From Enzyme to Preparative Cascade Reactions with Immobilized Enzymes: Tuning Fe(II)/?-Ketoglutarate-Dependent Lysine Hydroxylases for Application in Biotransformations

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