Expression and Characterization of a New Self-Sufficient P450 Monooxygenase (P450 AZC1) from Azorhizobium caulinodans

Oxyfunctionalization of non-activated carbon bonds by P450 monooxygenases has drawn great industrial attraction. Self-sufficient P450s containing catalytic heme and reductase domains in a single polypeptide chain offer many advantages since they do not require external electron transfer partners. Here, we report the first P450 enzyme identified and expressed from Azorhizobium caulinodans. Firstly, expression conditions of P450 AZC1 were optimized for enhanced expression in E.coli. The highest P450 content was obtained in E.coli Rosetta DE3 plysS when it was incubated in TB media supplemented with 0.75 mM IPTG, 0.5 mM ALA, and 0.75 mM FeCl₃ at 25 °C for 24 hours. Subsequently, the purified enzyme showed a broad substrate spectrum including fatty acids, linear and cyclic alkanes, aromatics, and pharmaceuticals. Finally, P450 AZC1 showed optimal activity at pH 6.0 and 40 °C and a broad pH and temperature profile, making it a promising candidate for industrial applications.