Application of the all-in-one electrode for in situ H2O2 generation in hydroxylation catalyzed by unspecific peroxygenase from Agrocybe aegerita.

• In situ and controllable generation of H 2 O 2 via the All-in-One (AiO) electrode. • New application of the AiO electrode in the electroenzymatic process. • AiO electrode-aided enzymatic hydroxylation catalyzed by the recombinant unspecific peroxygenase. • Total turnover number up to 450,000 mol−1 was obtained in a bioelectrochemical system. The versatile unspecific peroxygenase from the fungus Agrocybe aegerita (Aae UPO) is capable to perform hydroxylation of various substrates, even unactivated C H bonds. The technical application of Aae UPO is still limited due to an instability towards its co-substrate hydrogen peroxide (H 2 O 2). Electrochemical synthesis of H 2 O 2 is an attractive approach that provides controllable in situ generation of H 2 O 2. The aims of this study are to promote catalyst efficiency and develop an individually tailored system for H 2 O 2 -dependent reactions. Herein, an All-in-One electrode (AiO) system with a carbon felt cathode is used to generate H 2 O 2 in situ and combined for the first time with an enzymatic hydroxylation. The AiO electrode combines the counter and working electrode in a single rod structure. This shape provides convenient integration into conventional bioreactors, thus, converting them into bioelectrochemical systems (BES). Hydroxylation of 4-ethylbenzoic acid (EBA) catalyzed by the recombinant Aae UPO (r Aae UPO) was chosen as the model reaction. Total turnover number (TTN) up to 450,000 mol mol−1 and turnover frequency (TOF) up to 7.7 s −1 were achieved using the AiO electrode system. The H 2 O 2 productivity was identified as the limiting factor in the hydroxylation of EBA. However, with numbering-up or surface enlargement, this process could have great potential as an optimizable platform for H 2 O 2 -dependent enzymatic reactions. [Display omitted] [ABSTRACT FROM AUTHOR]