Since ancient times, people have been attracted by dyes and they were a symbol of power. Some of the oldest dyes are indigo and its derivative Tyrian purple, which were extracted from plants and snails, respectively. These ‘indigoid dyes’ were and still are used for coloration of textiles and as a food additive. Traditional Chinese medicine also knows indigoid dyes as pharmacologically active compounds and several studies support their effects. Further, they are interesting for future technologies like organic electronics. In these cases, especially the indigo derivatives are of interest but unfortunately hardly accessible by chemical synthesis. In recent decades, more and more enzymes have been discovered that are able to produce these indigoid dyes and therefore have gained attention from the scientific community. In this study, group E monooxygenases (styrene monooxygenase and indole monooxygenase) were used for the selective oxygenation of indole (derivatives). It was possible for the first time to show that the product of the enzymatic reaction is an epoxide. Further, we synthesized and extracted indigoid dyes and could show that there is only minor by-product formation (e.g. indirubin or isoindigo). Thus, group E monooxygenase can be an alternative biocatalyst for the biosynthesis of indigoid dyes.

Herein, different dehydrogenases (DH) were characterized by applying a novel two-step enzyme assay. We focused on the NAD(P)-dependent phenylacetaldehyde dehydrogenases because they produce industrially relevant phenylacetic acids, but they are not well studied due to limited substrate availability. The first assay step comprises a styrene oxide isomerase (440 U mg) which allows the production of pure phenylacetaldehydes (>70 mmol L) from commercially available styrene oxides. Thereafter, a DH of interest can be added to convert phenylacetaldehydes in a broad concentration range (0.05 to 1.25 mmol L). DH activity can be determined spectrophotometrically by following cofactor reduction or alternatively by RP-HPLC. This assay allowed the comparison of four aldehyde dehydrogenases and even of an alcohol dehydrogenase with respect to the production of phenylacetic acids (up to 8.4 U mg). FeaB derived from Escherichia coli K-12 was characterized in more detail, and for the first time, substituted phenylacetaldehydes had been converted. With this enzyme assay, characterization of dehydrogenases is possible although the substrates are not commercially available in sufficient quality but enzymatically producible. The advantages of this assay in comparison to the former one are discussed.

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Four phenylacetaldehyde dehydrogenases (designated as FeaB or StyD) originating from styrene-degrading soil bacteria were biochemically investigated. In this study, we focused on the Michaelis-Menten kinetics towards the presumed native substrate phenylacetaldehyde and the obviously preferred co-substrate NAD+. Furthermore, the substrate specificity on four substituted phenylacetaldehydes and the co-substrate preference were studied. Moreover, these enzymes were characterized with respect to their temperature as well as long-term stability. Since aldehyde dehydrogenases are known to show often dehydrogenase as well as esterase activity, we tested this capacity, too. Almost all results showed clearly different characteristics between the FeaB and StyD enzymes. Furthermore, FeaB from Sphingopyxis fribergensis Kp5.2 turned out to be the most active enzyme with an apparent specific activity of 17.8 ± 2.1 U mg-1. Compared with that, both StyDs showed only activities less than 0.2 U mg-1 except the overwhelming esterase activity of StyD-CWB2 (1.4 ± 0.1 U mg-1). The clustering of both FeaB and StyD enzymes with respect to their characteristics could also be mirrored in the phylogenetic analysis of twelve dehydrogenases originating from different soil bacteria.