Immobilization of a purine nucleoside phosphorylase from Aeromonas hydrophila on an open tubular capillary for biocatalytic applications

Nucleoside phosphorylases (NPs; E.C. 2.4.2) catalyze the reversible cleavage of the glycosidic bond of (deoxy)ribonucleosides in the presence of inorganic orthophosphate (Pi) to generate the nucleobase (B) and α-D-(deoxy)ribose-1-phosphate [(d)R-1-P]. If a second nucleobase is added to the reaction medium the formation of a new nucleoside can result (transglycosylation). Thus, NPs can be used for chemoenzymatic synthesis of both natural and unnatural nucleosides as an alternative to conventional chemical methods which are generally plagued by low stereoselectivity, multi-step procedures and modest yields. We have recently cloned and over-expressed a purine nucleoside phosphorylase (PNP) from A. hydrophila (AhPNP, encoded by deoD gene), tested its substrate specificity, and used it for few synthetic applications, also as immobilized biocatalyst. Here we described the development of a biochromatographic system based on this PNP as a tool to speed up the screening of nucleoside libraries aimed at biocatalytic applications. To this end, AhPNP has been immobilized on a Fused Silica Open Tubular Capillary (FSOTC) via a Schiff base chemistry. The resulting bioreactor has been typified by the determination of kinetic constants (Km and Vmax) for a natural substrate (inosine) in phosphorolysis reaction and then assayed versus all natural purine (deoxy)ribonucleosides. Characterization of the bioreactor has been set up through a bidimensional chromatographic system with the sample on-line transfer from the bioreactor to the analytical column for the separation and quantification of the substrate and product. Comparison with non-immobilized enzyme showed that the developed bioreactor is a reliable and robust tool for the medium-high throughput screening of nucleosides. Moreover, AhPNP-based bioreactor retained 100% of activity after 1 month and 50 reactions. Characterization and validation of the biochromatographic system with non natural substrates is in progress.