Unravelling the role of ultrasonic energy in the enhancement of enzymatic kinetics

The enzymatic hydrolysis of anabolic androgenic steroids excreted to urine as glucuronide conjugates has been recently reported to be improved by applying ultrasonic energy to the reaction medium. The hydrolysis time using β-glucuronidase from Escherichia coli K12 was reduced by a factor of six when ultrasonic energy was employed to enhance the enzymatic kinetic. In this study, the effect of ultrasonic energy on the enzymatic hydrolysis kinetic parameters, as well as on the enzymatic activity of β-glucuronidase from E. coli K12 was assessed. The study was conducted using the compounds 4-nitrophenyl-β-d-glucuronide and 4-nitrophenol. Experimental data suggested that the reaction follows the Michaelis–Menten kinetics type. In addition it was found that the ultrasonic energy affects the initial velocity of reaction, which is higher when ultrasound waves are employed when compared to the classical method of incubation at 55 °C. Moreover the values of Vmax and kcat are higher for the ultrasonic essay (Vmax(US) = 17.1 ± 0.8 μM min−1; kcat(US) = 340,523 min−1; Vmax(55 °C) = 14.8 ± 0.7 μM min−1; kcat(55 °C) = 295,187 min−1) whilst the Michaelis–Menten constant obtained for the two methodologies showed similar values (KM(US) = 94.7 ± 7.2 μM; KM(55 °C) = 92.5 ± 6.6 μM). Deactivation of the enzyme was also observed under ultrasonic energy, which was found particularly evident for experimental conditions with excess of substrate; enzymatic activity half-life time increased with the increase of the E/S ratio. This research work seems to support the idea that the use of ultrasonic energy affects the transition state of the enzymatic reaction and that mass transfer processes are also most likely enhanced.