Biotechnological applications of cytochromes P450 show difficulties, such as low activity, thermal and/or solvent instability, narrow substrate specificity and redox partner dependence. In an attempt to overcome these limitations, an exploitation of novel thermophilic P450 enzymes from nature via uncultured approaches is desirable due to their great advantages that can resolve nearly all mentioned impediments. From the metagenomics library of the Binh Chau hot spring, an open reading frame (ORF) encoding a thermostable cytochrome P450—designated as P450-T3—which shared 66.6% amino acid sequence identity with CYP109C2 of Sorangium cellulosum So ce56 was selected for further identification and characterization. The ORF was synthesized artificially and heterologously expressed in Escherichia coli C43(DE3) using the pET17b system. The purified enzyme had a molecular weight of approximately 43 kDa. The melting temperature of the purified enzyme was 76.2 °C and its apparent half-life at 60 °C was 38.7 min. Redox partner screening revealed that P450-T3 was reduced well by the mammalian AdR-Adx4-108 and the yeast Arh1-Etp1 redox partners. Lauric acid, palmitic acid, embelin, retinoic acid (all-trans) and retinoic acid (13-cis) demonstrated binding to P450-T3. Interestingly, P450-T3 also bound and converted testosterone. Overall, P450-T3 might become a good candidate for biocatalytic applications on a larger scale.

A predicted alkali‐thermophilic cytochrome P450‐T2 from the DNA database of Binh Chau hot spring was synthesized, expressed and purified. The properties of the enzyme, including the optimum pH and temperature, melting temperature and half‐life at 50 °C were characterized. P450‐T2 accepts electrons from the BmCPR‐Fdx2 system. l‐Mimosine and emodin may be its putative substrates.