(600ag) Assays for Enzymes Used in the Chemoenzymatic Synthesis of Heparin

The goal of this project is to develop assays for the chemoenzymatic synthesis of bioengineered heparin. Heparin is a widely used anticoagulant drug that currently comes from animal sources, such as porcine intestine. Heparin has been subject to contamination in the past, especially in a worldwide contamination crisis in 2008. A bioengineered heparin would increase heparin availability and improve drug safety. In such a process, heparosan, produced by E. coli fermentation, is chemically converted to N-sulfoheparosan, which is then further modified by O-sulfotransferases (OSTs) and C5 epimerase to produce bioengineered heparin.  2-OST, 6-OST-1, and 3-OST-1 transfer sulfo groups from 3’phosphoadenosine-5’phosphosulfate (PAPS) to N-sulfoheparosan affording bioengineered heparin.  An assessment of catalytic activity is required to control these enzymatic steps. Radiometric OST assays are expensive and unsafe to apply at the industrial scale.  Assays being developed use fluoresecently labeled oligosaccharide substrates and offer reduced cost, improved safety and convenience for monitoring OSTs used in our bioengineered heparin process.