(German title: Nutzung neuartiger, regioselektiver Halogenasen für Biotransformationen)

The provision of halogenated compounds for chemical processes, both as end products and as intermediates, plays an important role. There is increasing interest in the synthesis of halogenated compounds using halogenating enzymes. Halogenases are of great importance for such processes, as they enable selective and specific halogenation of a substrate in biotransformations and thus offer a favorable perspective for white biotechnology processes. In this sense, the substitution of classic toxic halogenating agents such as thionyl halides or sulfuryl halides in the chemical synthesis of halogenated intermediates by these enzymes is conceivable. Furthermore, there is particular interest in such processes from the pharmaceutical industry, as halogenated natural products often have a higher activity than non-halogenated ones, and from chemical synthesis chemistry in the provision of reactive intermediates. The background to the project was to demonstrate a proof-of-concept for the enzymatic synthesis of the tryptophan halogenated in positions 5 and 6.

The recombinantly expressed tryptophan-5 and tryptophan-6 halogenases were initially obtained in high purity using metal ion affinity chromatography. Due to the FADH2 dependence of the halogenases, a flavin reductase (FLR) had to be additionally provided to carry out the halogenation reaction, which reduced FAD to FADH2 under NADH consumption. The FLR was successfully isolated from a recombinant E. coli organism and provided in sufficient quality for the halogenation reaction. For an economical design of the halogenation step, it is essential to use the necessary enzymes in immobilized form for the process so that they can be recycled and to increase their service life. To this end, the service life and kinetics of the native enzymes were initially investigated and optimized and transferred to immobilized systems (Trp-5-halogenase), resulting in a significant improvement in service life. Furthermore, co-substrate recycling was successfully established, which is a mandatory prerequisite for a sustainable design of the halogenation process. As part of the project, initial preliminary investigations were carried out into the development of a continuous halogenation process, which should generate a higher space-time yield than the batch process.