A peroxygenase-catalysed hydroxylation of organosilanes is reported. The recombinant peroxygenase from Agrocybe aegerita (AaeUPO) enabled efficient conversion of a broad range of silane starting materials in attractive productivities (up to 300 mM h⁻¹), catalyst performance (up to 84 s⁻¹ and more than 120 000 catalytic turnovers). Molecular modelling of the enzyme-substrate interaction puts a basis for the mechanistic understanding of AaeUPO selectivity.

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Heme-dependent oxygenases (i.e. P450 monooxygenases and peroxygenases) are highly selective catalysts for the selective oxyfunctionalisation or organic compounds. Both enzyme classes exhibit mechanistic similarities (i.e. using so-called compound I (CpdI) as active oxidation species) and differences in how CpdI is formed. From the differences also practical differences arise which may influence the scalability, economic attractiveness and environmental impact of P450 monooxygenase- or peroxygenase-catalysed reactions. In this contribution we propose a range of performance indicators to compare the potential of both enzyme classes.

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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.@en

In this study, we developed a new bienzymatic reaction to produce enantioenriched phenylethanols. In a first step, the recombinant, unspecific peroxygenase from Agrocybe aegerita (rAaeUPO) was used to oxidise ethylbenzene and its derivatives to the corresponding ketones (prochiral intermediates) followed by enantioselective reduction into the desired (R)- or (S)-phenylethanols using the (R)-selective alcohol dehydrogenase (ADH) from Lactobacillus kefir (LkADH) or the (S)-selective ADH from Rhodococcus ruber (ADH-A). In a one-pot two-step cascade, 11 ethylbenzene derivatives were converted into the corresponding chiral alcohols at acceptable yields and often excellent enantioselectivity.

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This paper outlines the immobilization of the recombinant dimeric unspecific peroxygenase from Agrocybe aegerita (rAaeUPO). The enzyme was quite stable (remaining unaltered its activity after 35 h at 47^◦C and pH 7.0). Phosphate destabilized the enzyme, while glycerol stabilized it. The enzyme was not immobilized on glyoxyl-agarose supports, while it was immobilized albeit in inactive form on vinyl-sulfone-activated supports. rAaeUPO immobilization on glutaraldehyde pre-activated supports gave almost quantitative immobilization yield and retained some activity, but the biocatalyst was very unstable. Its immobilization via anion exchange on PEI supports also produced good immobilization yields, but the rAaeUPO stability dropped. However, using aminated agarose, the enzyme retained stability and activity. The stability of the immobilized enzyme strongly depended on the immobilization pH, being much less stable when rAaeUPO was adsorbed at pH 9.0 than when it was immobilized at pH 7.0 or pH 5.0 (residual activity was almost 0 for the former and 80% for the other preparations), presenting stability very similar to that of the free enzyme. This is a very clear example of how the immobilization pH greatly affects the final biocatalyst performance.

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The chemoenzymatic oxidative decarboxylation of glutamic acid to the corresponding nitrile using the vanadium chloroperoxidase from Curvularia inaequalis (CiVCPO) as HOBr generation catalysts has been investigated. Product inhibition was identified as major limitation. Nevertheless, 1630000 turnovers and k_cat of 75 s⁻¹ were achieved using 100 mM glutamate. The semi-preparative enzymatic oxidative decarboxylation of glutamate was also demonstrated.

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