Self-sufficient cytochromes P450 of the sub-family CYP116B have gained great attention in biotechnology due to their ability to catalyze challenging reactions toward a wide range of organic compounds. However, these P450s are often unstable in solution and their activity is limited to a short reaction time. Previously it has been shown that the isolated heme domain of CYP116B5 can work as a peroxygenase with H2 O2 without the addition of NAD(P)H. In this work, protein engineering was used to generate a chimeric enzyme (CYP116B5-SOX), in which the native reductase domain is replaced by a monomeric sarcosine oxidase (MSOX) capable of producing H2 O2 . The full-length enzyme (CYP116B5-fl) is characterized for the first time, allowing a detailed comparison to the heme domain (CYP116B5-hd) and CYP116B5-SOX. The catalytic activity of the three forms of the enzyme was studied using p-nitrophenol as substrate, and adding NADPH (CYP116B5-fl), H2 O2 (CYP116B5-hd), and sarcosine (CYP116B5-SOX) as source of electrons. CYP116B5-SOX performs better than CYP116B5-fl and CYP116B5-hd showing 10- and 3-folds higher activity, in terms of p-nitrocatechol produced per mg of enzyme per minute. CYP116B5-SOX represents an optimal model to exploit CYP116B5 and the same protein engineering approach could be used for P450s of the same class.
Keywords: CYP116; H2O2; cytochromes P450; domain switch; peroxide shunt; peroxygenase.
© 2023 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.