Purification and characterization of alpha-amylase from safflower (Carthamus tinctorius L.) germinating seeds

C R Biol. 2009 May;332(5):426-32. doi: 10.1016/j.crvi.2009.01.002. Epub 2009 Feb 27.

Abstract

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+).

MeSH terms

  • Carthamus tinctorius / enzymology*
  • Carthamus tinctorius / growth & development
  • Cations, Divalent / pharmacology
  • Chromatography, Affinity
  • Chromatography, Agarose
  • Germination
  • Hydrogen-Ion Concentration
  • Hydrolysis
  • Molecular Weight
  • Plant Proteins / isolation & purification*
  • Plant Proteins / metabolism
  • Seeds / enzymology
  • Starch / metabolism
  • Temperature
  • alpha-Amylases / isolation & purification*
  • alpha-Amylases / metabolism

Substances

  • Cations, Divalent
  • Plant Proteins
  • Starch
  • alpha-Amylases