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    Elucidating the unusual reaction kinetics of glucuronyl C5-epimerase

    Author
    Vaidyanathan, Deepika
    View/Open
    180377_Vaidyanathan_rpi_0185E_11781.pdf (5.402Mb)
    Other Contributors
    Dordick, Jonathan S.; Linhardt, Robert J.; Koffas, Mattheos A. G.; Wang, Chunyu;
    Date Issued
    2020-08
    Subject
    Chemical engineering
    Degree
    PhD;
    Terms of Use
    This electronic version is a licensed copy owned by Rensselaer Polytechnic Institute, Troy, NY. Copyright of original work retained by author.;
    Metadata
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    URI
    https://hdl.handle.net/20.500.13015/2627
    Abstract
    Chemoenzymatic synthesis of heparin, a multienzyme process, represents an emerging alternative to animal sourced heparan sulfate and heparin. Glucuronyl C5-epimerase is the first enzyme that acts on the substrate polysaccharide, N-sulfoheparosan, chemically prepared from heparosan isolated from capsule of E. coli K5. Since the epimerization reaction is associated with no easily measured physical or chemical change, direct assays to determine the activity of C5-epimerase has been a challenge. As a consequence, understanding of its reaction mechanism and its application in heparin and heparan sulfate biosynthesis is quite difficult. The goal of this thesis is to elucidate the kinetics and reaction mechanism of C5-epimerase within the context of the chemoenzymatic scheme. Polysaccharide substrates directly relevant to the production of bioengineered heparin is used. Novel NMR-based online and offline assays were developed that leverages epimerase mediated hydrogen-deuterium exchange. Apparent kinetic parameters were determined for both the forward and pseudo-reverse reactions. Atypical sigmoidal behavior was observed for the forward reaction. We have also elucidated the specificity and action pattern of C5-epimerase using polysaccharide substrates. We demonstrate that C5-epimerase specificity relates directly to the location of the N-acetylglucosamine (GlcNAc) residues within the chain. Critical information gleamed from this research leads to better understanding the fundamental mechanism of C5-epimerase and ultimately enables the optimization of large-scale production of bioengineered heparin;
    Description
    August 2020; School of Engineering
    Department
    Dept. of Chemical and Biological Engineering;
    Publisher
    Rensselaer Polytechnic Institute, Troy, NY
    Relationships
    Rensselaer Theses and Dissertations Online Collection;
    Access
    Restricted to current Rensselaer faculty, staff and students. Access inquiries may be directed to the Rensselaer Libraries.;
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