Hydrogen-driven hydrocarbon oxidation by cytochrome P450 enzymes

P450BM3 (CYP102A1) from Bacillus megaterium is a 119 kDa, 1,046-residue polypeptide with the FAD/FMN reductase domain fused to the C-terminus of the haem domain and, as such it is catalytically self-sufficient; only NADPH and oxygen are required for monooxygenase activity. P450BM3 is a sub-terminal fatty acid hydroxylase, but generations of CYP102A1 engineering allowed them to be used in, e.g. drug metabolism and alkane oxidation. This thesis describes protein engineering of P450BM3 and altering reaction conditions to enhance C1 – C8 alkane oxidation activities, with the long-term goal of oxidising methane, and using the hydrogen-driven cofactor regeneration system to drive these reactions. Catalytic hydrocarbon oxidation under mild conditions is highly desirable in fuel synthesis and energy applications. Methane is a greenhouse gas and its effect can be minimised if methane is selectively oxidised to methanol, which can be used as a liquid fuel or feedstock. The R47L/Y51F, KT2, I401P and A330P mutants, which previously showed higher activities for a wide range of substrates, were used as templates to build a library of mutants. The R47L/Y51F/KT2/A330P mutant (RT2/AP) showed total turnover number (TTN) of 680 ± 10 under atmospheric pressure at ambient temperature for propane oxidation, and the TTN improved by 16-fold under 5 bar propane pressure (TTN is defined as the maximum number of moles of substrate converted per mole of P450BM3). TTN values of 14,250 ± 1,370 (KU4/AP) and 920 ± 50 (KU3/AP) were observed under 5 bar propane and 8 bar ethane, respectively, at ambient temperature. The effect of adding an inert perfluorocarboxylic acid (PFC), which resembles the structures of natural substrates and constrains small alkanes to bind closer to the haem, was investigated. The R47L/Y51F/N70S/M237L/A328V mutant (RL/YF/NMA) with PFC11 gave a TTN of 13,590 ± 30 under 5 bar propane at ambient temperature. Higher TTNs of 26,320 ± 1,010 for propane and 1,440 ± 70 for ethane oxidation were observed for the RL/YF/NMA mutant at 4 °C due to improved aqueous alkane solubility. Octane and propane oxidations were performed using a hydrogen-driven NADP+ recycling system without changing the selectivity of products, although the observed propane oxidation activity was 15% of the glucose/GDH cofactor recycling system.