Process Development through Solvent Engineering in the Biocatalytic Synthesis of the Heterocyclic Bulk Chemical ε‐Caprolactone

Abstract

For an alternative synthetic approach toward the heterocyclic industrial chemical ε‐caprolactone, which is based on a biocatalytic oxidation of readily available cyclohexanol with air in aqueous media (using an alcohol dehydrogenase and a Baeyer–Villiger monooxygenase as enzyme components), a solvent engineering has been carried out identifying isooctane as a suitable co‐solvent. Biotransformations in an aqueous‐isooctane biphasic solvent system were found to proceed faster at both investigated substrate concentrations of 40 and 80 mm, respectively, compared with the analogous enzymatic reactions in pure aqueous medium. In addition, in all cases quantitative conversions were observed after a reaction time of 23 h when using isolated enzymes. The achievements indicate a high compatibility of isooctane [10%(v/v)] with the enzymes as well as the potential for an in situ removal of the organic reaction components, thus decreasing inhibition and/or destabilization effects of these organic components on the enzymes used. In contrast, so far, the use of recombinant whole‐cells gave less satisfactory results, which might be due to limitations of the permeation of, for example, the substrate through the cell membrane.

High‐Throughput Feasible Screening Tool for Determining Enzyme Stabilities against Organic Solvents Directly from Crude Extracts

“…Furthermore, the T m values are in good accordance with the residual activity measured in crude extract after 4 h (Figure S6), as well as with the conversions obtained in biotransformations, under the assumption that the CHMO is the limiting biocatalyst in the biotransformation (Scheme ) . This assumption could be confirmed by determining the melting temperature of the ADH.…”

“…This proof of concept, of measuring thermodynamic enzyme stabilities of a recombinant protein, even in crude extracts, was also confirmed by a spiking experiment with 10 % EtOAc (Figure ); this is known from previous studies to have a highly destabilizing and deactivating effect on the CHMO …”

Process design of a continuous biotransformation with in situ product removal by cloud point extraction

“…Considering the flow regime, batch‐wise processes are mainly reported [ 15,31–36 ] for extractive biocatalysis, whereas only few authors realized a continuous process [ 7,37,38 ] ; although the continuous operation generally results in higher productivity and more uniform product quality. [ 39 ] Ritter and Smirnova [ 7 ] have shown a successful realization of a continuous ISPR process in a stirred extraction column for penicillin G (PenG) hydrolysis as a model reaction (here, homogeneous catalysis was used).…”

Dielectrophoretic analysis of the impact of isopropyl alcohol on the electric polarisability of Escherichia coli whole-cells

“…Anrade et al and Reimer et al investigated the impact of immobilisation matrices and cofactor release of E. coli on (bio)catalytic reactions. They determined the impact of the cofactor loss indirectly by monitoring the enzymatic activity [6,7].…”