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Quantitative detection method for content of chondroitin sulfate polysaccharide in fermentation liquor

A quantitative detection method and chondroitin sulfate technology, applied in the field of analysis and detection, can solve the problems of medium component interference and inability to quantitatively detect fermentation broth, etc., and achieve the effect of improving accuracy and high detection requirements.

Pending Publication Date: 2022-03-18
SINOCHEM HEALTH IND DEV CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The present invention provides a quantitative detection method for the content of chondroitin in the fermentation broth in view of the current lack of standard samples of chondroitin and the interference of medium components and the inability to quantitatively detect the content of chondroitin in the fermentation broth

Method used

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  • Quantitative detection method for content of chondroitin sulfate polysaccharide in fermentation liquor
  • Quantitative detection method for content of chondroitin sulfate polysaccharide in fermentation liquor
  • Quantitative detection method for content of chondroitin sulfate polysaccharide in fermentation liquor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] This example provides the analysis of the induced expression, purification and enzymatic hydrolysis of chondroitin sulfate lyase AsChnAC.

[0051] Chondroitin sulfate lyase AC can cut the GalNAc-β-1,4-GlcA glycosidic bond of chondroitin sulfate-like polysaccharides, which has bond specificity for enzyme-catalyzed reactions. For details, see Yin FX, Wang FS, Sheng JZ (2016) Uncovering the Catalytic Direction of Chondroitin AC Exolyase: FROM THE REDUCING END TOWARDS THE NON-REDUCING END. J Biol Chem 291(9): 4399-406 doi: 10.1074 / jbc.C115.708396. The strain expressing chondroitin sulfate lyase AsChnAC was obtained from Shandong University. For details, see CHEN L, SHI C Q, Ye F X, etal. Cloning and Characterization of a Chondroitin AC Exolyase from Arthrobacter sp.SD-04[J].Molecular Biotechnology, 2019, 61(10):791-800. Cultivate the strain carrying AsChnAC expressing chondroitin sulfate lyase overnight and transfer it to 2L of fresh LB medium, culture with shaking at 37°C...

Embodiment 2

[0058] This example provides the standard curve drawing of chondroitin sulfate C.

[0059] Weigh chondroitin sulfate C (brand: McLean, article number: C832332-1g, 90%, derived from sharks) and dissolve it in ddH 2 O, prepared as 20mg / ml mother solution, dilute chondroitin sulfate C to 0.08mg / ml, 0.2mg / ml, 0.4mg / ml, 0.6mg / ml, 0.8mg / ml, 2.0mg / ml, each concentration Set the gradient in 3 parallels, mix 100 μl sample with 300 μl anthrone reaction reagent (containing 0.1% anthrone and 80% concentrated sulfuric acid), put the mixture in a water bath at 100°C for 15 min, and ice bath for 15 min, measure OD620, and draw a standard curve with OriginPro , to calculate the linear regression equation.

[0060] Result analysis:

[0061] Concentrated sulfuric acid hydrolyzes polysaccharides into monosaccharides, and the monosaccharides react with anthrone to form a dark green complex, which has a maximum absorbance value at OD620, and there is an obvious linear relationship between the ab...

Embodiment 3

[0063] This example provides the detection method of the present invention to measure the content of chondroitin in the fermentation broth, and the standard curve is shown in Example 2.

[0064] Microbial fermentation to produce chondroitin sulfate: pick Escherichia coli K4ΔkfoE (refer to Liu J, Yang A, Liu J, Ding X, Liu L, Shi Z (2014) KfoE encodes a fructosyltransferase involved incapsular polysaccharide biosynthesis in Escherichia coli K4.Biotechnol Lett36 ( 7): 1469-77 doi: 10.1007 / s10529-014-1502-9) monoclonal in 3mL LB culture medium, shake overnight at 37°C; transfer the above bacteria solution to 50mL LB culture medium in an amount of 1%, respectively, Shake culture at 37°C for about 20 hours; centrifuge the bacterial solution at 12,000 rpm for 10 minutes to collect the supernatant.

[0065] Standard solution: Weigh chondroitin sulfate C and dissolve it in LB culture medium to prepare a 1mg / mL solution.

[0066] Sample preparation: Take 800 μl of the above-mentioned ...

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Abstract

The invention discloses a quantitative detection method for the content of chondroitin sulfate polysaccharide in fermentation liquor, and belongs to the field of analysis and detection. The method comprises the following steps: taking chondroitin sulfate C as a standard sample, combining an enzymolysis method with an anthrone-sulfuric acid method, removing small-molecular impurities in fermentation liquor through ultrafiltration before enzymolysis, then carrying out enzymolysis on chondroitin sulfate polysaccharide into oligosaccharide by using chondroitin sulfate lyase, and then removing macromolecular impurities through ultrafiltration to obtain the chondroitin sulfate polysaccharide oligosaccharide. And determining the sugar content of the obtained filtrate by using an anthrone-sulfuric acid method. The method is coupled with a chemical analysis method of enzyme-specific decomposition of the chondroitin sulfate polysaccharide, small-molecule and macromolecular impurities causing quantitative interference in complex components can be removed only through two steps, and the content of the chondroitin sulfate polysaccharide in a complex culture solution can be simply, conveniently and accurately measured by combining an anthrone-sulfuric acid method.

Description

technical field [0001] The invention belongs to the field of analysis and detection, and in particular relates to a quantitative detection method for the content of chondroitin sulfate polysaccharides in fermentation broth. Background technique [0002] Chondroitin Sulfate (Chondroitin Sulfate, CS) is a kind of acidic glycosaminoglycan macromolecular polysaccharide widely present in animal connective tissue. Uronic acid (GlcA) and N-acetylgalactosamine (GalNAc) are alternately linked by β-1,3 and β-1,4 glycosidic bonds (4-GlcA-β-1,3-GalNAc-β-1) composed of repeating disaccharide units. According to the different sulfation sites, chondroitin sulfate polysaccharides can be divided into non-sulfated chondroitin (CS-O), chondroitin sulfate A (CS-A), chondroitin sulfate B (CS-B), Types such as chondroitin sulfate C (CS-C) and chondroitin sulfate E (CS-E), among which, chondroitin sulfate derived from animals is mostly CS-A and CS-C. Chondroitin Sulfate is called "Human Gold", ...

Claims

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Application Information

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IPC IPC(8): G01N33/15
CPCG01N33/15
Inventor 周贤轩魏晓珍李文静狄莹莹陈婷婷汪振洋
Owner SINOCHEM HEALTH IND DEV CO LTD
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