A method for efficiently producing chondroitin sulfate a by artificial enzymatic method

A technology of chondroitin sulfate and chondroitin, which is applied in the field of bioengineering, can solve the problems of small reaction scale, high cost, and long catalytic reaction time, and achieve the effects of quality and safety assurance, improved synthesis efficiency, and uniform product structure

Active Publication Date: 2021-09-28
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] According to previous literature reports, it takes about 48 hours to complete the complete conversion of 100 mg chondroitin (Zhou, et al. (2018). A microbial-enzymatic strategy for producing chondroitin sulfate glycosaminoglycans. Biotechnology and Bioengineering, 115(6), 1561-1570. ), the catalytic reaction lasts too long, and the reaction scale is small, so it cannot be really applied in practice
At the same time, many current studies are still stuck in the use of mammalian cells for the active expression of C4ST, which is expensive and cannot be used for actual large-scale production

Method used

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  • A method for efficiently producing chondroitin sulfate a by artificial enzymatic method
  • A method for efficiently producing chondroitin sulfate a by artificial enzymatic method
  • A method for efficiently producing chondroitin sulfate a by artificial enzymatic method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Construction of recombinant Pichia pastoris GS115 / lin1, GS115 / lin2, GS115 / lin3, GS115 / lin4

[0050] 1. Optimization of the gene encoding chondroitin 4-O-sulfotransferase

[0051] (1) First remove the N-terminal 1-62 amino acids from the sequence of GenBank accession number NP_067414.2, then perform codon optimization and artificial synthesis, and assemble and connect the synthesized original sequence to the pPIC9K plasmid to obtain the pPIC9K-Δ62C4ST plasmid. The plasmid was linearized with fast cutting enzyme SalI and then transformed into Pichia pastoris GS115 to obtain recombinant strain S001;

[0052] (2) The SUMO tag protein sequence was fused to the N-terminal of the Δ62C4ST sequence by fusion PCR technology, and then the fused sequence SUMOC4 was connected to the pPIC9K vector by Gibson assembly to obtain the pPIC9K-SUMOC4ST plasmid, which was linearized with the fast cutting enzyme SalI Transformed into Pichia pastoris GS115 after transformation to o...

Embodiment 2

[0067] Embodiment 2: the comparison of two kinds of catalyst systems chondroitin sulfate A transformation rate

[0068] The engineering strains GS115 / lin1, GS115 / lin2, GS115 / lin3, and GS115 / lin4 constructed in Example 1 were respectively streaked on the YPD plate for activation, and then a single colony was inoculated in the YPD medium for 16 hours at 30°C, and then pressed for 10 % inoculum size Inoculated in BMGY medium, cultured at 30°C for 24h. After the cultivation, collect the bacteria, resuspend and wash the bacteria twice with physiological saline, and finally resuspend the bacteria in BMMY medium for cultivation at 30°C, add 1% methanol every 24 hours to induce expression, and induce the culture for 96 hours Afterwards, the supernatant was collected by centrifugation, and the enzyme activities of C4ST and AST IV were determined. Using freely expressed C4ST and AST IV as controls, the relative enzyme activity was calculated, and the results were as follows: figure 2...

Embodiment 3

[0073] Example 3: Steps for obtaining the bifunctional protein lin3

[0074] Inoculate a single colony of the recombinant strain GS115 / lin3 in 5mL YPD liquid medium, culture at 30°C for 24h, then transfer 10% of the inoculum to a shaker flask containing 50mL of BMMY medium, and culture at 30°C for 24h The bacteria were collected by centrifugation, washed twice with sterile saline, resuspended in 50 mL of BMGY medium, and cultured at 25°C for 96 hours, during which 1% volume of methanol was added every 24 hours to induce protein expression.

[0075] After shaking the flask, the supernatant of the fermentation broth was collected by centrifugation, which was the crude enzyme solution of the bifunctional protein lin3. The bifunctional protein lin3 was eluted and purified through a nickel column under the conditions of phosphate buffer (pH 7.0) containing 300mM imidazole and 500mM NaCl. .

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Abstract

The invention discloses a method for efficiently producing chondroitin sulfate A by artificial enzyme method, which belongs to the technical field of bioengineering. This method expresses a bifunctional protein chondroitin 4‑O‑sulfotransferase C4ST and aryl sulfotransferase AST IV through microbial heterologous expression, which is used to catalyze the synthesis of chondroitin sulfate A from chondroitin, combined with site-directed mutagenesis and The optimization of the components of the reaction system accelerates the synthesis efficiency of chondroitin sulfate.

Description

technical field [0001] The invention relates to a method for efficiently producing chondroitin sulfate A by artificial enzymatic method, which belongs to the technical field of bioengineering. Background technique [0002] Chondroitin sulfate A (CSA) is a proteoglycan widely distributed in cartilage tissue with important biological functions. The backbone of chondroitin sulfate A is a linear polysaccharide formed by alternating links of D-glucuronic acid and N-acetylgalactosamine. Chondroitin sulfate is formed by the sulfonation modification of chondroitin by different types of sulfotransferases. According to the position of sulfonation modification, chondroitin sulfate can be divided into the following four types: Chondroitin sulfate A (4-O- sulfonated), chondroitin sulfate C (6-O-sulfonated), chondroitin sulfate D (2,6-di-O-sulfonated), and chondroitin sulfate E (4,6-di-O-sulfonated) . Chondroitin sulfate has excellent biocompatibility, and its application range is very...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12P19/26C12N1/19C12N9/10C12N15/81C12R1/84
CPCC07K2319/00C12N9/13C12N15/815C12N2800/22C12P19/26C12Y208/02001C12Y208/02005
Inventor 康振陈坚堵国成李江华金学荣
Owner JIANGNAN UNIV
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