Unspecific peroxygenase from Agrocybe aegerite (AaeUPO) is a remarkable catalyst for the oxyfunctionalization of non-activated C−H bonds under mild conditions. It exhibits comparable activity to P450 monooxygenase but offers the advantage of using H₂O₂ instead of a complex electron transport chain to reductively activate O₂. Here, we demonstrate the successful oxidation of cyclohexane to cyclohexanol/cyclohexanone (KA-oil) using sol-gel encapsulated AaeUPO. Remarkably, cyclohexane serves both as a solvent and a substrate in this system, which simplifies product isolation. The ratio of cyclohexanone to cyclohexanol using this approach is remarkably higher compared to the oxidation using free AaeUPO in aqueous media using acetonitrile as a cosolvent. The utilization of sol-gel encapsulated AaeUPO offers a promising approach for oxyfunctionalization reactions and improves the chances for this enzyme to be incorporated in the same pot with other chemical transformations.

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This review article critically compares two widely used types of catalysis, chemo- and biocatalysis, and provides insights on their greenness according to specified parameters. A comparative analysis of the environmental impact of chemo- and biocatalytic oxyfunctionalisation reactions based on published experimental data reveals that both methods produce comparable amounts of waste, with the majority stemming from the solvent used. However, it is emphasised that the synthesis of the catalysts themselves, including biocatalysts, should also be considered when assessing their environmental impact. The review underscores the complexity of assessing the environmental impact of catalytic oxyfunctionalisation reactions. The article also discusses the relationship between solvent properties and the energy demands for chemical transformations and downstream processing, underlining that the choice of solvent can significantly influence the environmental impact of a catalytic process. Additionally, the review highlights the importance of considering the recyclability of reagents and the secondary CO2 emissions caused by the energy requirements of the reaction when evaluating the environmental impact of a catalytic process. Each chemo- and biocatalysis produce a certain environmental impact, the greenness of either method is dependent on several factors, including the type of waste generated, the recyclability of reagents, and secondary CO2 emissions. This review therefore recommends using consistent metrics and a comprehensive life cycle assessment approach to evaluate this environmental impact, and highlights the importance of considering the synthesis of the catalysts themselves.@en

The hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. In this study, we have engineered a highly regioselective fungal peroxygenase for the ω-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutation near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward subterminal hydroxylation with a drop in the over-oxidation activity. While crystallographic soaking experiments and molecular dynamic simulations shed light on this unique oxidation pattern, the selective biocatalyst was produced by Pichia pastoris at 0.4 g L⁻¹ in a fed-batch bioreactor and used in the preparative synthesis of 1.4 g of (ω-1)-hydroxytetradecanoic acid with 95 % regioselectivity and 83 % ee for the S enantiomer.

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Utilisation of fatty acids generally relies on pre-existing functional groups such as the carboxylate group or C=C-double bonds. Addition of new functionalities into the hydrocarbon part opens up new possibilities for fatty acid valorisation. In this contribution we demonstrate the synthetic potential of a peroxygenase mutant AaeUPO−Fett for selective fatty acid oxyfunctionalisation. The ω-1 hydroxy fatty acid (esters) produced are further transformed into lactones, alcohols, esters and amines via multi-enzyme cascades thereby paving the way for new fatty acid valorisation pathways.

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Unspecific peroxygenases (UPOs) are promising biocatalysts for oxyfunctionalisation reactions, owing to their simplicity of handling, stability and robustness. A limitation of using UPOs on a large scale is their deactivation in the presence of even rather modest concentrations of H₂O₂, requiring a constant and controlled supply of low amount of H₂O₂. Herein, we report an organometallic complex [Cp*Ir(pica)NO₃] {pica=picolinamidate=κ²-pyridine-2-carboxamide ion (−1)} 1 capable of efficiently regenerating FMNH₂ from FMN (TOF=350 h⁻¹, 298 K), driven by NaHCOO; FMNH₂, in turn, spontaneously reacts with O₂ leading to H₂O₂. After having studied the compatibility of 1 with the UPO from Agrocybe aegerita (rAaeUPO PaDa-I) and individuated the best experimental conditions, we applied such a hybrid catalytic tandem in some hydroxylation, epoxidation and sulfoxidation reactions. Best performances were obtained by using a 1/rAaeUPO molar ratio of 50. TONs for the biocatalyst of up to 18933 were obtained for the transformation of ethylbenzene derivatives into (R)-1-phenylethanols (ee>99 %). 1/rAaeUPO was found to oxidise also cis-methyl styrene (TON=13488), leading exclusively (1R,2S)-cis-methyl styrene oxide (ee>99 %), cyclohexane (TON=1634) and thioanisole (TON=1369).

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Catalysts are often used in challenging chemical reactions to accelerate the reaction rate, increase reaction efficiency, reduce energy consumption, and minimise waste production. In biocatalysis, enzymes or whole cells are used as catalysts with the advantage of reactivity, selectivity and mild reaction condition over chemocatalysis. Nowadays, with the increasing variety of enzymes, biocatalysis exhibits more and more applicability potential as an alternative tool for chemical reactions. This thesis focuses on two categories of challenging chemical reactions: oxyfunctionalisation and decarboxylation reactions, where two enzyme families have been investigated. Unspecific peroxygenases (UPOs) exhibit remarkable catalytic activity by facilitating the specific incorporation of oxygen atoms into both C-H and C=C bonds through hydroxylation and epoxidation reactions, respectively. This biocatalytic ability occurs under mild reaction conditions, rendering UPOs highly versatile and attractive for various synthetic applications. Fatty acid photodecarboxylases (FAPs) demonstrate the capacity to effectively catalyse the cleavage of carboxylic groups from substrates, leading to the formation of the corresponding alka(e)nes when subjected to illumination. This photoenzymatic reaction offers a sustainable and environmentally friendly pathway for the conversion of fatty acids into valuable hydrocarbon products by harnessing light as an energy source. In chapter 1, we show a critical and quantitative comparison between chemocatalysis and biocatalysis in oxyfunctionalisation reactions and an overview of decarboxylation reactions. For oxyfunctionalisation reactions, this thesis is focusing on both classic hydroxylation and epoxidation reactions. For instance, further derivatisation fatty acids generally relies on pre-existing functional groups such as the carboxylate group or C=C-double bonds. However, the enzymatic conversions of saturated, non-activated fatty acids remain relatively underdeveloped, primarily owing to the inherent difficulty of C-H activation. In chapter 2, we demonstrate the application of a peroxygenase mutant AaeUPO-Fett for selective fatty acid hydroxylation. The primary products (i.e. hydroxy fatty acids) are interesting building blocks for lactone and polyester synthesis. Besides, when the produced w-1 hydroxy fatty acid (esters) are transformed, further synthetic possibilities arise as demonstrated by the fatty acid decarboxylation, Baeyer-Villiger oxidation and reductive amination reactions. Thereby, the utilisation of peroxygenase-promoted enzymatic cascades has emerged as a versatile toolbox for the conversion of recalcitrant saturated fatty acids into valuable products and essential building blocks... @en

The fatty acid photodecarboxylase from Chlorella variabilis NC64 A (CvFAP) catalyses the light-dependent decarboxylation of fatty acids. Photoinactivation of CvFAP still represents one of the major limitations of this interesting enzyme en route to practical application. In this study we demonstrate that the photostability of CvFAP can easily be improved by the administration of medium-chain length carboxylic acids such as caprylic acid indicating that the best way of maintaining CvFAP stability is ‘to keep the enzyme busy’.

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Light-driven biocatalytic processes are notoriously hampered by poor penetration of light into the turbid reaction media. In this study, wirelessly powered light-emitting diodes are found to represent an efficient and scalable approach for process intensification of the photobiosynthetic production of diesel alkanes from renewable fatty acids.

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Oxyfunctionalisation reactions in neat substrate still pose a challenge for biocatalysis. Here, we report an alginate-confined peroxygenase-CLEA to catalyse the enantioselective epoxidation of cis-β-methylstyrene in a solvent-free reaction system achieving turnover numbers of 96 000 for the biocatalyst and epoxide concentrations of 48 mM.

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