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 E-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. (c) 2018 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.