Several cytochrome P450 alkane hydroxylases of the CYP153 family were recently identified and functionally expressed in Pseudomonas putida GPol2 [van Beilen JB, Funhoff EG, van Loon A, Just A, Kaysser L, Bouza M, et al. Cytochrome P450 alkane hydroxylases of the CYP153 family are common in alkane-degrading eubacteria lacking integral membrane alkane hydroxylases. Appl Environ Microbiol 2006;72:59-65]. Eight recombinants containing divergent CYP153's were cultivated on a 1.5-1.8 L scale with octane or decane/dodecane as sole carbon source. Growth rates between 0.06 and 0.23 h(-1) were obtained and analysis of cell free extracts showed that the CYP153's were expressed to a maximum of 11% of total protein. The hydroxylation activities towards various substrates translate to maximal CYP153 turnover numbers between < 0.1 and 58 min(-1). Aliphatic alkanes were exclusively hydroxylated on terminal carbon atoms. Epoxidation experiments with styrene, octene and cyclohexene showed turnover numbers that were slightly lower than the hydroxylation activities, with the exception of CYP153A11 and CYP153A14. The results provide insight in structural features that determine the substrate specificity of these enzymes. (c) 2006 Elsevier Inc. All rights reserved.