Preparation method and application of chondroitin sulfate ABC zymoprotein

A technology of chondroitin sulfate and enzyme protein, which is applied in botany equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of high cost, limited research on heterologous recombination expression, cumbersome preparation process, etc., and meet the production conditions not high effect

Active Publication Date: 2019-05-21
BEIJING POLYTECHNIC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, research on the heterologous recombination expression of ChSase ABC is very limited. In the prior art, the proportion of ChSase ABC enzyme protein expressed in the active protein state after using the prokaryotic expression system is very low. The denatured protein needs to undergo denaturation and renaturation process, and the preparation process is cumbersome. ,higher cost

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  • Preparation method and application of chondroitin sulfate ABC zymoprotein
  • Preparation method and application of chondroitin sulfate ABC zymoprotein

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Embodiment 1

[0049] Embodiment 1, recombinant plasmid pET-GroES-RBS-ChSase ABC I, pET-ChSase ABC I-RBS-GroES, Construction and expression of pET-GroES-T7-ChSase ABC I

[0050] 1. The specific process of expression vector pET-GroES-RBS-ChSase ABC I construction is as follows:

[0051] 1.1 Design and synthesis of primers

[0052] The DNA sequences of Proteus vulgaris chondroitin sulfate ABC enzyme and molecular chaperone protein GroES were obtained through Genbank query, and the upstream and downstream primers used were:

[0053] Upstream primer P1:

[0054] 5'-CG GGATCC AGGAGATATACCATGGCCACCAGCAATCCTGCATT-3' (SEQ ID NO: 2) (underlined bases are Bam H Restriction site of I),

[0055] Downstream primer P2:

[0056] 5'-CCG CTCGAG TTATCAAGGGAGTGGCGAGAGTTTG-3' (SEQ ID NO: 3) (underlined base is xho Restriction site of I), after amplification, they were respectively introduced into Bam H I and xho I restriction site.

[0057] Upstream primer P1:

[0058] 5'-GGAATTC CATATG ...

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Abstract

The invention provides a preparation method of ChSase ABC zymoprotein. The method comprises the steps that a chondroitin sulfate ABC enzyme gene and a molecular chaperone GroES gene are cloned respectively, then the two protein genes are connected to a same expression vector, and a recombinant vector containing chondroitin sulfate ABC zymoprotein and molecular chaperone GroES is obtained; the recombinant vector is transformed into a host cell for expression to obtain a transformant containing chondroitin sulfate ABC zymoprotein and molecular chaperone GroES; the transformant is cultured, physical crushing is carried out, and the chondroitin sulfate ABC zymoprotein is prepared. The invention further provides the ChSase ABC zymoprotein and application thereof. The ChSase ABC zymoprotein hashigh enzyme activity, can be used for producing low-molecular-weight chondroitin sulfate, the generation conditions of the chondroitin sulfate are not high, and the method is simple, convenient and easy to control.

Description

technical field [0001] The invention relates to a preparation method and application of a chondroitin sulfate ABC enzyme in the field of genetic engineering and fermentation engineering. Background technique [0002] Chondroitin sulfate lyase (chondroitinase or chondroitin sulfateyase, referred to as "ChSase") is a kind of glycosaminoglycans that can degrade chondroitin sulfate, chondroitin, hyaluronic acid and other glycosaminoglycans into unsaturated disaccharides (ΔDi and oligosaccharides) Lyase. [0003] With the in-depth research on ChSase, it is found that chondroitin sulfate ABC enzyme (referred to as "ChSase ABC") has a wide range of application values. Researchers use ChSase ABC to detect chondroitin sulfate and produce low molecular weight chondroitin sulfate. Bao Lunjun, Yang Jiancheng, etc. used ChSase ABC enzymatic hydrolysis-high performance liquid chromatography to detect hyaluronic acid in shark fins. Using ZORBAX sugar analysis column, the ultraviolet dete...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): C12N15/60C12N9/88C12N15/70C12N1/21
Inventor李晔陈振娅周朝辛秀兰袁其朋
OwnerBEIJING POLYTECHNIC