BACKGROUNDIt is widely accepted that the poor thermostability of Baeyer–Villiger monooxygenases limits their use as biocatalysts for applied biocatalysis in industrial applications. The goal of this study was to investigate the biocatalytic oxidation of 3,3,5‐trimethylcyclohexanone using a thermostable cyclohexanone monooxygenase from Thermocrispum municipale (TmCHMO) for the synthesis of branched ϵ‐caprolactone derivatives as building blocks for tuned polymeric backbones. In this multi‐enzymatic reaction, the thermostable cyclohexanone monooxygenase was fused to a phosphite dehydrogenase (PTDH) in order to ensure co‐factor regeneration.RESULTSUsing reaction engineering, the reaction rate and product formation of the regio‐isomeric branched lactones were improved and the use of co‐solvents and the initial substrate load were investigated. Substrate inhibition and poor product solubility were overcome using continuous substrate feeding regimes, as well as a biphasic reaction system with toluene as water‐immiscible organic solvent. A maximum volumetric productivity, or space–time‐yield, of 1.20 g L‐1 h‐1 was achieved with continuous feeding of substrate using methanol as co‐solvent, while a maximum product concentration of 11.6 g L‐1 was achieved with toluene acting as a second phase and substrate reservoir.CONCLUSIONThese improvements in key process metrics therefore demonstrate progress towards the up‐scaled Baeyer–Villiger monooxygenase‐biocatalyzed synthesis of the target building blocks for polymer application. © 2018 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Although Baeyer–Villiger monooxygenases (BVMOs) have gained attention in recent years, there are few cases of their upscaled application for lactone synthesis. A thermostable cyclohexanone monooxygenase from Thermocrispum municipale (TmCHMO) was applied to the oxidation of 3,3,5-trimethylcyclohexanone using a glucose dehydrogenase (GDH) for cofactor regeneration. The reaction progress was improved by optimizing the biocatalyst loading, with investigation into oxygen limitations. The product concentration and productivity were increased by keeping the substrate concentration below the inhibitory level via continuous substrate feeding (CSF). This substrate feeding strategy was evaluated against two biphasic reactions using either toluene or n-butyl acetate as immiscible organic solvents. A product concentration of 38 g L–1 and a space-time yield of 1.35 g L–1 h–1 were achieved during the gram-scale synthesis of the two regioisomeric lactones by applying the CSF strategy. These improvements contribute to the large-scale application of BVMOs in the synthesis of branched building blocks for polymer applications.

The cover image, by Marie AF Delgove et al., is based on the Research Article Application of a thermostable Baeyer–Villiger monooxygenase for the synthesis of branched polyester precursors, DOI: 10.1002/jctb.5623. image