Application of a thermostable Baeyer–Villiger monooxygenase for the synthesis of branched polyester precursors

Abstract

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.

High performing immobilized Baeyer-Villiger monooxygenase and glucose dehydrogenase for the synthesis of ε-caprolactone derivative

“…The soluble and immobilized biocatalysts were applied to the oxidation of 3,3,5trimethylcyclohexanone. Similarly to our previous studies with TmCHMO and this substrate, [15] it was necessary to control the pH during the reaction since each molecule of the substrate that was converted resulted in the formation of one molecule of D-gluconolactone which was hydrolyzed to gluconic acid and consequently increased the acidity of the reaction (Figure 1). Auto-titration of the reaction by addition of NaOH at 1 M ensured a constant pH throughout the reaction course.…”

“…[10] Additionally, several BVMOs have been identified as relevant biocatalyst for the synthesis of lactone as monomers for polymeric materials, for example, ε-caprolactone, either from whole-cell [11] or via a cascade reaction, [12] lauryl lactone, [13] a nitrile-substituted ε-caprolactone as precursor for polyamide, [14] and β,δ-trimethyl-ε-caprolactone (TMCL). [15,16] Alkyl substituted lactones are particularly interesting for the synthesis of polyesters with low glass transition temperature (Tg < 0 °C in general). [17] This property enables applications such as biodegradable plasticizers [18] or encapsulating agents for coating formulations [19] with polymers from TMCL for example.…”

“…13.22) was identified as being particularly relevant for the preparation of lactones as polymeric building blocks due to its high thermostability, good resistance to organic solvents, and broad substrate scope towards cyclic ketones. [27,28] Although TmCHMO has already been applied for the synthesis of ε-caprolactone derivatives from 3,3,5-trimethylcyclohexane, using either a self-sufficient fused biocatalyst [15] or a glucose dehydrogenase to regenerate the NADPH co-factor, [16] this enzyme has not yet been immobilized. Immobilization of whole-cells or isolated enzymes is indeed known to increase the operational stability of enzymes.…”

Trimethyl-ε-caprolactone synthesis with a novel immobilized glucose dehydrogenase and an immobilized thermostable cyclohexanone monooxygenase

“…The reaction course can be seen in Figure 4. A continuous substrate feeding strategy was used in order to avoid substrate inhibition which has been observed for this enzyme [18,43]. Even though the substrate was continuously added, certain amount of it was accumulated at the beginning of the reaction.…”

“…However, their implementation is hindered by some drawbacks that may come with biocatalysts and specially with monooxygenases [16,17]. BVMOs have been suffering from low operational stability, substrate and product inhibition and the use of the costly NADPH cofactor [11,12,18]. These limitations can be tackled mainly by three strategies: protein engineering [19], reaction engineering [20] and immobilization [21,22].…”