Response surface design for the optimization of enzymatic detection of mercury in aqueous solution using immobilized urease from vegetable waste

Soluble and alginate immobilized urease was utilized for detection and quantitation of mercury in aqueous samples. Urease from the seeds of pumpkin, being a vegetable waste, was extracted and purified to apparent homogeneity (sp. activity 353 U/mg protein; A280/A260 = 1.12) by heat treatment at 48 ± 0.1 °C and gel filtration through Sephadex G-200. Homogeneous enzyme preparation was immobilized in 3.5% alginate leading to 86% immobilization, no leaching of enzyme was found over a period of 15 days at 4 °C. Urease catalyzed urea hydrolysis by soluble and immobilized enzyme revealed a clear dependence on the concentration of Hg2+. Inhibition caused by Hg2+ was non-competitive (Ki = 1.2 × 10−1 μM for soluble and 1.46 × 10−1 μM for alginate immobilized urease.). Time-dependent inhibition both in presence and in absence of Hg2+ ion revealed a biphasic inhibition in activity. For optimization of this process response surface methodology (RSM) was utilized where two-level-two-full factorial (22) central composite design (CCD) has been employed. The regression equation and analysis of variance (ANOVA) were obtained using MINITAB® 15 software. Predicted values thus obtained were closed to experimental value indicating suitability of the model. 3D response surface plot, iso-response contour plot and process optimization curve were helpful to predict the results by performing only limited set of experiments.