The advent of Green Chemistry in the 20^th century paved the way for the steady increase in biocatalyst applications, revealing the undisputed advantages that can be reached by adopting enzymes as catalysts. An excellent example is represented by the selective oxyfunctionalization of inert C-H, C-C and C=C bonds where the enzymes can help to overcome the limitations of traditional chemical approaches. In particular, unspecific peroxygenases (UPOs) are the most promising, as they can indeed oxidize these functionalities by employing hydrogen peroxide (H₂O₂) as the sole co-substrate. However, massive hurdles like cost-intensive production and sensitivity towards hydrogen peroxide still hinder UPOs practical application at the industrial level.
Therefore, one of the main goals of this Ph.D. thesis was to improve the enzyme stability and to enable its recyclability via enzyme immobilization. After an in-dept study of different carrier-bound and carrier-free methods, a valid immobilization procedure was developed and optimized. The enzyme was successfully immobilized on an amino carrier via covalent binding reaching an immobilization yield of 55% and a 15-fold higher half-life time. Further studies proved the applicability of the developed procedure in both rotating bed reactor and continuous flow system. At the same time, the hydrogen peroxide-mediated deactivation issues can be elegantly mitigated thanks to its photocatalytic in situ production. For the first time, the immobilized PaDa-I was combined with the photocatalyst graphitic carbon nitride (g-C₃N₄). Such a worthwhile combination led to promising results both in terms of high total turnover number (334,500) and increased enzyme stability.
Turning to the research conducted during a secondment at the University of Graz, the focus was shifted towards the optimization of the modular photo-electron shuttling (MPS) system developed for the regeneration of nicotinamide cofactors. In a nutshell, the conducted research furtherly proved a high flexibility of the MPS system by combining the recombinant Synechococcus elongatus PCC 7942 LkADH mutant with D- and L-HicDHs. 2-Oxoisocaproic acid and phenylpyruvate were used as model substrates, and in both cases a product formation >80% as well as an e.e. >99% was reached.