Optimization, Production, and Partial Purification of Thermostable -Amylase Produced by Marine Bacterium Bacillus sp. NRC12017

Abstract

α-Amylase biosynthesis was illustrated under stress circumstances of high temperature and high salinity in aerobically cultivated culture of a newly isolated moderately thermophilic bacterium of spore-forming Bacillus sp. NRC12017 in medium containing starch, peptone and yeast extract. Maximum yield (18.41 U/ml) was took place at pH 6.5 with 200 µl inoculum size at 45°C and an incubation time of 3 days. The ideal volume of the fermentation broth was found to be 15 ml in 100 ml Erlenmeyer flask, supplementation of starch at 2.5% and peptone plus yeast extract at 0.7% brought about the greatest production of α-amylase. Two fractions of α-amylase activities, designated FIII and FIV, were refined from culture filtrate utilizing ammonium sulfate (60 and 80%) and showed the main band at 30 KDa. Both fractions had the highest activity at 250 µl starch in a reaction mixture and pH 6.0-7.0. FIII and FIV showed that a temperature of 50 and 55°C and a reaction time of 20 and 30 min were the best available conditions for their activities, respectively, both fractions were stable up to 65°C and the activity was decreased drastically to 3.22 and 3.26 % when heated at 70°C. Concerning pH stability, a broad range of pH stability (5.0-11.0) was obtained by FIII and FIV.