Abstrait

Purification and characterization of -amylase from germinating chickpea (Cicer arietinum L.) seed

Niranjan Kumar Sana, Md.Shanawazur Rahman, Md.Mehedi Hasan, Bidhan Chandra Sarkar, Md.Entazul Huque, Ranajit Kumar Shaha

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 -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.