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Assessing Peroxygenase-Mediated Oxidations in the Presence of High Concentrations of Water-Miscible Co-Solvents

Hilberath, T. (author), van Troost, A.G.L. (author), Alcalde, Miguel (author), Hollmann, F. (author)

The use of water-miscible organic co-solvents in biocatalysis is a simple procedure for obtaining higher enzymatic activities toward hydrophobic substrates. However, effects on activity and stability have to be carefully evaluated, also with regard to the type and concentration of the respective co-solvent. In this contribution, we investigated...

journal article 2022

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Biocatalytic Aromaticity-Breaking Epoxidation of Naphthalene and Nucleophilic Ring-Opening Reactions

Zhang, W. (author), Li, Huanhuan (author), Younes, S.H.H. (author), Gómez De Santos, Patricia (author), Tieves, F. (author), Grogan, Gideon (author), Pabst, Martin (author), Alcalde, Miguel (author), Whitwood, Adrian C. (author), Hollmann, F. (author)

Aromatic hydroxylation reactions catalyzed by heme-thiolate enzymes proceed via an epoxide intermediate. These aromatic epoxides could be valuable building blocks for organic synthesis giving access to a range of chiral trans-disubstituted cyclohexadiene synthons. Here, we show that naphthalene epoxides generated by fungal peroxygenases can...

journal article 2021

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Solvent-Free Photobiocatalytic Hydroxylation of Cyclohexane

Hobisch, Markus (author), van Schie, M.M.C.H. (author), Kim, Jinhyun (author), Røjkjær Andersen, Kasper (author), Alcalde, Miguel (author), Kourist, Robert (author), Park, Chan Beum (author), Hollmann, F. (author), Kara, S. (author)

The use of neat reaction media, that is the avoidance of additional solvents, is the simplest and the most efficient approach to follow in biocatalysis. Here, we show that unspecific peroxygenase from Agrocybe aegerita (AaeUPO) can hydroxylate the neat model substrate cyclohexane. H₂O₂ was photocatalytically generated in...

journal article 2020

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Evolved Peroxygenase-Aryl Alcohol Oxidase Fusions for Self-Sufficient Oxyfunctionalization Reactions

Gomez De Santos, Patricia (author), Lazaro, Sofia (author), Viña-Gonzalez, Javier (author), Hoang, Manh Dat (author), Sánchez-Moreno, Israel (author), Glieder, Anton (author), Hollmann, F. (author), Alcalde, Miguel (author)

Fungal peroxygenases are deemed emergent biocatalysts for selective C-H bond oxyfunctionalization reactions. In this study, we have engineered a functional and stable self-sufficient chimeric peroxygenase-oxidase fusion. The bifunctional biocatalyst carried a laboratory-evolved version of the fungal peroxygenase fused to an evolved fungal...

journal article 2020

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Protection strategies for biocatalytic proteins under plasma treatment

Yayci, Abdulkadir (author), Dirks, Tim (author), Kogelheide, Friederike (author), Alcalde, Miguel (author), Hollmann, F. (author), Awakowicz, Peter (author), Bandow, Julia E. (author)

In plasma-driven biocatalysis, enzymes are employed to carry out reactions using species generated by non-thermal plasmas as the precursors. We have previously demonstrated that this is feasible in principle, but that the approach suffers from the short lifetime of the biocatalyst under operating conditions. In this work, protection...

journal article 2020

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Directed evolution of unspecific peroxygenase in organic solvents

Martin-Diaz, Javier (author), Molina-Espeja, Patricia (author), Hofrichter, Martin (author), Hollmann, F. (author), Alcalde, Miguel (author)

Fungal unspecific peroxygenases (UPOs) are efficient biocatalysts that insert oxygen atoms into nonactivated C–H bonds with high selectivity. Many oxyfunctionalization reactions catalyzed by UPOs are favored in organic solvents, a milieu in which their enzymatic activity is drastically reduced. Using as departure point the UPO secretion...

journal article 2021

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A Peroxygenase-Alcohol Dehydrogenase Cascade Reaction to Transform Ethylbenzene Derivatives into Enantioenriched Phenylethanols

Xu, X. (author), Brasselet, H.L.Y. (author), Jongkind, E.P.J. (author), Alcalde, Miguel (author), Paul, C.E. (author), Hollmann, F. (author)

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...

journal article 2022

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Pilot-scale production of peroxygenase from agrocybe aegerita

Tonin, F. (author), Tieves, F. (author), Willot, S.J. (author), Van Troost, Anouska (author), van Oosten, R.J.C. (author), Breestraat, Stefaan (author), Van Pelt, Sander (author), Alcalde, Miguel (author), Hollmann, F. (author)

The pilot-scale production of the peroxygenase from Agrocybe aegerita (rAaeUPO) is demonstrated. In a fed-batch fermentation of the recombinant Pichia pastoris, the enzyme was secreted into the culture medium to a final concentration of 0.29 g L-1 corresponding to 735 g of the peroxygenase in 2500 L of the fermentation broth after 6 days. Due...

journal article 2021

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An alginate-confined peroxygenase-CLEA for styrene epoxidation

Nintzel, Friederike E.H. (author), Wu, Y. (author), Planchestainer, Matteo (author), Held, Martin (author), Alcalde, Miguel (author), Hollmann, F. (author)

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.

journal article 2021

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Hybrid Organometallic and Enzymatic Tandem Catalysis for Oxyfunctionalisation Reactions

Domestici, C. (author), Wu, Y. (author), Hilberath, T. (author), Alcalde, Miguel (author), Hollmann, F. (author), Macchioni, Alceo (author)

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...

journal article 2023

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Peroxygenase-Promoted Enzymatic Cascades for the Valorisation of Fatty Acids

Wu, Y. (author), Paul, C.E. (author), Hilberath, T. (author), Jongkind, E.P.J. (author), Zhang, Wuyuan (author), Alcalde, Miguel (author), Hollmann, F. (author)

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...

journal article 2023

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Peroxygenase-Catalysed Selective Oxidation of Silanes to Silanols

Xu, X. (author), van Hengst, J.M.A. (author), Mao, Yejia (author), Martinez, Mireia (author), Roda, Sergi (author), Floor, Martin (author), Guallar, Victor (author), Paul, C.E. (author), Alcalde, Miguel (author), Hollmann, F. (author)

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...

journal article 2023

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Nuclear Waste and Biocatalysis: A Sustainable Liaison?

Zhang, W. (author), Liu, H. (author), van Schie, M.M.C.H. (author), Hagedoorn, P.L. (author), Alcalde, Miguel (author), Denkova, A.G. (author), Djanashvili, K. (author), Hollmann, F. (author)

It is well-known that energy-rich radiation induces water splitting, eventually yielding hydrogen peroxide. Synthetic applications, however, are scarce and to the best of our knowledge, the combination of radioactivity with enzyme-catalysis has not been considered yet. Peroxygenases utilize H2O2 as an oxidant to promote highly selective...

journal article 2020

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Selective Peroxygenase-Catalysed Oxidation of Toluene Derivates to Benzaldehydes

Wang, Y. (author), Teetz, Niklas (author), Holtmann, Dirk (author), Alcalde, Miguel (author), van Hengst, J.M.A. (author), Liu, Xiaoxiao (author), Wang, Mengfan (author), Qi, Wei (author), Zhang, Wuyuan (author), Hollmann, F. (author)

Biocatalytic oxidation reactions of toluene derivates to the corresponding aldehydes are typically challenged by regio- and chemoselectivity issues. In this contribution we address both challenges by a combined reactant- and reaction engineering approach. We demonstrate that the peroxygenase-catalysed transformation of ring-substituted...

journal article 2023

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Towards preparative peroxygenase-catalyzed oxyfunctionalization reactions in organic media

Fernandez Fueyo, E. (author), Ni, Y. (author), Gomez Baraibar, A. (author), Alcalde, Miguel (author), van Langen, L.M. (author), Hollmann, F. (author)

The peroxygenase from Agrocybe aegerita (AaeUPO) has been evaluated for stereoselective oxyfunctionalization chemistry under non-aqueous reaction conditions. The stereoselective hydroxylation of ethylbenzene to (R)-1-phenylethanol was performed in neat substrate as reaction medium together with the immobilized biocatalyst and <sup>tert</sup...

journal article 2016

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Peroxygenase-catalyzed oxyfunctionalization reactions promoted by the complete oxidation of methanol

Ni, Y. (author), Fernandez Fueyo, E. (author), Baraibar, AG (author), Ullrich, R (author), Hofrichter, Martin (author), Yanase, H (author), Alcalde, Miguel (author), van Berkel, WJH (author), Hollmann, F. (author)

Peroxygenases catalyze a broad range of (stereo)selective oxyfunctionalization reactions. However, to access their full catalytic potential, peroxygenases need a balanced provision of hydrogen peroxide to achieve high catalytic activity while minimizing oxidative inactivation. Herein, we report an enzymatic cascade process that employs methanol...

journal article 2016

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Selective Synthesis of the Human Drug Metabolite 5′-Hydroxypropranolol by an Evolved Self-Sufficient Peroxygenase

Gomez De Santos, Patricia (author), Cañellas, Marina (author), Tieves, F. (author), Younes, S.H.H. (author), Molina-Espeja, Patricia (author), Hofrichter, Martin (author), Hollmann, F. (author), Guallar, Victor (author), Alcalde, Miguel (author)

Propranolol is a widely used beta-blocker that is metabolized by human liver P450 monooxygenases into equipotent hydroxylated human drug metabolites (HDMs). It is paramount for the pharmaceutical industry to evaluate the toxicity and activity of these metabolites, but unfortunately, their synthesis has hitherto involved the use of severe...

journal article 2018

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Selective aerobic oxidation reactions using a combination of photocatalytic water oxidation and enzymatic oxyfunctionalizations

Zhang, W. (author), Fernandez Fueyo, E. (author), Ni, Y. (author), van Schie, M.M.C.H. (author), Gacs, Jenö (author), Renirie, R. (author), Wever, Ron (author), Mutti, Francesco G. (author), Rother, Dörte (author), Alcalde, Miguel (author), Hollmann, F. (author)

Peroxygenases offer an attractive means to address challenges in selective oxyfunctionalization chemistry. Despite this, their application in synthetic chemistry remains challenging due to their facile inactivation by the stoichiometric oxidant H₂O₂. Often atom-inefficient peroxide generation systems are required, which...

journal article 2018

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Selective Oxyfunctionalisation Reactions Driven by Sulfite Oxidase-Catalysed In Situ Generation of H₂O₂

van Schie, M.M.C.H. (author), Kaczmarek, Alexander T. (author), Tieves, F. (author), Gomez de Santos, Patricia (author), Paul, C.E. (author), Arends, I.W.C.E. (author), Alcalde, Miguel (author), Schwarz, Günter (author), Hollmann, F. (author)

H₂O₂ can be accepted by several peroxygenases as a clean oxidant, able to supply both the necessary electrons and oxygen atom at the same time. The biocatalysts, in turn, are able to catalyse an array of interesting oxygen insertion reactions at enantio- and regio-selectivities hard to attain with classical chemical...

journal article 2020

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Enantioselective Sulfoxidation of Thioanisole by Cascading a Choline Oxidase and a Peroxygenase in the Presence of Natural Deep Eutectic Solvents

Li, Yongru (author), Ma, Yunjian (author), Li, Peilin (author), Zhang, Xizhen (author), Ribitsch, Doris (author), Alcalde, Miguel (author), Hollmann, F. (author), Wang, Yonghua (author)

A bienzymatic cascade for selective sulfoxidation is presented. The evolved recombinant peroxygenase from Agrocybe aegeritra catalyses the enantioselective sulfoxidation of thioanisole whereas the choline oxidase from Arthrobacter nicotianae provides the H2 O2 necessary via reductive activation of ambient oxygen. The reactions are performed...

journal article 2020