Isolation, Purification and Characterisation of α- Amylase from Maize seedlings

Abstract

In the present study, crude extract of the enzyme α-Amylase was obtained from 7-days old maize (Zea mays L.) seedlings. Conventional protein purification techniques such as ammonium sulphate precipitation, ion exchange chromatography using strong acidic cation exchange resin- seralite SRC – 120, followed by gel filteration on Sephadex G-100 column was employed. And the percent yield of α-amylase obtained was 51.95. SDS PAGE and gel filtration chromatography on Sephadex G 100 column was used to determine the molecular weight of the purified amylase. The purified enzyme was nearly homogeneous and its molecular weight was found to be 42,000 Daltons. The optimum pH and the temperature of the purified enzyme was 6.5 and 30⁰C respectively. Showed 100% activity with Starch as a substrate. The Km and Vmax determined by M-M graph was found to be 9.5 x10^-7 moles and 50 µmoles/minute in enzyme respectively. The Km and Vmax values by the double reciprocal plot (Lineweaver-Burk plot) were found to be 12.5x10-7moles, 0.09µmoles/minute. The activity was linear up to 30 mins. The enzyme was activated by Ca²⁺ and inhibited by Hg²⁺, Pb³⁺.