Process intensification for cytochrome P450 BM3‐catalyzed oxy‐functionalization of dodecanoic acid
| Autor: | Alexander Dennig, Moritz B. Buergler, Bernd Nidetzky |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
Biocatalysis
Protein Engineering and Nanobiotechnology Immobilized enzyme Recombinant Fusion Proteins Bioengineering process control Applied Microbiology and Biotechnology Article hydroxylation Catalysis Reaction rate Hydroxylation ARTICLES chemistry.chemical_compound Bacterial Proteins Cytochrome P-450 Enzyme System process engineering Glucose dehydrogenase process intensification NADPH-Ferrihemoprotein Reductase cytochrome P450 BM3 Lauric Acids Substrate (chemistry) Enzymes Immobilized Combinatorial chemistry Oxygen chemistry mono‐oxygenase Yield (chemistry) Organic synthesis NADP Biotechnology |
| Zdroj: | Biotechnology and Bioengineering |
| ISSN: | 1097-0290 0006-3592 |
| Popis: | 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. Cytochrome P450‐catalyzed oxy‐functionalization reactions of fatty acid substrates represent a class of synthetically important bio‐transformations. Here, the authors identified key engineering parameters for the hydroxylation of dodecanoic acid by soluble and immobilized forms of cytochrome P450 BM3 and demonstrated their integration into a coherent strategy for process intensification. Metrics of process performance (as shown in the figure) revealed a highly efficient enzymatic hydroxylation. Reaction scale up to 500 ml enabled production of gram amounts of purified product. |
| Databáze: | OpenAIRE |
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