Factorial design for the optimization of enzymatic detection of cadmium in aqueous solution using immobilized urease from vegetable waste

Free as well as alginate immobilized urease was utilized for detection and quantitation of cadmium (Cd2+) in aqueous samples. Urease from the seeds of pumpkin (Cucumis melo), 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. The homogeneous enzyme preparation was immobilized in 3.5% alginate leading to 86% immobilization and no leaching of the enzyme was found over a period of 15 days at 4 °C. Urease catalyzed urea hydrolysis by both soluble and immobilized enzyme revealed a clear dependence on the concentration of Cd2+. The inhibition caused by Cd2+ was non-competitive (Ki = 1.41 × 10−5 M). The time dependent inhibition both in the presence and in absence of Cd2+ ion revealed a biphasic inhibition in the activity. A Response Surface Methodology (RSM) for the parametric optimization of this process was performed using two-level-two-full factorial (22), central composite design (CCD). The regression coefficient, regression equation and analysis of variance (ANOVA) was obtained using MINITAB® 15 software. The predicted values thus obtained were closed to the experimental value indicating suitability of the model. In addition to this 3D response surface plot and isoresponse contour plot were helpful to predict the results by performing only limited set of experiments.