This study explores a chemoenzymatic cascade to synthesise chiral β-hydroxy ketones by integrating the selective oxyfunctionalisation capabilities of peroxygenases with the carbon-carbon bond-forming progress of organocatalysts. Initial results with simple organocatalysts demonstrated poor performance due to mutual inactivation of the biocatalyst and organocatalyst. However, the use of more complex prolinamide derivatives improved the reaction efficiency and enantioselectivity, enabling a one-pot, one-step synthesis process. This methodology was further optimised to produce high yields of enantiomerically pure aldol products and was shown to be extendable to other substituted toluenes and aldol donors.@en

Green waste, especially of municipal origin, is currently used as a material only to a limited extent. However, the large material flows could also be used in a more economical way if they were integrated into biorefinery concepts. Besides the production of basic and fine chemicals, green waste could also be used as source of industrial relevant enzymes. Here, the purification and characterization of peroxidases from common lawn grass species Lolium perenne and Festuca arundinacea are reported. The purified peroxidase fractions as well as crude extracts were investigated for the removal of common wastewater pollutants such as phenol, m-cresol, and 2,4-dichlorophenol by oxidative polymerization. The grass-derived peroxidases showed the highest affinity towards 2,4-dichlorophenol, followed by m-cresol and phenol. A crude extract of real lawn grass was able to remove over 95 % of 0.5 mM 2,4-dichlorophenol within 20 min.

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In September 2015, the United Nations General Assembly established the 2030 Agenda for Sustainable Development, which includes 17 Sustainable Development Goals (SDGs) [...].

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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 toluenes proceeds highly regioselectively in benzylic position. Furthermore, neat reaction conditions not only enable attractive product concentrations (up to 185 mM) but also result in highly chemoselective oxidations to the aldehyde level.

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Enzyme catalysis, made tremendous progress over the last years in identification of new enzymes and new enzymatic reactivity’s as well as optimization of existing enzymes. However, the performance of the resulting processes is often still limited, e.g., in regard of productivity, realized product concentrations and the stability of the enzymes. Different topics (like limited specific activity, unfavourable kinetics or limited enzyme stability) can be addressed via enzyme engineering. On the other hand, there is also a long list of topics that are not addressable by enzyme engineering. Here typical examples are unfavourable reaction thermodynamics, selectivity in multistep reactions or low water solubility. These challenges can only be addressed through an adaption of the reaction system. The procedures of process intensification (PI) represent a good approach to reach most suitable systems. The general objective of PI is to achieve significant benefits in terms of capital and operating costs as well as product quality, waste, and process safety by applying innovative principles. The aim of the review is to show the current capabilities and future potentials of PI in enzyme catalysis focused on enzymes of the class of oxidoreductases. The focus of the paper is on alternative methods of energy input, innovative reactor concepts and reaction media with improved properties.@en

Peroxygenases are an emerging new class of enzymes allowing selective oxyfunctionalisation reactions in a cofactor-independent way different from well-known P450 monooxygenases. Herein, we focused on recent developments from organic synthesis, molecular biotechnology and reaction engineering viewpoints that are devoted to bring these enzymes in industrial applications. This covers natural diversity from different sources, protein engineering strategies for expression, substrate scope, activity and selectivity, stabilisation of enzymes via immobilisation, and the use of peroxygenases in low water media. We believe that peroxygenases have much to offer for selective oxyfunctionalisations and we have much to study to explore the full potential of these versatile biocatalysts in organic synthesis.

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Process intensification aims at enabling bridging the gap between fundamental research such as identification of new catalysts and reactions and their implementation in industrial environments. Especially the field of biocatalysis has seen some tremendous improvements and the development of new tools and approaches to bridge this gap. In this contribution we highlight some recent developments as selected case studies.

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In chemical process engineering, process intensification (PI) has proven itself as a method that resulted very often in processes with an at least doubled process performance. In recent years, the PI techniques have found more and more applications in biotechnology. Exemplary continuous processes, single-use reactors, electrobiotechnology and hybrid techniques are discussed here.

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Unspecific peroxygenases have recently gained significant interest due to their ability to catalyse the hydroxylation of non-activated C−H bonds using only hydrogen peroxide as a co-substrate. However, the development of preparative processes has so far mostly concentrated on benzylic hydroxylations using liquid substrates. Herein, we demonstrate the application of a peroxygenase for the hydroxylation of the inert, gaseous substrate butane to 2-butanol in a bubble column reactor. The influence of hydrogen peroxide feed rate and enzyme loading on product formation, overoxidation to butanone and catalytic efficiency is investigated at 200 mL scale. The process is scaled up to 2 L and coupled with continuous extraction. This setup allowed the production of 115 mmol 2-butanol and 70 mmol butanone with an overall total turnover number (TTN) of over 15.000, thereby demonstrating the applicability of peroxygenases for preparative hydroxylation of such inert, gaseous substrates at mild reaction conditions.

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In general, hydrogen peroxide is a stable and relatively mild oxidant and it can be regarded as the ultimate "green" reagent because water and oxygen are the only by-products. Besides the direct application of H₂O₂ in chemical processes more and more enzymatic syntheses based on hydrogen peroxide were developed. Different types of reactions can be addressed by using a hydrogen-peroxide driven biocatalysis (e.g. hydroxylations, epoxidations, sulfoxidations, halogenations, Baeyer-Villiger oxidations, decarboxylations). H₂O₂-driven reactions can often be used to substitute NAD(P)H dependent reactions. Therefore, laborious cofactor regeneration systems can be avoided by using H₂O₂-dependent enzymes. The tremendous increase in the number of publications dealing with this type of reactions clearly demonstrates the progress in this area in recent years. The described innovations range from new enzymes and types of reaction to novel reaction engineering approaches. This review aims to give the scope of possible advantageous applications of peroxyzymes and a critical discussion of their current limitations. The versatile reactions, the ecological advantageous and the great progress in the discovery and engineering of novel enzymes make a technical use feasible.

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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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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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Old yellow enzymes are able to catalyze asymmetric C=C reductions. A mediated electroenzymatic process to regenerate the NADPH in combination with an old yellow enzyme was investigated. Due to the fact that the overall process was affected by a broad set of parameters, a design of experiments (DoE) approach was chosen to identify suitable process conditions. Process conditions with high productivities of up to 2.27 mM/h in combination with approximately 90% electron transfer efficiency were identified.

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Monooxygenases are promising catalysts because they in principle enable the organic chemist to perform highly selective oxyfunctionalisation reactions that are otherwise difficult to achieve. For this, monooxygenases require reducing equivalents, to allow reductive activation of molecular oxygen at the enzymes' active sites. However, these reducing equivalents are often delivered to O₂ either directly or via a reduced intermediate (uncoupling), yielding hazardous reactive oxygen species and wasting valuable reducing equivalents. The oxygen dilemma arises from monooxygenases' dependency on O₂ and the undesired uncoupling reaction. With this contribution we hope to generate a general awareness of the oxygen dilemma and to discuss its nature and some promising solutions.

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A catalytic, enzyme-initiated (aza-) Achmatowicz reaction is presented. The involvement of a robust vanadium-dependent peroxidase from Curvularia inaequalis allows the simple use of H₂O₂ and catalytic amounts of bromide.

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