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Chondrosulphatase screening, identification and optimized expression

A technology of chondroitin sulfate enzyme and amino acid, which is applied in the field of bioengineering, can solve the problems of complex process technology, high price, unfavorable industrial scale-up, etc., and achieve the effect of simple process and post-treatment, wide application value, and high-efficiency biosynthesis

Active Publication Date: 2019-06-21
NANJING HANXIN PHARMA TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most chondroitin sulfate lyases from microorganisms are intracellular enzymes with low enzyme activity
Tao Ke et al. (Screening of chondroitin sulfate lyase ABC-producing bacteria and research on fermentation process [J]. Chinese Journal of Antibiotics, 2004, (29) 3:138-140.) once reported the use of a strain of Proteus pangzii to ferment The process of producing ChSase is relatively complicated, and in the later stage of fermentation, the enzyme activity of ChSase decreases significantly, and the highest enzyme activity in the whole process is only 322.17U / L
Su Xin et al. (Study on the preparation of chondroitin sulfate lyase by fermentation method and its separation and purification [J]. Journal of Microbiology, 2005, (25) 4:64-67.) reported a strain Aeromonas temperatus, although it has a high enzyme-producing ability, it needs to go through a purification process of 1-step dialysis and 4-step column chromatography. The process is relatively complicated and the cost is high, which is not conducive to industrial scale-up
In addition to fermentation source microorganisms for enzyme production, Li et al. (Expression, purification and characterization of GAPDH-ChSase ABC I from Proteus vulgaris in Escherichia coli [J]. Protein Expression and Purification, 2016, (128): 36-41.) also reported ChSase derived from Proteus was expressed heterologously in Escherichia coli. In order to increase the expression of ChSase, it was reported that glyceraldehyde-3-phosphate dehydrogenase was co-expressed, but the enzyme activity after purification was reduced by 3.1 times. Therefore, Not conducive to industrial scale-up
At present, there is no product supply in China, almost all of which are imported, and the price is very expensive, which limits the application of chondroitin sulfate enzyme in the preparation of low molecular weight chondroitin sulfate and medicine. Therefore, it is of great significance to screen chondroitin sulfate enzyme with high enzyme activity

Method used

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  • Chondrosulphatase screening, identification and optimized expression
  • Chondrosulphatase screening, identification and optimized expression
  • Chondrosulphatase screening, identification and optimized expression

Examples

Experimental program
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Effect test

Embodiment 1

[0019] Embodiment 1: Screening and identification of chondroitin sulfate enzyme

[0020] Collect soil samples, sewage or silt from the coast, river, farmer's market, slaughterhouse and canteen, etc., take an appropriate amount of soil samples, extract with normal saline, after enrichment and culture, gradient dilution and coating on a screening plate, culture at 37 °C After 3 days, colonies with transparent circles were selected for re-screening. The selection medium (g / L) used was: chondroitin sulfate 4.5, ammonium chloride 2.5, sodium chloride 0.95, magnesium sulfate 1.0, dipotassium hydrogen phosphate 1.0, agar 20.0, bovine serum albumin 5.0, pH 7.0. Compare and select the colony with the largest ratio of the transparent circle to the strain diameter in the re-screening plate, inoculate the colony into a liquid fermentation medium for culture, extract the genome of the flora for metagenomic sequencing, analyze and screen out the chondroitinase with the highest possibility ...

Embodiment 2

[0021] Example 2: Optimal expression of chondroitinase in Escherichia coli and Bacillus subtilis

[0022] Construction of the expression system:

[0023] The CS DNA fragment was amplified by PCR using primers CS(pBRSF)-F / CS(pBRSF)-R (using artificially synthesized CSDNA as a template), and the resulting CS DNA PCR product was assembled with the backbone pBRSFDuet-1 (using primer pBRSF-F / pBRSF-R amplification) to generate pBRSFDuet-1-CS recombinant plasmid.

[0024] The CS DNA fragment was amplified by PCR using primers CS(pHT)-F / CS(pHT)-R (using artificially synthesized CS DNA as a template), and the resulting CS DNA PCR product was assembled with the backbone pHT01 (using primers pHT01-F / pHT01-R amplification) to generate the pHT01-CS recombinant plasmid.

[0025] Construction of recombinant bacteria:

[0026] The recombinant plasmid pBRSFDuet-1-CS was transferred to Escherichia coli MG1655, Escherichia coli DH5α, Escherichia coli W3110, and Escherichia coli BL21, respec...

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Abstract

The invention belongs to the technical field of bioengineering and discloses chondrosulphatase screening, identification and optimized expression. An amino acid sequence of chondrosulphatase screenedand identified from the nature is similar to that of chondrosulphatase reported by NCBI, wherein the maximum similarity is 85%; by optimized expression in escherichia coli and bacillus subtilis, efficient biosynthesis of high-enzyme-activity chondrosulphatase is realized, wherein the maximum enzyme activity is 11976.5U / L; a whole process and aftertreatment are simpler relatively. A potential and extensive application value in preparation of products containing low-molecular-weight chondroitin sulfate in fields of medicines, cosmetics and biology is achieved, a foundation is laid for efficientfermentation preparation of high-enzyme-activity chondrosulphatase through a microbial system, and suitableness for industrial production and application is realized.

Description

technical field [0001] The invention relates to the screening, identification and optimized expression of a chondroitin sulfate enzyme, which belongs to the technical field of bioengineering. Background technique [0002] Chondrosulphatase (ChSase for short) is a lyase that can degrade glycosaminoglycans such as chondroitin sulfate, dermatan sulfate and hyaluronic acid into unsaturated disaccharides and oligosaccharides. ChSase is divided into ChSase ABC, ChSase AC, ChSase B and ChSase C according to the different substrates. Chondroitin sulfate enzyme has important application value in the fields of biochemical industry and medicine. In basic research, it can be used as a tool enzyme for the quality research of chondroitin sulfate and the efficient preparation of chondroitin sulfate oligosaccharides with various biological activities; In medicine, chondroitin sulfate is used as a medicinal enzyme, which can degrade mucus in cystic fibrosis, promote regeneration of nerve ax...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/70C12N15/75C12P19/26
CPCC12N15/70C12P19/26C12N9/88C12N15/75C12P21/02C12N9/16C12Y301/06004
Inventor 张昊宁陈松汤传根王璟
Owner NANJING HANXIN PHARMA TECH CO LTD