NDLI: In vitro synthesis of heparosan using recombinant Pasteurella multocida heparosan synthase PmHS2

Content Provider	Springer Nature Link
Author	Chavaroche, Anaïs A. E. Springer, Jan Kooy, Floor Boeriu, Carmen Eggink, Gerrit
Copyright Year	2009
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.
Starting Page	1881
Ending Page	1891
Page Count	11
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	85
Issue Number	6
e-ISSN	14320614
Language	English
Publisher	Springer-Verlag
Publisher Date	2009-09-16
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	Heparosan synthase Pasteurella multocida Glycosyltransferase Glycosaminoglycan Nucleotide sugar Polymerase Microbial Genetics and Genomics Microbiology Biotechnology
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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In vitro synthesis of heparosan using recombinant Pasteurella multocida heparosan synthase PmHS2

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