Isolation, Purification and Characterization of Alpha Amylase from Citrus sinensis

Several cultivars of Citrus sinensis were screened for amylase activity. The cultivar locally called Kinnoo which exhibited the maximum amylase activity, was selected for isolation, purification and characlerization of amylase. The extracted Alpha amylase was initially purified for Anunonium Sulphate [(NH4)2SO4] precipitation. Further purification was done by ion exchange chromatography on OEAE Cellulose and gel filtration chromatography on Sephadex G-150. The native molecular weight determined by gel filtration chromatography was 48,300 Daltons and subunit molecular weight determined by SOS-PAGE was 48,000 Daltons. Both values were approximately the same, which indicated the enzyme was a monomer. Alpha amylase was found to be homogenous by SOS-PAGE. Kinetic studies of the purified enzyme showed an optimum pH of 6.5 and optimum temperature of 40 °C. The Km value for the substrate of the enzyme was determined as 1.9x10^-5 g/mI. The enzyme showed thermo stability up to 70°C.