Process intensification for cytochrome P450 BM3‐catalyzed oxy‐functionalization of dodecanoic acid

Selective oxy‐functionalization of nonactivated C‐H bonds is a long‐standing “dream reaction” of organic synthesis for which chemical methodology is not well developed. Mono‐oxygenase enzymes are promising catalysts for such oxy‐functionalization to establish. Limitation on their applicability arises from low reaction output. Here, we showed an integrated approach of process engineering to the intensification of the cytochrome P450 BM3‐catalyzed hydroxylation of dodecanoic acid (C12:0). Using P450 BM3 together with glucose dehydrogenase for regeneration of nicotinamide adenine dinucleotide phosphate (NADPH), we compared soluble and co‐immobilized enzymes in O(2)‐gassed and pH‐controlled conversions at high final substrate concentrations (≥40mM). We identified the main engineering parameters of process output (i.e., O(2) supply; mixing correlated with immobilized enzyme stability; foam control correlated with product isolation; substrate solubilization) and succeeded in disentangling their complex interrelationship for systematic process optimization. Running the reaction at O(2)‐limited conditions at up to 500‐ml scale (10% dimethyl sulfoxide; silicone antifoam), we developed a substrate feeding strategy based on O(2) feedback control. Thus, we achieved high reaction rates of 1.86g·L(−1)·hr(−1) and near complete conversion (≥90%) of 80mM (16g/L) C12:0 with good selectivity (≤5% overoxidation). We showed that “uncoupled reaction” of the P450 BM3 (~95% utilization of NADPH and O(2) not leading to hydroxylation) with the C12:0 hydroxylated product limited the process efficiency at high product concentration. Hydroxylated product (~7g; ≥92% purity) was recovered from 500ml reaction in 82% yield using ethyl‐acetate extraction. Collectively, these results demonstrate key engineering parameters for the biocatalytic oxy‐functionalization and show their integration into a coherent strategy for process intensification.