Immobilization of Deoxyadenosine Kinase from Dictyostelium discoideum (DddAK) and Its Application in the 5’-Phosphorylation of Arabinosyladenine and Arabinosyl-2-fluoroadenine

摘要

Deoxyadenosine kinase from Dictyostelium discoideum (DddAK) phosphorylates its natural substrate (2’-deoxyadenosine, dAdo) as well as the arabinosyladenine analogues vidarabine (araA) and fludarabine (F-araA) to their corresponding 5’-monophosphates. DddAK has been here immobilized by ionic interaction on an aminated epoxy-functionalized support (Sepabeads~(TM) ECEP), and cross-linked with oxidized dextran. The final activity recovery was 33–42 %, depending on the protein loading. Immobilization enhanced the stability of DddAK at pH 10 and, to a lesser extent, at 45℃. Phosphorylation of dAdo, araA and F-araA catalyzed by immobilized DddAK was always nearly quantitative in less than 12 hours. Fludarabine monophosphate was synthesized from F-araA (95% conversion, 20 g/L) using immobilized DddAK in fully aqueous medium, thus showing that this biocatalyst could be used for developing a preparative phosphorylation process greener and more efficient than POCl3-based phosphorylation.