Immobilized purine nucleoside phosphorylase from Aeromonas hydrophila as an on-line enzyme reactor for biocatalytic applications

We described the development of a biochromatog. system which uses a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP) for the evaluation of the substrate specificity on nucleoside libraries. AhPNP has been covalently immobilized on a fused silica Open Tubular Capillary (OTC) via Schiff base chem. The resulting bioreactor has been characterized by the detn. of kinetic consts. (Km and Vmax) for a natural substrate (inosine) and then assayed vs. all natural purine (deoxy)​ribonucleosides and a small library of 6-​substituted purine ribosides. Characterization of the bioreactor has been carried out through a bidimensional chromatog. system with the sample online transfer from the bioreactor to the anal. column for the sepn. and quantification of substrate and product. Comparison with the sol. enzyme showed that the AhPNP-​based bioreactor is reliable as the same ranking order, with respect to the std. activity assay, was obtained. The stability of the IMER was also assessed and the system was found to be stable up to 60 reactions.