5500
Eman Ahmed Hassan Elmansy
Production And Purification Of Α-Amylase From Thermohalophilic Bacteria Isolated From Different Local Marine Environments
α-Amylase, Starch hydrolysis, Bacillus sp. NRC12017, Bacillus sp. NRC22017, thermo-halophilic bacteria
Two bacterial strains produce α-amylase were obtained from red sea sediment in Safaga and Wadi El-Natron. Based on microbiological, biochemical tests and 16S rRNA gene sequences the isolates were identified as Bacillus sp. NRC12017 and Bacillus sp. NRC22017 and were later used for further studies. The ideal conditions of different bioprocess variables that supported microbial growth and maximal αamylase production have been studied employing one factor at a time approach and were found to be as follows: maximum α-amylase yield (18.41 and 15.14 U/ml) from Bacillus sp. NRC12017 and Bacillus sp. NRC22017, respectively was achieved at pH 6.5 and 6.0 with inoculum size of 200 and 500 µl at 45°C and an incubation period of 72 hrs. The optimum volume of the fermentation medium was found 15 and 20 ml in 100 ml Erlenmeyer flask, respectively. Supplementation of starch at 2.5% and peptone plus yeast extract at 0.7% resulted in the maximum production of α-amylase from Bacillus sp. NRC12017. While the best concentration of starch as a sole carbon source and meat extract plus yeast extract concentration that provided the highest enzyme production from Bacillus sp. NRC22017 were found to be 2% and 1.05% (w/v), respectively. The produced αamylases under the obtained optimal conditions were partially purified by ammonium sulfate. This step caused an increase in specific activity of α-amylases from both strains. Two fractions of α-amylase activities were purified from culture filtrate utilizing ammonium sulfate (60 and 80%), designated FIII, FIV for active fractions from Bacillus sp. NRC12017 and FIIIa, FIVa for active fractions from Bacillus sp. NRC22017 and showed the main band at 30 KDa. The optimum conditions for α-amylase activity from both strains were found to be as follows: the optimum incubation time for FIII, FIIIa, and FIVa was 20 min and for FIV was 30 min. Activities of (FIII, FIVa) and (FIV, FIIIa) were maximum at 50 and 55°C, respectively. All fractions had maximal activity at 250 µl starch concentration in a reaction mixture. Optimum pH of both FIII and FIV was 6.0-7.0, while the ideal pH for FIIIaand FIVa activities was 7.0. Regarding temperature and pH stability, all obtained fractions were stable up to 65°C and showed pH stability over a wide range of pH (5.0-11.0).
2018
M.Sc
Ain Shams
Science