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.

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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 oxyfunctionalization reactions but are also irreversibly inactivated in the presence of too high H2O2 concentrations. Therefore, there is a need for efficient in situ H2O2 generation methods. Here, we show that radiolytic water splitting can be used to promote specific biocatalytic oxyfunctionalization reactions. Parameters influencing the efficiency of the reaction and current limitations are shown. Particularly, oxidative inactivation of the biocatalyst by hydroxyl radicals influences the robustness of the overall reaction. Radical scavengers can alleviate this issue, but eventually, physical separation of the enzymes from the ionizing radiation will be necessary to achieve robust reaction schemes. We demonstrate that nuclear waste can also be used to drive selective, peroxygenase-catalyzed oxyfunctionalization reactions, challenging our view on nuclear waste in terms of sustainability.

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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 methods. The sensitivity of most peroxygenases towards H₂O₂, however, requires this oxidant to be generated in situ. Here, we suggest the application of (modified) sulfite oxidases to couple the oxidation of sulfites to the reduction of oxygen. This enables us to use calcium sulfite, an industrial waste product from scrubbing flue gases, as an electron donor to reduce oxygen. This will supply the required peroxide in a controlled manner and enables us to perform these challenging reactions at the expense of simple salts.

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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 situ by nitrogen-doped carbon nanodots (N−CNDs) and UV LED illumination. AaeUPO entrapment in alginate beads increased enzyme stability and facilitated the reaction in neat cyclohexane. N−CNDs absorption in beads containing AaeUPO created a 2-in-1 heterogeneous photobiocatalyst that was active for up to seven days under reaction conditions and produced cyclohexanol, 2.5 mM. To increase productivity, the bead size and the photocatalyst-to-enzyme ratio have been identified as promising targets for optimisation.@en

An increasing number of biocatalytic oxidation reactions rely on H ₂ O ₂ as a clean oxidant. The poor robustness of most enzymes towards H ₂ O ₂ , however, necessitates more efficient systems for in situ H ₂ O ₂ generation. In analogy to the well-known formate dehydrogenase to promote NADH-dependent reactions, we here propose employing formate oxidase (FOx) to promote H ₂ O ₂ -dependent enzymatic oxidation reactions. Even under non-optimised conditions, high turnover numbers for coupled FOx/peroxygenase catalysis were achieved.

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Photoenzymatic cascades can be used for selective oxygenation of C−H-Bonds under mild conditions circumventing the hydrogen peroxide mediated peroxygenase inactivation via in situ H₂O₂ generation. Here, we report the “on demand” production of hydrogen peroxide via methanol assisted reduction of molecular oxygen using UV-illuminated titanium dioxide (Aeroxide P25) combined with the enantioselective hydroxylation of ethylbenzene to (R)-1-phenylethanole catalyzed by the Unspecific Peroxygenase from Agrocybe Aegerita. For the application of the system it is important to understand the influence of the reaction parameters to be able to optimize the system. Therefore, we systematically investigated product formation and enzyme inactivation as well as ROS formation (H₂O₂, ^.OH and ^.O₂⁻) applying different light intensities and temperatures. As a result, Turnover Numbers up to 220 000, photonic efficiencies up to 13.6 % and production rates up to 0.9 mM h⁻¹ were achieved.

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Various enzymes utilize hydrogen peroxide as an oxidant. Such “peroxizymes” are potentially very attractive catalysts for a broad range of oxidation reactions. Most peroxizymes, however, are inactivated by an excess of H₂O₂. The electrochemical reduction of oxygen can be used as an in situ generation method for hydrogen peroxide to drive the peroxizymes at high operational stabilities. Using conventional electrode materials, however, also necessitates significant overpotentials, thereby reducing the energy efficiency of these systems. This study concerns a method to coat a gas-diffusion electrode with oxidized carbon nanotubes (oCNTs), thereby greatly reducing the overpotential needed to perform an electroenzymatic halogenation reaction. In comparison to the unmodified electrode, with the oCNTs-modified electrode the overpotential can be reduced by approximately 100 mV at comparable product formation rates.

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In biocatalysis we use enzymes to accelerate chemical reactions. The advantage of enzymes over other chemical catalysts is their excellent performance in respect to reactivity, regioselectivity and enantioselectivity. The gentle environment at which they can optimally function further enhances their applicability to provide more sustainable alternatives for our chemical processes. The amount of different enzymes available to us is increasing, as is the variety of reactions we can catalyse with them. Enzymes are divided in 7 classes, depending on the reactions they catalyse. The first class of enzymes, the one this thesis is focussing on, is the oxidoreductase family. As the name implies, these enzymes catalyse redox reactions, the specific transfer of electrons from or to a certain reactant. To close this redox-balance, these enzymes naturally rely on coenzymes, organic structures which are needed in a stoichiometric amount. As stoichiometric addition of these compounds would greatly strain the aspired reactions, both economically as ecologically, these coenzymes are conventionally regenerated using a second enzyme system and co-substrate. Though this practice is established, it does further complicate the reaction schemes and adds waste streams to the reaction. We therefore aim a replacing these systems with new alternatives. Within the enzyme class of oxidoreductases, this thesis focuses on the subclass of oxidases and oxygenases, which all rely on oxygen in their reaction mechanisms. These enzymes are i.a. able to catalyse the regio- and enantioselective insertion of heteroatoms into molecular structures, even on inactivated bonds. These are reactions which are challenging, if not impossible, to perform using “classical” chemical methods. @en

The selective oxidation of trans-2-hexen-1-ol to the corresponding aldehyde using a recombinant aryl alcohol oxidase from Pleurotus eryngii (PeAAOx) is reported. Especially using the two liquid phase system to overcome solubility and product inhibition issues enabled to achieve more than 2.200.000 catalytic turnovers for the production enzyme as well as molar product concentrations, pointing towards an economic feasible reaction.

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Peroxygenases are very interesting catalysts for specific oxyfunctionalization chemistry. Instead of relying on complicated electron transport chains, they rely on simple hydrogen peroxide as the stoichiometric oxidant. Their poor robustness against H2O2 can be addressed via in situ generation of H2O2. Here we report that simple graphitic carbon nitride (g-C3N4) is a promising photocatalyst to drive peroxygenase-catalyzed hydroxylation reactions. The system has been characterized by outlining not only its scope but also its current limitations. In particular, spatial separation of the photocatalyst from the enzyme is shown as a solution to circumvent the undesired inactivation of the biocatalyst. Overall, very promising turnover numbers of the biocatalyst of more than 60.000 have been achieved.@en

Two-component-diffusible-flavomonooxygenases are versatile biocatalysts for selective epoxidation-, hydroxylation- or halogenation reactions. Their complicated molecular architecture can be simplified using photochemical regeneration of the catalytically active, reduced FADH ₂ prosthetic group. In this contribution we provide the proof-of-concept and characterization for the direct regeneration of the styrene monooxygenase from Pseudomonas.

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A recently discovered photodecarboxylase from Chlorella variabilis NC64A ( CvFAP) bears the promise for the efficient and selective synthesis of hydrocarbons from carboxylic acids. CvFAP, however, exhibits a clear preference for long-chain fatty acids thereby limiting its broad applicability. In this contribution, we demonstrate that the decoy molecule approach enables conversion of a broad range of carboxylic acids by filling up the vacant substrate access channel of the photodecarboxylase. These results not only demonstrate a practical application of a unique, photoactivated enzyme but also pave the way to selective production of short-chain alkanes from waste carboxylic acids under mild reaction conditions.

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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.

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The biocatalytic preparation of trans-hex-2-enal from trans-hex-2-enol using a novel aryl alcohol oxidase from Pleurotus eryngii (PeAAOx) is reported. As O2-dependent enzyme PeAAOx-dependent reactions are generally plagued by the poor solubility of O2 in aqueous media and mass transfer limitations resulting in poor reaction rates. These limitations were efficiently overcome by conducting the reaction in a flow-reactor setup reaching unpreceded catalytic activities for the enzyme in terms of turnover frequency (up to 38 s-1) and turnover numbers (more than 300000) pointing towards preparative usefulness of the proposed reaction scheme.@en

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 show little potential for large-scale implementation. Here, we show that visible-light-driven, catalytic water oxidation can be used for in situ generation of H₂O₂ from water, rendering the peroxygenase catalytically active. In this way, the stereoselective oxyfunctionalization of hydrocarbons can be achieved by simply using the catalytic system, water and visible light.

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Recent developments in microfluidic and nanofluidic technologies have resulted in development of new chip-based microfluidic calorimeters with potential use in different fields. One application would be the accurate high-throughput measurement of enzyme activity. Calorimetry is a generic way to measure activity of enzymes, but unlike conventional calorimeters, chip-based calorimeters can be easily automated and implemented in high-throughput screening platforms. However, application of chip-based microfluidic calorimeters to measure enzyme activity has been limited due to problems associated with miniaturization such as incomplete mixing and a decrease in volumetric heat generated. To address these problems we introduced a calibration method and devised a convenient protocol for using a chip-based microfluidic calorimeter. Using the new calibration method, the progress curve of alkaline phosphatase, which has product inhibition for phosphate, measured by the calorimeter was the same as that recorded by UV-visible spectroscopy. Our results may enable use of current chip-based microfluidic calorimeters in a simple manner as a tool for high-throughput screening of enzyme activity with potential applications in drug discovery and enzyme engineering.@en