Urea enzymatic hydrolysis reaction: Optimization by response surface methodology based on potentiometric measurements

Abstract

The enzymatic hydrolysis reaction of urea by urease is optimized in this work by the chemometric response surface methodology (RSM), based on an initial rate potentiometric measurement using an NH4+ ion-selective electrode (ISE). In this investigation, the ranges of critical variables determined by a preliminary “one at a time” (OVAT) procedure were used as input for the subsequent RSM chemometric analysis. The RSM quadratic response was found to be quite appropriate for modeling and optimization of the hydrolysis reaction as illustrated by the relatively high value of the determination coefficient (R2=90.1%), along with the satisfactory results obtained by the analysis of variance (ANOVA). All the evaluated analytical characteristics of the optimized method such as: the linear calibration curve, the upper and lower detection limits, the within-day precisions at low and at high levels, the assay recovery in pool serum media, along with the activation kinetic parameters, were also reported. Further, in order to check the quality of the optimization and the validity of the model, the assay of urea, both in aqueous laboratory and human serum samples, were performed. It has to be noted that the kinetic initial rate measurement method used in this work, permitted to overcome the general problem of NH4+ ISE low selectivity against Na+ and K+ interfering ions in real samples.