Expanding the scope of H2O2-driven biocatalysis

H2O2 is a relatively 'green' oxidant because its by-products are only H2O. In recent years, an increasing number of enzymatic synthesis methods based on H2O2 have been estab-lished. H2O2-driven reactions are usually applied as an alternative to NAD(P)H-dependent reactions to avoid complicated cofactor regeneration systems.

The aim of this thesis was to develop H2O2-driven peroxizymes-catalysed reactions. Four approaches were studied: (1) UPO-ADHs combinations for the synthesis of enantiomeri-cally pure (R)- and (S)-phenylethanol derivatives; (2) UPO-catalysed selective oxidation of silane to silanol; (3) VCPO-catalysed oxidative decarboxylation of glutamic acid to the corresponding nitrile at semi-preparative scale; (4) The investigation of the formate oxi-dase (FOx)-driven H2O2 generation system.

In Chapter 3, UPO-catalysed hydroxylation of ethylbenzene could only produce (R)-phe-nylethanol exclusively. We therefore developed a bienzymatic reaction to produce not only (R)- but also (S)-phenylethanols with the combination of a peroxygenase and com-plementary alcohol dehydrogenases. The results obtained are promising (10 samples, >91% ee). Reaction conditions for this one-pot two-step system would require further study and optimisation.

In Chapter 4, a peroxygenase-catalysed hydroxylation of organosilanes is reported. Aae-UPO enabled efficient conversion of a broad range of silane starting materials in attractive productivities (up to 300 mM h-1) and catalyst usage (up to 84 s-1 and more than 120,000 catalytic turnovers). As this enzymatic Si-H oxyfunctionalisation route is a completely new application of UPOs, there are still some limitations that need further research and inves-tigation.

In Chapter 5, the chemoenzymatic oxidative decarboxylation of glutamic acid to the cor-responding nitrile using the vanadium chloroperoxidase (CiVCPO) has been investigated. 1,630,000 turnovers and kcat of 75 s-1 were achieved using 100 mM glutamate. The semi-preparative enzymatic oxidative decarboxylation of glutamate was also demonstrated. Product inhibition was identified as a major limitation.

In Chapter 6, the formic acid oxidase (AoFOx) driven H2O2 generation system was used to drive the AaeUPO-catalysed hydroxylation of ethylbenzene derivatives. The investiga-tion of factors such as formate and enzyme spiking, pH, oxidase concentration, co-sol-vent, O2 supply and production inhibition did not solve the premature ending of the reac-tion. In our opinion, the nature of the electronic donor for AoFOx could be the break-through.

The results of this thesis contribute to the application of H2O2-driven peroxyzymes. The achievements and challenges noted in the thesis will promote the future implementation and popularisation of enzymatic oxyfunctionalisation reactions.