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

Moritz B. Buergler, Alexander Dennig, Bernd Nidetzky*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


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 O2-gassed and pH-controlled conversions at high final substrate concentrations (≥40mM). 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% dimethyl sulfoxide; silicone antifoam), we developed a substrate feeding strategy based on O2 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 O2 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.

Original languageEnglish
Pages (from-to)2377-2388
Number of pages12
JournalBiotechnology and Bioengineering
Issue number8
Publication statusPublished - 1 Aug 2020


  • cytochrome P450 BM3
  • hydroxylation
  • mono-oxygenase
  • process control
  • process engineering
  • process intensification

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology


Dive into the research topics of 'Process intensification for cytochrome P450 BM3-catalyzed oxy-functionalization of dodecanoic acid'. Together they form a unique fingerprint.

Cite this