Immobilized purine nucleoside phosphorylase from Aeromonas hydrophila as an on-line enzyme reactor for biocatalytic applications.
We described the development of a biochromatographic 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 chemistry. The resulting bioreactor has been characterized by the determination of kinetic constants (Km and Vmax) for a natural substrate (inosine) and then assayed versus 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 chromatographic system with the sample on-line transfer from the bioreactor to the analytical column for the separation and quantification of substrate and product. Comparison with the soluble enzyme showed that the AhPNP-based bioreactor is reliable as the same ranking order, with respect to the standard activity assay, was obtained. The stability of the IMER was also assessed and the system was found to be stable up to 60 reactions.
Department of Drug Sciences and Italian Biocatalysis Center, University of Pavia, Via Taramelli 12, 27100 Pavia Italy.
Enrica
E
Calleri
Department of Drug Sciences and Italian Biocatalysis Center, University of Pavia, Via Taramelli 12, 27100 Pavia Italy.
Daniela
D
Ubiali
Department of Drug Sciences and Italian Biocatalysis Center, University of Pavia, Via Taramelli 12, 27100 Pavia Italy.
Immacolata
I
Serra
Department of Drug Sciences and Italian Biocatalysis Center, University of Pavia, Via Taramelli 12, 27100 Pavia Italy.
Caterina
C
Temporini
Department of Drug Sciences and Italian Biocatalysis Center, University of Pavia, Via Taramelli 12, 27100 Pavia Italy.
Giulia
G
Cattaneo
Department of Chemistry and Italian Biocatalysis Center, University of Milano, Via Golgi 19, 20133 Milano Italy.
Giovanna
G
Speranza
Department of Chemistry and Italian Biocatalysis Center, University of Milano, Via Golgi 19, 20133 Milano Italy.
Carlo F
CF
Morelli
Department of Drug Sciences and Italian Biocatalysis Center, University of Pavia, Via Taramelli 12, 27100 Pavia Italy. Electronic address: gabriella.massolini@unipv.it.
Gabriella
G
Massolini
D016980:Aeromonas hydrophila / Q000737:chemistry* / Q000378:metabolism*; D055162:Biocatalysis; D019149:Bioreactors; D055767:Immobilized Proteins / Q000737:chemistry* / Q000378:metabolism*; D007288:Inosine / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity
D055767:Immobilized Proteins; D007288:Inosine; D011683:Purine-Nucleoside Phosphorylase
Nucleosides screening; On-line enzyme reactor; Phosphorolysis reaction; Purine nucleoside phosphorylase from Aeromonas hydrophila