Extraction and Characterization of Alpha Amylase from Phaseolus Aconitifolius.

Abstract

The Alpha amylase breaks down long chain carbohydrates and that's why it is very useful in food and pharmaceutical industries. The aim of the present work is to extract and characterize the Alpha amylase from germinated Mat beans, for optimum pH and temperature for activity and stability, effect of metal ions, optimum substrate concentration, Km and V max. Mat beans were germinated and then extracted with Acetate buffer (pH 5.0). The amylase assay was based on the reduction in blue color intensity resulting from enzyme hydrolysis of starch. For optimization of pH for stability, buffer solutions from pH range of 3.5 to 10.0 were prepared. For determination of optimum temperature, the range used was 35 o C to 65 o C. Substrate solutions in range of 0.1% to 4.0% were prepared for optimum substrate concentration. Mat beans germinated for 3 days show maximum enzymatic activity (65.79U/ml). 95% of the original activity was retained in the pH range 7.0 to 8.0. The maximum activity of an enzyme was found to be at 55 C. 10mM Ca 2+ stimulated the enzyme activity up to 123%. Optimum substrate concentration was found to be 1.5%. Km was found to be 2.0 mM, and Vmax 100.0 nm/min. Alpha amylase extracted from germinated Mat beans follows Michaelis-Menten equation. It can be concluded that Mat bean is a good source of Alpha amylase and this enzyme could find promising application of hydrolysis of starch.