Multienzymatic in situ hydrogen peroxide generation cascade for peroxygenase-catalysed oxyfunctionalisation reactions

Milja Pesic, Sébastien Jean Paul Willot, Elena Fernández-Fueyo, Florian Tieves, Miguel Alcalde, Frank Hollmann*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

16 Citations (Scopus)
45 Downloads (Pure)


There is an increasing interest in the application of peroxygenases in biocatalysis, because of their ability to catalyse the oxyfunctionalisation reaction in a stereoselective fashion and with high catalytic efficiencies, while using hydrogen peroxide or organic peroxides as oxidant. However, enzymes belonging to this class exhibit a very low stability in the presence of peroxides. With the aim of bypassing this fast and irreversible inactivation, we study the use of a gradual supply of hydrogen peroxide to maintain its concentration at stoichiometric levels. In this contribution, we report a multienzymatic cascade for in situ generation of hydrogen peroxide. In the first step, in the presence of NAD+ cofactor, formate dehydrogenase from Candida boidinii (FDH) catalysed the oxidation of formate yielding CO2. Reduced NADH was reoxidised by the reduction of the flavin mononucleotide cofactor bound to an old yellow enzyme homologue from Bacillus subtilis (YqjM), which subsequently reacts with molecular oxygen yielding hydrogen peroxide. Finally, this system was coupled to the hydroxylation of ethylbenzene reaction catalysed by an evolved peroxygenase from Agrocybe aegerita (rAaeUPO). Additionally, we studied the influence of different reaction parameters on the performance of the cascade with the aim of improving the turnover of the hydroxylation reaction.

Original languageEnglish
JournalZeitschrift fur Naturforschung - Section C Journal of Biosciences
Publication statusPublished - 2018

Bibliographical note

Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.


  • formate dehydrogenase
  • hydrogen peroxide generation
  • old yellow enzyme
  • oxyfunctionalisation
  • peroxygenase


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