Pressure-enhanced activity and stability of α-l-rhamnosidase and β-d-glucosidase activities expressed by naringinase

Naringinase is an enzyme complex, expressing α-l-rhamnosidase and β-d-glucosidase activities. The impact of high pressure and temperature on naringinase activity and stability were studied, in order to assess the potential of enzyme thermostability on glycosides hydrolyses. To a better understanding of these effects on naringinase enzyme complex, they were also evaluated over α-l-rhamnosidase and β-d-glucosidase activities, using specific substrates, p-nitrophenyl α-l-rhamnopyranoside (4-NRham) and p-nitrophenyl β-d-glucopyranoside (4-NGluc), respectively. Hydrolysis rate of 4-NRham and naringin increased with pressure from 0.1 to 150 MPa. uilibrium constants for α-l-rhamnosidase and β-d-glucosidase reactions, at pressures of 0.1–200 MPa and 40 °C, were determined and best fitted with the model of Baliga and Whalley equation. Accordingly, reaction volumes of 93 and 64 mL mol−1 were obtained for α-l-rhamnosidase and β-d-glucosidase reactions, respectively. Reaction rate constants were also determined at the same experimental conditions and well fitted to the models of Golinkin, Laidlaw, Hyne and of Burris and Laidler. A negative ΔV≠ of −7.7 ± 1.5 and −20.0 ± 5.2 mL mol−1 were obtained for α-l-rhamnosidase and naringinase reactions, correspondingly, which reflect the accelerating effect of pressure on the biocatalysis. er, the KM, kcat and kcat/KM values, on naringin hydrolysis under atmospheric (0.1 MPa) and high pressure (150 MPa) conditions at different temperatures (25–80 °C) were determined. A 3-fold and 4-fold increase on naringinase thermostability was observed under 150 MPa at 70 and 80 °C, respectively, compared to 0.1 MPa experiments. In addition, a 15-fold increase of kcat/kM values from experimental conditions of 0.1 MPa and 30 °C to 150 MPa and 70 °C was observed. In fact, high pressure showed to be a powerful tool to increase stability of naringinase against thermal denaturation. In conclusion, the effect of amplification of pressure effects on reaction rates by temperature could have a pragmatic use for accelerating enzymatic reactions.