Partial Purification of Alpha-Amylase Produced by Brevibacillus Borstelensis R1
Journal Title: International Journal of Engineering Sciences & Research Technology - Year 30, Vol 3, Issue 5
Abstract
Ideal purification should optimize both the purity and the concentration of the metabolite. Alpha-amylase is an extracellular enzyme. The precipitate collected from 70% (NH4)2SO4 salting-out was dissolved in required amount of 0.1M phosphate buffer (pH 6.8). The dialysis was conducted to get rid of the ions in the protein. After dialysis in the buffer for 24hrs, the sample was subjected to gel filtration. The fraction number 38th had shown highest α-amylase activity (3793±12U/ml) with protein concentration (4.8mg/ml). The fold purification obtained (3.9) when the sample was subjected to sephadex G-100 gel filtration. The specific activity increased from 202.6U/mg protein to 790.21U/mg protein. In ascending paper chromatography and thin layer chromatography the Rf value of test (maltose formed by the action of α-amylase on starch) was similar to standard maltose. Native polyacrylamide gel electrophoresis (PAGE) was conducted to determine the homogeneity of α-amylase at pH 8.3 in 12% slab gel. Specific staining was conducted to confirm the protein band as α-amylase.
Authors and Affiliations
K. Suribabu*1
Keywords
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