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 389961
Title In vitro synthesis of heparosan using recombinant Pasteurella multocida heparosan synthase PmHS2
Author(s) Chavaroche, A.A.E.; Springer, J.; Kooy, F.K.; Boeriu, C.G.; Eggink, G.
Source Applied Microbiology and Biotechnology 85 (2010)6. - ISSN 0175-7598 - p. 1881 - 1891.
Department(s) Bioprocess Engineering
BBP Bioconversion
BBP Sustainable Chemistry & Technology
Publication type Refereed Article in a scientific journal
Publication year 2010
Keyword(s) molecular-weight heparins - hyaluronan synthase - chemoenzymatic synthesis - capsular polysaccharide - identification - streptococcus - biosynthesis - acid - glycosyltransferases - polymers
Abstract In vertebrates and bacteria, heparosan the precursor of heparin is synthesized by glycosyltransferases via the stepwise addition of UDP-N-acetylglucosamine and UDP-glucuronic acid. As heparin-like molecules represent a great interest in the pharmaceutical area, the cryptic Pasteurella multocida heparosan synthase PmHS2 found to catalyze heparosan synthesis using substrate analogs has been studied. In this paper, we report an efficient way to purify PmHS2 and to maintain its activity stable during 6 months storage at -80¿°C using His-tag purification and a desalting step. In the presence of 1 mM of each nucleotide sugar, purified PmHS2 synthesized polymers up to an average molecular weight of 130 kDa. With 5 mM of UDP-GlcUA and 5 mM of UDP-GlcNAc, an optimal specific activity, from 3 to 6 h of incubation, was found to be about 0.145 nmol/µg/min, and polymers up to an average of 102 kDa were synthesized in 24 h. In this study, we show that the chain length distribution of heparosan polymers can be controlled by change of the initial nucleotide sugar concentration. It was observed that low substrate concentration favors the formation of high molecular weight heparosan polymer with a low polydispersity while high substrate concentration did the opposite. Similarities in the polymerization mechanism between PmHS2, PmHS1, and PmHAS are discussed
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