Production, Purification and Characterization of Alfa-amylase produced by Bacteria from Pharaonic Lake
Mahmoud M. Hazaa ,Shawky Z. Sabae, Areej Ibrahim F. T., Ayman S. Eldourghamy, H. Sayed
In the present study thirty bacteria isolates collected from Pharaonic lake, Menoufiya Governorate, Egypt. Out of which 12 isolates that were α-amylase producer. Isolate No. 1 was selected as the best enzyme producer, identified as Alloiococcus otitis and was used for further studies. Attempts were made to optimize the cultural condition for getting high yields of enzyme. The optimum level of pH for enzyme production was 8.0 on Bennett medium after 8 hours. Among carbon sources, maltose supported maximum production of α-amylase followed by lactose, sucrose, starch and manitol. Urea was the best nitrogen source for enzyme production, followed by L-asparagine. The purified fraction detected by the appearance of a single band corresponding to 38 k Da in SDS-PAGE, and the enzyme was purified 67.0-fold with amylase units 225 ml and specific activity of 234.4 U/mg. The pure enzyme was stable at all degrees of temperatures (20–70ºC). The maximum activity was at pH of 9.0. Starch was the best substrate. CaCl2, MgCl2 and KCl were the best activators. There is a continuous decreasing in the activity due to the increase of SDS and H2O2 concentrations. Enzyme was stable in presence of butanol, n-hexane and toluene.