New kinetic approach to the evolution of polygalacturonase (EC 3.2.1.15) activity in a commercial enzyme preparation under pulsed electric fields

The effect of pulsed electric fields (PEF) on polygalacturonase (PG) activity in an aqueous solution of a commercial enzyme preparation was studied. Monopolar square wave pulses of 4 μs were delivered to the solution, which circulated through tubular chambers with 2 stainless steel electrodes in continuous operation. The electric field intensities (E) and treatment time (t) ranged within 15 to 38 kV cm^-1 and 300 to 1100 μs, respectively. Although important reduction of the PG activity could be achieved (up to 76.5% at E = 38 kV cm^-1 and t = 1100 μs), the experimental data showed certain enhancement of PG activity under soft conditions (up to 110.9% at E = 15 kV cm^-1 and t = 300 μs). A kinetic mechanism involving 2 consecutive irreversible first-order steps was developed to describe and explain the experimental data. The tested model exhibited high accuracy in respect of the experimental data and the error of the model was lower than 4.4%. The kinetic rate constant of the first (κ₁) and second step (κ₂), ratio between the activities of intermediate and native forms of the enzyme (Λ), and other quantities related to the model, were obtained within a Bayesian framework. κ₁ resulted independent of the E applied and considerably greater in magnitude order (κ₁ mean = 6 μs^-1) with respect to κ₂, which was dependent on the applied E (mean values ranged from 489E-6 μs^-1 at E = 38 kV cm^-1 to 1215E-6 μs^-1 at 38 kV cm^-1). The dependency of κ₂ toward E was described using an exponential relationship.