Cytochrome P450 monooxygenases (CYPs) of the CYP153 family catalyse terminal hydroxylation of n‐alkanes. Alkane hydroxylating mutants of self‐sufficient CYP102A1 have also been described. We evaluated two CYP153s (a three‐component system and a fused self‐sufficient CYP), wild‐type CYP102A1 and nine CYP102A1 mutants, for the conversion of three cycloalkanes (C6, C7 and C8) and three n‐alkanes (C6, C8 and C10) using whole cells (WCs) and crude cell‐free extracts (CFEs). The aim was to identify substrate–enzyme combinations that give high product titres and space‐time yields (STYs). Comparisons were made using total turnover numbers (TTNs) and turnover frequencies (TOFs) to normalize for CYP expression. Reactions were carried out using high enzyme and substrate concentrations compatible with high STYs. Under these conditions CYP102A1 and the double R47L,Y51F mutant, although not regioselective, performed better on all substrates in terms of product titres over 8 h, and thus STYs and TTNs, than heavily mutated variants that have been reported to give very high TOFs. CYP153A6, with its ferredoxin (Fdx) and ferredoxin reductase (FdR), emerged as the superior catalyst for conversion of n‐alkanes. In addition to its excellent regioselectivity it also gave the highest final product titres and STYs in WC conversions of hexane and octane. Interaction with FdR and Fdx initially limited performance in CFEs, but with additional FdR and Fdx gave 1‐octanol titres of 50 mmol⋅LBRM−1 and TTNs exceeding 12,000 over 18 h, rivalling results reported with self‐sufficient CYPs. Selecting biocatalysts for application requires caution, since experimental conditions such as amount of substrate added and solubility as well as cofactor dependence and regeneration can have a profound effect on catalyst performance, while stability and efficiency with regard to cofactor usage (coupling efficiency) are at least as important as TOFs when high product titres and STYs are the target.
