Isolation, Purification, Characterization and the Possible Involvement of Histidine and Cysteine in the Catalytic Mechanism of Beta-amylase Sourced from Cassava (Manihot esculenta Crantz) Peel

Abstract

Beta-amylase is a starch hydrolyzing enzyme which is fondly used in both foods, pharmaceutical and brewing industries to convert starch into maltose. Hence, this study was carried out to isolate, purify, characterize and determine the possible involvement of histidine and cysteine in the catalytic mechanism of beta-amylase sourced from cassava (Manihot esculenta, Crantz) peels. Beta Amylase was obtained from cassava peels and purified by gel filtration and ion exchange chromatography. The homogeneity of the enzyme was established by polyacrylamide gel electrophoresis and its molecular weight by Sodium Dodecyl Sulphate Polyacrylamide Electrophoresis (SDS-PAGE) on 10% gel. The Michealis-Menten constant, Km, maximum velocity, Vmax and Kcat were obtained from Line-Weaver Bulk plot. From the plot of logVmax/km Vs pH, the apparent pk values of 5.21 and 6.58 were obtained. Effects of temperature, pH, salts concentration and temperature on stability of beta amylase activity at pH ranging from 5-8 were determined. The polyacrylamide gel electrophoresis in the presence and absence of SDS produced a single bond. The enzyme was found to have an optimum activity at pH 5 and 60^oC. The current work confirmed the presence of beta-amylase in cassava peels and was also found to be thermostable and thermoactive, good enough for some industrial applications.