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Purification and characterization of -amylase from germinating wheat (Triticum aestivum L.) seed

Niranjan Kumar Sana, Bidhan Chandra Sarkar, Goutam Kumar Sarker, Md.Entazul Huque, Shaha Ranajit Kumar

In germinating wheat (Triticum aestivum L) seeds at 42 hours the abundant amylolytic activity was found to be due to -amylase (-1-4-glucan maltohydrolase). The enzyme was purified to homogeneity by ammonium sulphate precipitation followed by gel filtration on Sephadex G-75, and DEAE-cellulose chromatography. The enzyme was found to be more active against starch (pea) and amylopectin than soluble starch used as substrate. The -amylase showedmaximumactivity at pH6.0 and at 45C.The enzyme was stable at a pHrange of 4.0-8.0 and at 30-60Cfor 15min. Themolecular weight of the enzyme was estimated to be 88kDa by Sephadex G-75 column chromatography and 89kDa by sodiumdodecylsulfate gel electrophoresis (SDS-PAGE). The Km value for -amylase with soluble starch as substrate was found to be 1.47mg/ml. The enzyme was completely inactivated by Cu2+, Hg2+, Pb2+, Urea andAg+ at 0.5mMconcentration and its activity was increased by the addition of Fe3+,Mn2+ and EDTA. The study indicates the importance of - amylase as a starch-degrading enzyme.