Designing biocatalytic redox reactions with oxidoreductases for organic chemistry

The focus of this thesis is to identify improvements of oxidoreductase reactions to bring them to preparative scale. Three approaches were studied: (1) photoregeneration of oxidised nicotinamide cofactors with LEDs as light source and flavins, (2) direct regeneration of Old Yellow Enzymes with light or metals, (3) heme-thiolate enzyme peroxygenases in neat substrate conditions.Flavins are the main actors of the first two approaches. These redox cofactors have been chosen for their photocatalytic properties as a photosensitiser and as a cofactor in the so-called flavoproteins.BT/Biocatalysi

Metals in Biotechnology: Cr-Driven Stereoselective Reduction of Conjugated C=C Double Bonds

Elemental metals are shown to be suitable sacrificial electron donors to drive the stereoselective reduction of conjugated C=C double bonds using Old Yellow Enzymes as catalysts. Both direct electron transfer from the metal to the enzyme as well as mediated electron transfer is feasible, although the latter excels by higher reaction rates. The general applicability of this new chemoenzymatic reduction method is demonstrated, and current limitations are outlined.BT/Biocatalysi

Peroxygenase-Catalysed Epoxidation of Styrene Derivatives in Neat Reaction Media

Biocatalytic oxyfunctionalisation reactions are traditionally conducted in aqueous media limiting their production yield. Here we report the application of a peroxygenase in neat reaction conditions reaching product concentrations of up to 360 mM.BT/Biocatalysi

Photobiocatalytic alcohol oxidation using LED light sources

The photocatalytic oxidation of NADH using a flavin photocatalyst and a simple blue LED light source is reported. This in situ NAD+ regeneration system can be used to promote biocatalytic, enantioselective oxidation reactions. Compared to the traditional use of white light bulbs this method enables very significant reductions in energy consumption and CO2 emission.BT/BiocatalysisBT/Biotechnolog

Photocatalysis to promote cell-free biocatalytic reactions

Cofactors assist enzymes to catalyze reactions and are indispensable and ubiquitous in nature, playing a central role in metabolic pathways. In biocatalysis, common redox cofactors such as nicotinamide, flavin and heme can be activated by light or synthetized to vary redox potentials, leading to different types of reactions for the formation of interesting chiral products, unattainable through classical chemical methods. This chapter will focus on light-driven cell-free biocatalytic reactions activated via their redox cofactors.Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care 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.BT/Biocatalysi

Light-Harvesting Dye-Alginate Hydrogel for Solar-Driven, Sustainable Biocatalysis of Asymmetric Hydrogenation

We report visible light-driven, asymmetric hydrogenation of C=C bonds using an ene-reductase from Thermus scotoductus SA-01 (TsOYE) and a light-harvesting dye (rose bengal, RB) co-immobilized in an alginate hydrogel. Highly efficient encapsulation of RB in alginate hydrogel was achieved using the intrinsic affinity between TsOYE and RB, which allowed for the construction of robust RB-TsOYE-loaded alginate capsules. In the absence of NADH, the photobiocatalytic system facilitated asymmetric reduction of 2-methylcyclohexenone to an enantiopure (R)-2-methylcyclohexanone (ee > 99%; max. conversion, 70.4%; turnover frequency, 1.54 min -1 turnover number, 300.2) under illumination. A series of stability tests revealed a significant enhancement of TsOYE's robustness in alginate hydrogel against heat and chemical denaturants. This study provides insight into a greener and sustainable approach of cofactor-free OYE catalysis for producing value-added chemicals using light energy. Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care 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.BT/Biocatalysi

Photochemical regeneration of flavoenzymes – An Old Yellow Enzyme case-study

Direct, NAD(P)H-independent regeneration of Old Yellow Enzymes represents an interesting approach for simplified reaction schemes for the stereoselective reduction of conjugated C=C-double bonds. Simply by illuminating the reaction mixtures with blue light in the presence of sacrificial electron donors enables to circumvent the costly and unstable nicotinamide cofactors and a corresponding regeneration system. In the present study, we characterise the parameters determining the efficiency of this approach and outline the current limitations. Particularly, the photolability of the flavin photocatalyst and the (flavin-containing) biocatalyst represent the major limitation en route to preparative application.BT/BiocatalysisOLD BT/Cell Systems Engineerin

Deazaflavins as photocatalysts for the direct reductive regeneration of flavoenzymes

Deazaflavins are potentially useful redox mediators for the direct, nicotinamide-independent regeneration of oxidoreductases. Especially the O2-stability of their reduced forms have attracted significant interest for the regeneration of monooxygenases. In this contribution we further investigate the photochemical properties of deazaflavins and investigate the scope and limitations of deazaflavin-based photoenzymatic reaction systems.Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care 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.BT/BiocatalysisBT/Biotechnolog

Natural Deep Eutectic Solvents as Performance Additives for Peroxygenase Catalysis

Natural deep eutectic solvents (NADES) are proposed as alternative solvents for peroxygenase-catalysed oxyfunctionalization reactions. Choline chloride-based NADES are of particular interest as they can serve as solvent, enzyme-stabiliser and sacrificial electron donor for the in situ H2O2 generation. This report provides the first proof-of-concept and basic characterisation of this new reaction system. Highly promising turnover numbers for the biocatalysts of up to 200,000 have been achieved.BT/Biocatalysi