Isolation of Cytochrome P-450cam from Alkaliphilic bacteriaKocuria sp. DL

Cytochrome P-450 is a terminal oxidase, involved in biotransformation of endogenous and exogenous substances. Cytochrome P-450cam is isozyme present in various bacteria. CYP-450cam was purified from alkaliphilic bacteria, Kocuria sp. DL, isolated from pristine alkaline Crater Lake, Lonar, (Maharashtra State), India. Identification of this bacterial strain was carried out by analysis of 16S rRNA gene sequence (Kocuria species DL strain). The content of CYP-450cam was found to be 2.68 n mol/mg of protein in 50 h grown culture under aerobic condition. Purification of CYP-450cam was carried out by applying analytical procedures at 4 oC. The Km values for both aminopyrine and acetanilide were 0.4 and 0.25 mM, respectively. The maximal velocity Vmax was 12.98 and 13.2 mM/mg of protein per min for aminopyrine and acetanilide, respectively. The optimal pH and temperatures were found to be 7.4 and 40 oC, respectively. The purification fold was found to be 6. The molecular mass was found to be 48 kDa. Thermal stability of purified CYP-450cam is up to 40 oC. This enzyme has shown the characteristic type-I and type-II substrate binding spectra.