Pseudomonas sp. VLB120 uses styrene as a sole source of carbon and energy. The first step in this metabolic pathway is catalyzed by an oxygenase (StyA) and a NADH-flavin oxidoreductase (StyB). Both components have been isolated from wild-type Pseudomonas strain VLB120 as well as from recombinant Escherichia coli. StyA from both sources is a dimer, with a subunit size of 47 kDa, and catalyzes the enantioselective epoxidation of CAC double bonds. Styrene is exclusively converted to S-styrene oxide with a specific activity of 2.1 U mg

The performance of biocatalytic reactions is often hampered by product and/or substrate toxicity and short-term reaction times due to instable biocatalysts. Microbes in biofilms show a remarkable resistance against biocides and form stable communities. In nature, especially in environments characterized by harsh conditions such as heavily contaminated sites, cells grow pre-dominantly in biofilms, which enable them to cope with physiological stress. This robustness was utilized to design a bioprocess concept based on catalytic biofilms for stable long-term transformations of toxic reactants. Sixty-nine bacterial strains have been screened to find organisms suitable for biofilm-based biotransformations. This included host strains important for recombinant enzyme expression and strains isolated from biofilters or contaminated soils. Nearly all organisms with bioremediation potential showed good biofilm forming capacities. Pseudomonas sp. strain VLB120DeltaC was chosen as a model organism due to its excellent biofilm forming capacity and its well-studied capability of catalyzing asymmetric epoxidations. A tubular reactor was used for the biotransformation of styrene to (S)-styrene oxide as a model reaction. The process was stable for at least 55 days at a maximal volumetric productivity of 16 g/(L(aq) day) and a yield of 9 mol%. In situ product extraction prevented product inhibition of the catalyst. Biofilm physiology and dynamics are characterized during the biotransformation and limitations and advantages of this reaction concept are discussed.

Electrochemical regeneration of nicotinamide cofactors has been discussed as a promising, clean, and sustainable technology since the 1980s. However, most concepts for the coupling of this technology to enzymes suffer from low productivities, insufficient stabilities, or are difficult to scale up. We have developed an electrochemical cell for the efficient regeneration of NAD(P)H, which can be coupled to a reduction reaction catalyzed by the thermophilic alcohol dehydrogenase from Thermus sp. Octane, as second organic phase avoided product inhibition and allowed for the production of (1S,3S)-3-methylcyclohexanol at a diastereomeric excess of 96 % from the corresponding racemic ketone with a productivity of 0.13 g L À1 h À1 and a current efficiency of 85 %. After 10 h, the experiment was actively terminated and the final product concentration reached was 1.32 g L À1. In our opinion this concept defines a new state of the art in electroenzymology and provides a strong basis for applications in organic synthesis.

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