Purification ofct-Amylasefrom Bacillus stearothermophilus by Ultrafiltration Membrane
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The objective of this experiment was to evaluate the optimum conditions of cc-amylase of Bacillus slearotherinophilus purification by ultrafiltration and the step of a-amylase purification from indigenous isolate. Culture filtrate from bioreactor was separated by microfiltration membrane with a pore sIze of 0.2 tm. The aupernatant was purifie again by ultrafiltration system membrane with cut off 30 000 Dalton. Treatments of cz-amylase purified by ultrafiltration were carried out at flow rate of 30,45 and 60 mI/minute, and concentrated by about 5, 10 and 15 tImes. The crude enzymes resulted From ultrafiltration were precipitated with acetone. The results showed that the optimum condition of ultrafiltration was using flow rate of 30 mI/minute and concentrated by about 10 times. At the optimum condition of ultrafiltration, the specific activity of ci-amylase was of 6 686.6 U/mg with 2.3 fold purification factor. The effect of flow rate decreased the total enzyme activity, specific activity and yield. The concentration disposal could decrease total activity and protein, but not always reduced specific activity of the enzyme. Purification of crude enzyme by ultrafiltration and acetone reduced the total activity, total protein and yield, but specific activity and purification factor increased. Ultrafiltration followed by acetone precipitation, gave enzyme specific activity of 18155.4 U/mg, purification factor of 6.3 and yield of 20%, respectively. Zymogram analysis using Native-Polyacrylamide Agarose Gel Electrophoresis indicated' ci-amylase of approximately 192 932.8 Da.