Biocatalysis community has witnessed a drastic increase in the number of studies for the use of enzymes in continuously operated flow reactors. This significant interest arose from the possibility of...
Unspecific peroxygenases (UPOs) are among the most studied enzymes in the last decade and their well-deserved fame owes to the enzyme’s ability of catalyzing the regio- and stereospecific hydroxylation of non-activated C–H bonds at the only expense of H2O2. This leads to more direct routes for the synthesis of different chiral compounds as well as to easier oxyfunctionalization of complex molecules. Unfortunately, due to the high sensitivity towards the process conditions, UPOs’ application at industrial level has been hampered until now. However, this challenge can be overcome by enzyme immobilization, a valid strategy that has been proven to give several benefits. Within this article, we present three different immobilization procedures suitable for UPOs and two of them led to very promising results. The immobilized enzyme, indeed, shows longer stability and increased robustness to reaction conditions. The immobilized enzyme half-life time is 15-fold higher than for the free AaeUPO PaDa-I and no enzyme deactivation occurred when incubated in organic media for 120 h. Moreover, AaeUPO PaDa-I is proved to be recycled and reused up to 7 times when immobilized.
The ability of unspecific peroxygenase (UPO) to hydroxylate a wide range of substrates with just H 2 O 2 as a cosubstrate has attracted a great deal of attention in biocatalytic research. The enzyme's intrinsic limitation to be inactivated by excess amounts of the oxidative cosubstrate has been tackled with in or ex situ hydrogen peroxide (H 2 O 2 ) provision strategies. In this paper, we present the application of the covalently immobilized UPO mutant PaDa-I in a rotating bed reactor for the hydroxylation of ethylbenzene in a two-liquid-phase system. By monitoring product formation in the organic phase and H 2 O 2 concentration in the aqueous phase, the multiphasic reaction was optimized. Over 58 h, up to 414 mM (R)-1-phenylethanol was accumulated in the organic phase, corresponding to a productivity of 436 mg L −1 h −1 and a selectivity for the alcohol product over the overoxidated ketone product of 62%. It was found that the overoxidation of (R)-1-phenylethanol to acetophenone resulted in part from the H 2 O 2 concentration in the aqueous phase but mainly from the concentration of the target alcohol. Therefore, a repetitive batch was performed over five times for 13 h with similar product concentrations and formation rates as in the conventional approach but a considerably higher selectivity of 79%.
Many biocatalytic redox reactions depend on the cofactor NAD(P)H, which may be provided by dedicated recycling systems. Exploiting light and water for NADPH-regeneration as it is performed, e.g. by cyanobacteria, is conceptually very appealing due to its high atom economy. However, the current use of cyanobacteria is limited, e.g. by challenging and timeconsuming heterologous enzyme expression in cyanobacteria as well as limitations of substrate or product transport through the cell wall. Here we establish a transmembrane electron shuttling system propelled by the cyanobacterial photosynthesis to drive extracellular NAD(P)H-dependent redox reactions. The modular photo-electron shuttling (MPS) overcomes the need for cloning and problems associated with enzyme-or substrate-toxicity and substrate uptake. The MPS was demonstrated on four classes of enzymes with 19 enzymes and various types of substrates, reaching conversions of up to 99 % and giving products with > 99 % optical purity.
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