Development of capillary bioreactors based on purine nucleoside phosphorylase from Aeromonas hydrophila 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 and α-D-(deoxy)ribose-1-phosphate (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 cloned and over-expressed a purine nucleoside phosphorylase (PNP) from A. hydrophila (encoded by deoD gene), tested its substrate specificity, and used it for few synthetic applications, also as immobilized biocatalyst. The aim of the present work is the development of a biochromatographic system based on this PNP as a tool to speed up the screening of new nucleoside libraries through a medium-high throughput approach and to characterize the catalytic efficiency of the biocatalyst. To this end, immobilization trials of PNP on different chromatographic supports (e.g. monolithic and/or Open Tubular Capillary) will be perfomed. The prepared bioreactors will be typified by the determination of kinetic constants (kM and Vmax), using the Michaelis-Menten kinetic model for a natural substrate (e.g. inosine) in phosphorolysis reaction and comparing the results with kinetic constants calculated for the in batch free enzyme in the same operative conditions. Experimentally, the characterization will be realized 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. The most promising bioreactor will be used to verify the system synthetic potential towards modified nucleosides.