PURIFICATION AND CHARACTERIZATION OF AMIDASE FROM BACILLUS SP. APB-6.SUDHA BATTA, POONAM KUMARI AND DUNI CHAND
Amidases (EC 188.8.131.52) hydrolyzes amides with the production of the corresponding carboxylic acids and ammonia and are the most widely used amide-hydrolyzing enzymes in industry. It holds a great potential for biotechnological applications in organic acids synthesis, bioremediation, waste water treatment, synthesis of pharmaceutical chemical like anti-cancerous and anti-HIV Hydroxamic acids etc. Here, we report the purification of amidase from Bacillus sp. APB-6 and after purification the enzyme was characterized for optimum activity. The enzyme was purified by about 21.7 fold with specific activity 19.5 U/mg protein. A purified amidase enzyme of 44kDa was obtained after initially precipitated with 60% Acetone. The acetone precipitated enzyme was then loaded on to the anion exchange column (EAECellulose) and then the protein was analysed by SDS-PAGE. The optimum temperature and pH of the enzyme was 55pC and 7.5 respectively. The Km and Vmax values for the purified amidase of APB-06 were 200mM and the Vmax was 100 μmoles/mg/min The enzyme was highly specific to substrate acetamide and less activity was detected in the presence of other amides, such as butyramide, cyanamide, nicotinamide and benzamide.
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