Purification and characterization of catechol 1, 2-dioxygenase from Rhodococcus sp. NCIM 2891

The main aim of this study was to purify Catechol 1, 2 dioxygenase from phenol utilizing Rhodococcus sp. NCIM 2891 which is basically an organism used wide for desulfurization purposes like desulfurization of diesel. The organism was cultivated in minimal medium supplemented with 500 mg/L phenol, as a sole carbon source. The temperature of incubation was 30oC at pH 7.0±0.2. The growth and phenol degradation studies show that organism was able to degrade the phenol completely within 120 h. The stationary phase of growth of the organism, however, started after 72 h. This implies that the degradation activities starts from late log phase and carries on till late stationary phase of growth. The enzyme responsible for phenol degradation was catechol 1, 2 dioxygenase. The optimal activity of catechol 1, 2 dioxygenase was observed at 500 mg/L phenol concentration. The enzyme was purified 2.34 fold by ion exchange chromatography. The Km was found to be 5 :mole with Vmax of 62.5 U/mg of protein. The optimal pH and temperature of the enzyme was 7.5 and 30oC respectively. The enzyme activity was completely inhibited by Cu, Hg and Fe metal ions. The SDS-PAGE analysis reveals that the enzyme has a molecular weight of 30 k Dalton.