Chemoenzymatic quantification for monitoring unpurified polysaccharide in rich medium

Applied Microbiology and Biotechnology
Yanying YuXianxuan Zhou

Abstract

The heparosan polysaccharide serves as the starting carbon backbone for the chemoenzymatic synthesis of heparin, a widely used clinical anticoagulant drug. The previous quantification methods for heparosan rely on time-consuming purification or expensive instruments not readily available for many labs. Here, a chemoenzymatic approach is developed to monitor the production of heparosan in rich medium without purification. After removing the interfering small molecules by ultrafiltration, heparosan was decomposed into oligosaccharides using heparin lyase III. The oligosaccharides were separated from large molecules by ultrafiltration and quantitatively determined by the anthrone-sulfuric acid assay using a spectrophotometer. Based on the different substrate specificity of heparin lyases, the study showed that the concentration of heparosan and heparin in a mixture was discriminatively determined by the two-step chemoenzymatic assay. Furthermore, the anthrone-sulfuric acid assay was observed to be more reliable than the phenol-sulfuric acid assay under these conditions. Besides heparosan and heparin, the chemoenzymatic assay may be adapted to quantify other types of polysaccharides if the specific lyases were available.

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Citations

Oct 5, 2020·Biotechnology Letters·Dandan WuXianxuan Zhou
Mar 9, 2021·Archives of Microbiology·Hui ShiXianxuan Zhou
Sep 3, 2021·Spectrochimica Acta. Part A, Molecular and Biomolecular Spectroscopy·Lun WuHai-Xue Kuang

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Methods Mentioned

BETA
nuclear magnetic resonance
NMR
electrophoresis

Software Mentioned

ImageJ
Masslynx

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