GLYCOSIDASES IN TISSUES OF SOME BRASSICACEAE - RADISH-ROOT BETA-AMYLASE - EXTRACTION, PURIFICATION AND CHARACTERIZATION

The enzyme beta-amylase (1,4-alpha-D-Glucan maltohydrolase, E.C. 3.2.1 .2) was isolated and purified to homogenity from the extract of health y radish roots (Raphanus sativus). The purification involved ammonium sulphate precipitation (100% saturation); DEAE-cellulose; hydroxylapat ite and Sephadex G-200 chromatography. The purity and homogenity of th e enzyme preparation were judged by gel filtration on Sephadex G-200 a nd disc electrophoresis. The amount of the original enzyme activity re maining was 23% after 195.2 times purification with specific activity 820 U/mg protein. The enzyme was active against starch, glycogen and c c-dextrin but it failed to hydrolyze sucrose, maltose and lactose. Its molecular weight was 58 880 daltons, as estimated by gel filtration o n Sephadex G-200. The K-m value was 2.85% for soluble starch at optimu m pH 5.0 and 45 degrees C. The enzyme was relatively heat-stable for 1 5 min at 30 and 40 degrees C, showing only about 8% loss of activity. The enzyme was completely inactivated by Cu+2 Fe+2 Ag+, Hg+2 but only moderately inhibited by p-chloromercuribenzoate. Strongly activated en zyme was obtained with EDTA, Zn+2, K+, Ca+2 and CO+2.