Single Step Purification of Novel Thermostable and Chelator Resistant Amylase from Bacillus Licheniformis RM44 by Affinity Chromatography

Bacillus licheniformis RM44 was isolated from hot spring near Karachi and screened forthe production of extracellular amylase Amy RM44. Amy RM44 was purified to homogeneityon a single step by affinity chromatography using insoluble corn starch. The molecular weightof Amy RM44 was estimated to be 66 kDa by SDS–PAGE and zymographic analysis. Nine foldpurification was achieved with the specific activity of 870 U/mg that provides the total yieldof the enzyme up to 31%. Studies on purified AmyRM44 characterization revealed that theoptimum temperature of enzyme was 100 ºC. Amy RM44 was proved to be highly thermostableas it retained 50% activity after 2 h at 100 ºC. Amy RM44 was stable over wide range of pHwith optimum activity at pH 5. Enzyme activity was not significantly inhibited by SDS andEDTA. Amy RM44 also exhibited its activity towards various carbohydrates such as dextrin,pullulan, α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin.