Purification and Biochemical Characterization of α-Amylase from Radish (Raphanus sativus L.) Seeds Using Response surface methodology

Abstract

The α-amylase from radish seeds has been purified to apparent homogeneity with specific activity of 821.66 U/mg of protein with 36 folds purification. The purified enzyme displayed single protein band on native PAGE (polyacrylamide gel electrophoresis) confirmed by activity staining. The SDS PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) revealed protein band of approximately 43 and 90 kDa. The calcium content of enzyme preparation was around 1.442 µg/mg of protein as analyzed by AAS (atomic absorption spectrophotometer). The energy of activation for enzyme was 3.82 kcal/moles with Km and Vmax values 4.8 mg/mL and 0.377, respectively, using starch as the substrate. The metal ions such as Ca2+ and Mg2+ showed augmented activity whereas Ni2+, Fe2+ and Cu2+ showed slight inhibition in the amylase activity. The thiol group reagents also activated the enzyme. Further, the effect of pH and temperature on α-amylase activity were analyzed and optimized utilizing response surface methodology. The value of regression coefficients was found significant (R2 = 92.62%) showing the suitability of the proposed model. The multiple regression and analysis of variance showed the individual and cumulative effect of pH and temperature on the activity of enzyme with optimum pH of 6.5 and temperature of 60 °C. Additionally, contour plot, 3D surface plot and optimization plot were used to predict the effect of each variable with minimum set of experiments.