Giulia Cattaneo

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.

Betulinic acid is a PPARγ antagonist that improves glucose uptake, promotes osteogenesis and inhibits adipogenesis

PPAR antagonists are ligands that bind their receptor with high affinity without transactivation activity. Recently, they have been demonstrated to maintain insulin-sensitizing and antidiabetic properties, and they serve as an alternative treatment for metabolic diseases. In this work, an affinity-based bioassay was found to be effective for selecting PPAR ligands from the dried extract of an African plant (Diospyros bipindensis). Among the ligands, we identified betulinic acid (BA), a compound already known for its anti-inflammatory, anti-tumour and antidiabetic properties, as a PPARγ and PPARα antagonist. Cell differentiation assays showed that BA inhibits adipogenesis and promotes osteogenesis; either down-regulates or does not affect the expression of a series of adipogenic markers; and up-regulates the expression of osteogenic markers. Moreover, BA increases basal glucose uptake in 3T3-L1 adipocytes. The crystal structure of the complex of BA with PPARγ sheds light, at the molecular level, on the mechanism by which BA antagonizes PPARγ, and indicates a unique binding mode of this antagonist type. The results of this study show that the natural compound BA could be an interesting and safe candidate for the treatment of type 2 diabetes and bone diseases.

Synthesis of Adenine Nucleosides by Transglycosylation using Two Sequential Nucleoside Phosphorylase-Based Bioreactors with On-Line Reaction Monitoring by using HPLC

Uridine phosphorylase from Clostridium perfringens (CpUP, EC 2.4.2.3) was immobilized covalently in an aminopropylsilica monolithic column (25 mm×4.6 mm) upon functionalization with glutaraldehyde. Imino bonds that result from the reaction between the enzyme and the support were reduced chemically to afford a 66 % yield (13 mg) determined spectrophotometrically. The CpUP immobilized enzyme reactor (IMER) was connected to a silica particle‐based IMER that contained a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP, EC 2.4.2.1), which was developed previously and used successfully for the fast synthesis of some purine ribonucleosides by a “one‐enzyme” transglycosylation. CpUP‐IMER and AhPNP‐IMER were connected to a HPLC system by a six‐way switching valve. In this set‐up, the synthesis of 2′‐deoxyadenosine (dAdo, 8), adenosine (Ado, 9), and arabinosyladenine (araA, 10) by a “two‐enzyme” transglycosylation is coupled directly to on‐line reaction monitoring. Under the optimized transglycosylation conditions (2:1 ratio sugar donor/base acceptor; 10 mm phosphate buffer; pH 7.25; temperature 37 °C, flow rate 0.1 mL min−1), defined by a 2(5‐2)III experimental design, the conversion of dAdo and Ado was approximately 90 %, and araA was synthesized in 20 % yield.