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Method for efficiently preparing micromolecular chondroitin sulfate and micromolecular hyaluronic acid by one step

A technology of chondroitin sulfate and hyaluronic acid, applied in the biological field, can solve the problems of affecting enzyme-related properties, low enzyme activity of ChSaseABC, and less research on recombinant expression of ChSaseABC

Active Publication Date: 2019-07-09
CHANGSHU INSTITUTE OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] At present, there are few studies on the recombinant expression of ChSase ABC. Although Prabhakar et al. constructed Escherichiacoli BL21(DE3)-pET-28a-csl ABC, only a small amount of soluble protein was obtained, and most of ChSase ABC exists in the form of inclusion bodies.
Although Li Ye et al. obtained the soluble expression of the ChSase ABC gene derived from P. vulgaris KCTC2579 in E. coli through the fusion tag glyceraldehyde-3-phosphate dehydrogenase GAPDH and maltose binding protein MBP, but the presence of the fusion tag affected the enzyme activity. Related properties
In addition, small molecular weight chondroitin sulfate is generally used in the fields of medicine and food, so ChSase ABC expressed in Escherichia coli is not suitable for the preparation of small molecular weight chondroitin sulfate
Gao Huiling et al. established a ChSase ABC secretory eukaryotic expression vector, which was expressed in a secreted form. However, the production cycle of human glioma cell TJ905 is long and the culture conditions are harsh, which is not suitable for industrial application.
[0011] The main problem with the fermentation production of ChSase ABC is that the enzyme activity of ChSase ABC produced by fermentation is low, and most of them are expressed intracellularly, and the crushing process will definitely increase the production cost

Method used

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  • Method for efficiently preparing micromolecular chondroitin sulfate and micromolecular hyaluronic acid by one step
  • Method for efficiently preparing micromolecular chondroitin sulfate and micromolecular hyaluronic acid by one step
  • Method for efficiently preparing micromolecular chondroitin sulfate and micromolecular hyaluronic acid by one step

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Embodiment 1: Construction of recombinant Bacillus subtilis

[0083] Amplification of the chondroitin sulfate ABC lyase gene: the genome of Proteus vulgaris ATCC33420 was used as a template to amplify the chondroitin sulfate ABC lyase gene fragment. The PCR amplification system was 1 μL of genomic DNA, 4 μL of each primer 1 and primer 2, 50 μL of KOD polymerase, ddH 2 O 41 μL, PCR reaction program: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 2 min; then annealing at 60°C for 30 s, extension at 72°C for 1.2 min, cycle 35 times; extension at 72°C for 1 min.

[0084] Amplification of the linearized pHY300PLK gene: using pHY300PLK as a template, amplify the linearized fragment of pHY300PLK that changes the restriction site. The PCR amplification system was pHY300PLK 1 μL, primer 3 and primer 4 each 4 μL, KOD polymerase 50 μL, ddH 2 O 41 μL, PCR reaction program: 94°C pre-denaturation for 4 min, 94°C denaturation for 2 min; then annealing at 62°C for 30 s,...

Embodiment 2

[0091] Example 2: Induced expression of chondroitin sulfate ABC lyase

[0092] Inoculate the constructed recombinant Bacillus subtilis str.l68-△spoOA-pHY300PLK-Pxyl-wapA-csl ABC in LB liquid medium containing tetracycline resistance, and culture overnight at 37°C; Received into 1L of tetracycline-containing fermentation medium, fermented for 2-4 hours to OD 660 When reaching 0.6, add 8g / L xylose to induce expression for 24h, and because the host in the present invention knocks out the key gene Bacillus subtilis required for spore growth, it is conducive to the secretion and expression of chondroitin sulfate ABC lyase, and the final fermentation The activity of chondroitin sulfate ABC lyase in the liquid supernatant can reach 24U / mL, which is the highest level at present.

Embodiment 3

[0093] Embodiment 3: the chondroitin sulfate ABC lyase enzyme activity assay of fermented liquid

[0094] Take 1mL of fermentation broth and centrifuge, take 0.1mL supernatant and 7.9mL 1g / L chondroitin sulfate (prepared with 0.02mol / LTris-HCL, pH7.5) respectively, add them to a 15mL colorimetric tube, and place at 37°C React in a water bath for 20 minutes, immediately place it in a boiling water bath and boil for 5 minutes, add inactivated fermentation broth supernatant to the control tube under the same conditions, and measure the light absorption value at 232nm. The enzyme activity unit U is defined as the amount of enzyme required to catalyze the formation of 1 μmol of unsaturated disaccharide per minute at 37°C.

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Abstract

The invention discloses a method for efficiently preparing micromolecular chondroitin sulfate and micromolecular hyaluronic acid by one step, and belongs to the technical field of bioengineering. According to the method, chondroitin sulfate ABC lyase derived from proteus vulgaris ATCC33420 is heterologously expressed, a signal peptide wapA is selected, and constitutive xylose promoter Pxyl and pHY300PLK vectors are used, and secreted expression of the chondroitin sulfate ABC lyase in bacillus subtilis is realized through induction by xylose; the micromolecular chondroitin sulfate and the micromolecular hyaluronic acid can be produced efficiently and continuously by a designed chondroitin lyase-enzyme membrane reactor. The method has the advantages that the food-grade bacillus subtilis serves as a host strain, and safety and reliability are realized; effective reference is provided for industrial green production of the micromolecular chondroitin sulfate and the micromolecular hyaluronic acid; energy conservation and emission reduction are realized, and economic and social benefits are remarkable.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the cloning and expression of a chondroitin sulfate lyase and its application in the preparation of chondroitin sulfate. Background technique [0002] Both Chondroitin Sulfate and Hyaluronic Acid are glycosaminoglycans. Chondroitin Sulfate is the sulfate ester salt of a copolymer of D-glucuronic acid and N-acetylgalactosamine. Carbon sugars are connected alternately by β-(1,4) and β-(1,3) glycosidic bonds, generally contain about 50-70 disaccharide units, and the molecular weight is between 10kDa-50kDa. Hyaluronic acid is a linear polysaccharide composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine linked by β-(1,3) glycosidic bonds; each disaccharide The unit is connected to another disaccharide unit through a β-(1,4) glycosidic bond; there can be as many as 25,000 disaccharide units, and the molecular weight is between 20,000 and 50,0...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/60C12N15/75C12N9/88C12P19/26C12P19/04C08B37/08C12R1/125
CPCC08B37/0003C08B37/0072C12N9/88C12N15/75C12P19/04C12P19/26C12Y402/02004
Inventor 吴凌天卢成慧高华朱益波杨福林熊欢黄晖荣卢艳
Owner CHANGSHU INSTITUTE OF TECHNOLOGY
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