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

: Selective oxy-functionalization of non-activated C-H bonds is a long-standing "dream reaction" of organic synthesis for which chemical methodology is not well developed. Monooxygenase 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 NADPH, we compared soluble and co-immobilized enzymes in O2 -gassed and pH-controlled conversions at high final substrate concentrations (≥ 40 mM). We identified the main engineering parameters of process output (i.e., O2 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 O2 -limited conditions at up to 500 ml scale (10% DMSO; silicone antifoam), we developed substrate feeding strategy based on O2 feedback control. Thus, we achieved high reaction rates of ~3 g L-1 h-1 and near complete conversion (≥ 90%) of 80 mM (16 g L-1 ) C12:0 with good selectivity (≤ 5% overoxidation). We showed that "uncoupled reaction" of the P450 BM3 (~95% utilization of NADPH and O2 not leading to hydroxylation) with the C12:0 hydroxylated product limited the process efficiency at high product concentration. Hydroxylated product (~7 g; ≥ 92% purity) was recovered from 500 ml 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. This article is protected by copyright. All rights reserved.