This study reports the synthesis of 3-phenylcatechol at the preparative scale using a continuous segmented flow tube-in-tube reactor (TiTR). 2-Hydroxybiphenyl 3-monooxygenase (HbpA) was applied as a biocatalyst for the hydroxylation reaction, which is dependent on the substrate 2-hydroxybiphenyl, NADH, and oxygen. While the regeneration of the cofactor NADH was guaranteed by formate dehydrogenase (FDH), oxygen was supplied via the membrane surface from the outside of the reactor system. The oxygen transfer rate through the membrane of the TiTR was determined to be 24 μmol O2 min–1 mL–1 emphasizing the potential of the TiTR as promising technology for realizing gas-dependent enzymatic reactions. Residence time and total turnover number have been identified as key limiting parameters. It was possible to scale-up this system by extending the TiTR by additional residence time units. This allowed synthesis of 1 g of 3-phenylcatechol at a high space time yield of 14.5 g L–1 h–1.

Biofilme werden aufgrund diverser Eigenschaften wie Biozidtoleranz und Selbstimmobilisierung für Anwendungen in der chemischen Synthese, beispielsweise für Biotransformationen immer interessanter.Biofilms become more and more interesting for applications in chemical synthesis (biotransformations) due to their enhanced robustness and self-immobilization.

The alcohol dehydrogenase from Thermus sp. ATN1 (TADH) was characterized biochemically with respect to its potential as a biocatalyst for organic synthesis. TADH is a NAD(H)-dependent enzyme and shows a very broad substrate spectrum producing exclusively the (S)-enantiomer in high enantiomeric excess (>99%) during asymmetric reduction of ketones. TADH is active in the presence of 10% (v/v) water-miscible solvents like 2-propanol or acetone, which permits the use of these solvents as sacrificial substrates in substrate-coupled cofactor regeneration approaches. Furthermore, the presence of a second phase of a water-insoluble solvent like hexane or octane had only minor effects on the enzyme, which retained 80% of its activity, allowing the use of these solvents in aqueous/organic mixtures to increase the availability of low-water soluble substrates. A further activity of TADH, the production of carboxylic acids by dismutation of aldehydes, was investigated. This reaction usually proceeds without net change of the NAD+/NADH concentration, leading to equimolar amounts of alcohol and carboxylic acid. When applying cofactor regeneration at high pH, however, the ratio of acid to alcohol could be changed, and full conversion to the carboxylic acid was achieved.

•Simple continuous flow-through reactor for reduction of FAD.•Principle of electrochemical plate and frame filter press cell.•Exceptionally large surface areas using porous, three-dimensional reticulated vitreous carbon electrodes.•Flavin adeninedinucleotide (FAD) was reduced at rates up to 93 mM h−1.Technical reactor limitations and low productivities have been shown to limit the implementation of electroenzymatic syntheses beyond lab-scale. One possible solution is a continuous flow-through reactor based on electrochemical plate and 6 frame filter press cells, as proposed in this study. With the aim of maximizing electroenzymatic productivities, the developed reactor set-up allows high electrochemical cofactor regeneration rates using porous, three-dimensional reticulated vitreous carbon electrodes with exceptionally large surface areas up to 19,685 m2 m−3. This system provides increased mass transfer rates and flavin adeninedinucleotide (FAD) was reduced at rates up to 93 mM h−1. The electrochemical FAD reduction was coupled to the styrene monooxygenase (StyA) catalyzed (S)-epoxidation of styrene. Electroenzymatic productivities increased with FAD reduction rates up to 1.3 mM h−1. This set-up now set the stage for efficient in vitro FAD regeneration and allows a broad electrochemical application of flavin dependent enzymes as biocatalysts.Download full-size image