| Summary: | Fe(II)/α-ketoglutarate-dependent dioxygenases (KDOs) catalyze a broad range of selective C–H oxidation reactions. However, the difficult production of KDOs in recombinant <i>E. coli</i> strains and their instability in purified form have so far limited their application in preparative biotransformations. Here, we investigated the immobilization of three KDOs (<i>Ca</i>KDO, <i>Cp</i>KDO, <i>Fj</i>KDO) that catalyze the stereoselective hydroxylation of the L-lysine side chain using two one-step immobilization techniques (HaloTag<sup>®</sup>, EziG™). The HaloTag<sup>®</sup>-based immobilisates reached the best results with respect to residual activity and stability. In preparative lab-scale experiments, we achieved product titers of 16 g L<sup>−1</sup> (3<i>S</i>)-hydroxy-L-lysine (<i>Ca</i>KDO) and (4<i>R</i>)-hydroxy-L-lysine (<i>Fj</i>KDO), respectively, starting from 100 mM L-lysine. Using a HaloTag<sup>®</sup>-immobilized lysine decarboxylase from <i>Selenomonas ruminantium</i> (<i>Sr</i>LDC), the (3<i>S</i>)-hydroxy-L-lysine from the <i>Ca</i>KDO-catalyzed reaction was successfully converted to (2<i>S</i>)-hydroxy-cadaverine without intermediate product purification, yielding a product titer of 11.6 g L<sup>−1</sup> in a 15 mL consecutive batch reaction. We propose that covalent in situ immobilization is an appropriate tool to access the preparative potential of many other KDOs.
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