alpha-Amylase from germinated barley hordeum vulgare [cv. Giza 125] seeds

ABSTRACT

The activity of alpha-amylase during germination of barley [cv. Giza 125], increased from day 0 to day 10, where it exhibited its highest level, followed by a gradual decrease in activity till day 16. alpha-Amylase A2 was purified to apparent homogeneity from 10-day-old germinated barley [cv. Giza 125] by two steps of purification via DEAE-Sepharose ion exchange and Sephacryl S-200 gel permeation chromatographies with a recovery of 24% and 12 fold purification. This amylase, having a molecular weight of 26 kDa was found to be monomeric. The pH and temperature optima of a-amylase A2 was found to be 5 and 50°C, respectively. alpha-Amylase was stable at temperatures up to 50°C for 30 min incubation. The substrate specificity study showed that the enzyme act on the substrate which had alpha-1,4-and alpha-l,6-linkages as glycogen. K[m] and V[max] values were 0.87% starch and 2.85 micro mol reducing sugar liberated/min, respectively. Ca[2+], Li[+], Ba[2+] and Zn[2+] were found to have activating effect and Ni[2+] and Hg[2+] completely inactivated the activity. EDTA, sodium citrate and sodium oxalate had different inhibition effects on the enzyme activity except sodium oxalate at 1 mM which had activation effect. alpha-Nature and endo-action of this amylase was identified by its ability to reduce the viscosity of starch solution