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Enzymatic method for preparing chondroitin sulfate

A technology of chondroitin sulfate and chondroitin, applied in the field of biomedicine, can solve the problems of unfavorable industrial production, complex animal cell culture, high cost, etc., and achieve the effect of huge industrial application value

Inactive Publication Date: 2017-05-31
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, C4ST and C6ST are mainly extracted from animal cells, but animal cell culture is complicated and costly, which is not conducive to industrial production

Method used

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  • Enzymatic method for preparing chondroitin sulfate
  • Enzymatic method for preparing chondroitin sulfate
  • Enzymatic method for preparing chondroitin sulfate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 PAPS regeneration system

[0033] The concentration of PAPS regeneration system components is as follows: the concentration of PNPS is 3mM, the concentration of PAP is 20μM, ASST IV 10mg, and 1-200mM Tris-HCl of pH 5-9 is used as the base solution. C4ST or C6ST was added as a sulfate group donor for chondroitin sulfation.

Embodiment 2

[0034] Preparation of embodiment 2 tool enzyme ASST IV, C4ST, C6ST

[0035] (1) Shake flask expression of recombinant Escherichia coli

[0036] Using the inducible vector pET series vector pET26b as the vector, construct pET26b-ASSTIV, pET26b-C4ST, pET26b-C6ST respectively, and transform Escherichia coli to obtain transformants. Recombinant expression vectors carrying genes encoding ASST IV, C4ST, and C6ST were respectively constructed with pUC series vectors, and transformed into Escherichia coli to obtain transformants as controls.

[0037] A single colony of the recombinant strain constructed with the pET series vector was picked and placed in 3 mL of LB medium (adding ampicillin at a final concentration of 50 μg / mL), and cultured overnight at 37° C. at 200 rpm. Transfer to 50mL TB medium (adding ampicillin with a final concentration of 50μg / mL) at a ratio of 1% (V / V), culture at 37°C, 200rpm for 2h to OD 600nmAt about 0.6-0.8, add IPTG with a final concentration of 0.1 m...

Embodiment 3

[0044] The preparation of embodiment 3 CSA

[0045] Using fermented chondroitin as raw material, construct a catalytic reaction system, including 20mM Tris-HCl (pH7.0), 3mMPNPS, 20μM PAP, 10mgASST IV (enzyme activity unit 0.1-100nmol / min·mg·protein), 5mg / mL Chondroitin and 20 μg C4ST (enzyme activity unit: 0.1-100 pmol / min·mg·protein) were reacted at 37°C for 20 hours and then heated at 100°C for 5 minutes to terminate the reaction.

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PUM

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Abstract

The invention discloses an enzymatic method for preparing chondroitin sulfate and belongs to the medical field. Chondroitin is used as a substrate, chondroitin sulfate A with bioactivity is formed through catalysis of chondroitin 4-sulfate transferase for microbial cell heterologous expression or chondroitin sulfate C with bioactivity is formed through catalysis of chondroitin 6-sulfate transferase, and a PAPS sulfuric donor and a regeneration system are provided by acyl sulfate transferase. A new way is provided for synthesis and industrial production of biological active substances, namely, chondroitin sulfate A and chondroitin sulfate C.

Description

technical field [0001] The invention relates to a method for preparing chondroitin sulfate by enzymatic method, which belongs to the field of biomedicine. Background technique [0002] Chondroitin sulfate (Chondroitin sulfate, CS) is a mucopolysaccharide formed by glucuronic acid and N-acetylgalactosamine alternately through β1→3, β1→4 glycosidic bonds, and accompanied by a certain degree of sulfated modification of N-acetylgalactosamine. . Among them, the chondroitin sulfated at the 4th position of N-acetylgalactosamine is called chondroitin sulfate A (CSA), and the chondroitin sulfated at the 6th position is called chondroitin sulfate C (chondroitin sulfate). C, CSC). CSA and CSC widely exist on the cell surface and in the extracellular matrix, and play a significant role in maintaining the stability of neural stem cells, neuron migration, neurite elongation, and dendritic regeneration, and are widely used in the treatment of osteoarthritis and other diseases. In additi...

Claims

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Application Information

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IPC IPC(8): C12P19/26C12N1/21C12N1/19C12R1/19C12R1/84
CPCC12P19/26C12N9/13
Inventor 康振陈坚堵国成周正雄
Owner JIANGNAN UNIV
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