Original Article
Niranjan Kumar Sana, Md.Shanaw
Abstract
In germinating chickpea seeds after 72 hours, the abundant amylolytic activity was found to be due to á-amylase. The enzyme was purified from germinating chickpea (Cicer arietinumL.) seed by successive 30-50%ammoniumsulphate fractionation followed byDEAE-cellulose and SephadexG-75 gel filtration chromatography to the homogenous state as confirmed by slab gel electrophoresis.The enzymewas purified 68.3-fold with a yield of 72.4% of the total activity. The purified enzyme was found to be a glycoprotein with an apparent molecular mass of 42 and 45 kDa as estimated by sodiumdodecylsulfate-polyacrylamide gel electrophoresis and gel permeation chromatography, respectively. The purified amylase fromgerminating chickpea seems to be ï¡-type as confirmed by EDTA and glycoprotein in nature. The glycoprotein was found to contain 2.7% sugar. Amylolytic activity of this enzyme was 96% and 63% for amylose and amylopectin, respectively. The enzyme has no effect on maltose and maltotetraose. The optimum pH and temperature of the purified á-amylase were 7.0 and 370C respectively.Metallic ions like Ca2+, Fe3+,Mn2+ and Na+ increased amylase activity while Cu2+, Fe2+ and Zn2+ strongly; Hg+, Ag+, Mg2+ and K+ moderately and Li+, Cd+ slightly inhibited the enzymatic activity.With increasing concentration of EDTA and urea, the activity of the purified enzyme decreased sharply. The Km value of this enzyme was found to be 0.28%for starch as substrate.