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

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

Purification and characterization of two cysteine proteases from germinated barley seeds

Different stages during barley [Hordium vulgare cv. Giza 126] gennination showed endoproteolytic activity. The changes of endoproteolytic activity and protein were detected with different stages during germination. In presence of gibberellic acid [GA 3], no changes could be detected in the profile of protease activity except on day 4 where the pinteolytic activity was increased to a 1.25 fold over its value in absence of GA3. During purification of H. vulgare protease, ion exchange chromatography on DEAE-cellulose led to five separate forms [from P1 to P5]. Proteases P3 and PS with the highest specific activities were pure after chromatography on Sephacryl 5-200. The molecular weights of P3 and PS were 25,000 and 30,000, respectively. By SDS-PAGE, the P3 and PS were composed of a single band of molecular weights of 24,000 and 30,000, respectively, indicating that the two proteases are brobably monomers. Barley P3 and P5 exhibited pH optima at 3.5 and 4.0, respectively. Km values for barley P3 and P5 were estimated to be 5.4 and 4.2 mg azocaseine/ml, respectively. Varying protease activity was detected for P3 and P5 when supplied with various proteins as substrates. P3 and P5 were found to have temperature optima at 30 and 40°C, respectively. P3 and P5 were stable up to 40°C and retained 45% and 35% of their activities at 60°C, respectively. The stability of P5 toward metal ions inactivation was considerably higher than the stability of P3. Only inhibitors of cysteine proteases significantly inhibited P3 and P5, while DTT as a reducing agent enhanced P3 and P5 activities. These results indicated unequivocally that P3 and P5 are cysteine proteases. With the purification of barley P3 and PS, the physiological roles of these specific proteases in germinating barley and the regulation of their expression by GA 3 can be addressed