Chemoenzymatic synthetic methods are being explored for the large-scale production of heparin. D-Glucuronyl C5-epimerase plays a vital role in the synthesis of heparin and heparan sulfate (HS), as it catalyzes the conversion of D-glucuronic acid to L-iduronic acid in the heparin precursor, which in turn allows the action of 2-O-sulfotransferase (2-OST). When expressed heterologously in <i>E. coli</i>, the C5-epimerase, being a eukaryotic enzyme, is unstable and prone to misfolding and aggregation. We are exploring various methods to decrease protein aggregation and increase solubility. This involves site-selective and domain-based protein engineering that results in effective mature protein and engineered protein analog production. In addition, coupling the C5-epimerase with 2-OST results in rapid synthesis of HS and heparin precursors with high yield and structural fidelity. As a result, we are advancing a new methodology for replacing animal sourced HS and heparin.

Engineering D-glucuronyl C5-epimerase for increased enzyme solubility and activity

250th National Meeting (2015)

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