Purification and characterization of alpha-amylase from safflower (Carthamus tinctorius L.) germinating seeds.
alpha-Amylase (alpha-1-4 D-glucan glucanohydrolase EC 3.2.1.1) crude extract was obtained from safflower (Carthamus tinctorius L.) cotyledons excised from 5-day-old dark grown seedlings. The enzyme was purified by precipitating the crude extract with ammonium sulphate at 20-60% saturation, and then by subjecting this fraction to affinity chromatography on a beta-cyclodextrin-Sepharose 6B column. The active fraction was dialysed and concentrated. An overall purification of about 131 folds with an activity yield of 81.25% was achieved. The molecular mass of purified enzyme determined by SDS-PAGE was 35 kD. When the purified alpha-amylase was subjected to gel electrophoresis followed by negative staining, only one band of active protein was detected. Its maximal activity was in the pH 6.0 and at a temperature of 55 degrees C. This enzyme was activated by Ca(2+) and inhibited by Fe(2+).
Ben Elarbi
Mosbah
M
Département de biologie, Tunis, Tunisia. mosbaharbi@yahoo.fr
Khemiri
Halima
H
Jridi
Taoufik
T
Ben Hamida
Jeannette
J
D031187:Carthamus tinctorius / Q000201:enzymology* / Q000254:growth & development; D002413:Cations, Divalent / Q000494:pharmacology; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D018525:Germination; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008970:Molecular Weight; D010940:Plant Proteins / Q000302:isolation & purification* / Q000378:metabolism; D012639:Seeds / Q000201:enzymology; D013213:Starch / Q000378:metabolism; D013696:Temperature; D000516:alpha-Amylases / Q000302:isolation & purification* / Q000378:metabolism
D002413:Cations, Divalent; D010940:Plant Proteins; D013213:Starch; D000516:alpha-Amylases