of glycoside hydrolase CmNAGase and its clone expression and application, which can solve the problems of high cost of waste treatment, generation of by-products, serious environmental pollution, etc.

Active Publication Date: 2019-07-19
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chemical method is difficult to control the reaction process, accompanied by by-products, and the biological activity of the product is poor.
In addition, a large amount of strong acid and strong alkali are involved in the production process, which seriously pollutes the environment and the cost of three wastes is high.

Method used

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  • Glucoside hydrolase CmNAGase and cloning expression and applications thereof
  • Glucoside hydrolase CmNAGase and cloning expression and applications thereof
  • Glucoside hydrolase CmNAGase and cloning expression and applications thereof

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Experimental program
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Embodiment 1

[0033] strain source

[0034] (1) The strain C. meiyuanensis SYBC-H1 used in the present invention was screened by our laboratory and preserved in China General Microorganism Collection (CGMCC 3438) and American Type Microorganism Collection (ATCC BAA-2140).

Embodiment 2

[0035] Example 2 Cloning of the glycoside hydrolase gene CmNAGase

[0036] (1) The gene of glycoside hydrolase CmNAGase, the nucleotide sequence of which is shown in SEN ID NO:1.

[0037] (2) PCR amplification primers CmF-5'-CATATGATGAGCCGTCCCGCCGGATC-3' and CmR-5'-CTCGAGTCAGGCGCCCACCTGCACCG-3' were designed to amplify the full length of the gene.

[0038] (3) The reaction conditions for the above PCR amplification are: pre-denaturation at 95°C for 5 minutes; 30 cycles at 94°C for 30 sec, 65°C for 30 sec, and 72°C for 40 sec; and finally extension at 72°C for 10 min.

[0039] (4) The PCR amplification product was double-digested with restriction endonucleases NdeI and XhoI, and the digested product was recovered.

[0040] (5) The vector pET-28(+)a was double digested with restriction enzymes NdeI and XhoI, and the vector backbone (about 5400bp) was recovered.

[0041] (6) Ligate the digested product of the above step with the vector backbone of step 4 to obtain the recombina...

Embodiment 3

[0042] The cloning expression of embodiment 3 glycoside hydrolase CmNAGase gene, comprises the following steps:

[0043] (1) The recombinant plasmid pET-28a(+)-Cmnagase was introduced into Escherichia coli BL21(DE3) to obtain Escherichia coli BL21(DE3) containing the recombinant plasmid pET-28a(+)-Cmnagase, which was named as recombinant bacteria.

[0044] (2) Select a single clone of recombinant bacteria (expressing pET-28a(+)-Cmnagase containing His tag), inoculate it into 5 mL LB medium containing 100 μg / mL kanamycin in a shaking shaker at 37°C and 200rmp Incubate overnight.

[0045] (3) Inoculate the above bacterial solution into 100mL LB liquid medium containing 100μg / mL kanamycin at a volume ratio of 1:100, and culture it at 37°C and 200rmp shaking until OD 600 Reach 0.6-0.8;

[0046](4) Then add IPTG (inducing agent) to a concentration of 1mmol / L, shake and culture at 20°C and 200rmp for 20h.

[0047] (5) Collect the above bacterial solution into a centrifuge tube, c...

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Abstract

The present invention discloses glucoside hydrolase CmNAGase and cloning expression and applications thereof. A strain Chitnitolyticbacter meiyuanensis SYBC-H1 which is screened from soil is subjectedto genome sequencing analysis, CmNAGase is cloned, a recombinant strain is constructed, CmNAGase protein is expressed, HIS-TAG is purified, and the invention identifies that the recombinant protein has the properties of glycoside hydrolase, remains good enzymatic activity under high temperature environment, belongs to an endonuclease, and can transfer an acetylglucosaminethe to the sugar chain ofchitobiose, several chitriose, chitotetraose, chitopentaose and chitohexaose separately so as to increase the length of the sugar chain. Through enzyme engineering and synthetic biology, molecular modification can be carried out to enable the enzymatic reaction direction of the enzyme to go towards the direction of increasing sugar chain, so that a foundation for synthesizing chitosan oligosaccharide is laid.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a glycoside hydrolase CmNAGase and its clone expression and application. Background technique [0002] Chitin, also known as chitin and chitin, is a polymer linked by N-acetylglucosamine through β-1,4-glycosidic bonds. It is the polysaccharide with the highest content on the earth except cellulose. Chitin widely exists in shrimp and crab shells, insect exoskeletons and fungal cell walls, and its final hydrolyzed product N-acetylglucosamine can protect cartilage tissue and bone joints. Chitinase can prevent and treat fungal diseases of plants, and is widely used in food, medicine, agriculture, cosmetics and other industries. [0003] The method for preparing N-acetylglucosamine (GlcNAc) with chitin as substrate can be mainly divided into chemical method and enzymatic method. At present, chemical method is mostly used in industry, that is, after removing acetyl group...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/70C12N1/21C12P19/26C12R1/19
CPCC12N9/2402C12N15/70C12P19/26C12Y302/01052Y02A50/30
Inventor 陈可泉莫晓芳张阿磊杨赛魏衍鹏周宁王莹莹欧阳平凯
Owner NANJING UNIV OF TECH
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