Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Record number 371568
Title Quantification and characterization of enzymatically produced hyaluronan with fluorophore-assisted carbohydrate electrophoresis
Author(s) Kooy, F.K.; Muyuan Ma, ; Beeftink, H.H.; Eggink, G.; Tramper, J.; Boeriu, C.G.
Source Analytical Biochemistry 384 (2009)2. - ISSN 0003-2697 - p. 329 - 336.
DOI https://doi.org/10.1016/j.ab.2008.09.042
Department(s) Bioprocess Engineering
AFSG Biobased Products
VLAG
Publication type Refereed Article in a scientific journal
Publication year 2009
Keyword(s) polyacrylamide-gel electrophoresis - synthase - oligosaccharides - microanalysis - acid - fragments - sulfate - assay - face
Abstract Hyaluronan (HA) is a polysaccharide with high-potential medical applications, depending on the chain length and the chain length distribution. Special interest goes to homogeneous HA oligosaccharides, which can be enzymatically produced using Pasteurella multocida hyaluronan synthase (PmHAS). We have developed a sensitive, simple, and fast method, based on fluorophore-assisted carbohydrate electrophoresis (FACE), for characterization and quantification of polymerization products. A chromatographic pure fluorescent template was synthesized from HA tetrasaccharide (HA4) and 2-aminobenzoic acid. HA4-fluor and HA4 were used as template for PmHAS-mediated polymerization of nucleotide sugars. All products, fluorescent and nonfluorescent, were analyzed with gel electrophoresis and quantified using lane densitometry. Comparison of HA4- and HA4-fluor-derived polymers showed that the fluorophore did not negatively influence the PmHAS-mediated polymerization. Only even-numbered oligosaccharide products were observed using HA4-fluor or HA4 as template. The fluorophore intensity was linearly related to its concentration, and the limit of detection was determined to be 7.4 pmol per product band. With this assay, we can now differentiate oligosaccharides of size range DP2 (degree of polymerization 2) to approximately DP400, monitor the progress of polymerization reactions, and measure subtle differences in polymerization rate. Quantifying polymerization products enables us to study the influence of experimental conditions on HA synthesis
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