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BT/Biocatalysis

Biocatalysis has an enormous impact on chemical synthesis. The waves in which biocatalysis has developed, and in doing so changed our perception of what organic chemistry is, were reviewed 20 and 10 years ago. Here we review the consequences of these waves of development. Nowadays, hydrolases are widely used on an industrial scale for the benign synthesis of commodity and bulk chemicals and are fully developed. In addition, further enzyme classes are gaining ever increasing interest. Particularly, enzymes catalysing selective C-C-bond formation reactions and enzymes catalysing selective oxidation and reduction reactions are solving long-standing synthetic challenges in organic chemistry. Combined efforts from molecular biology, systems biology, organic chemistry and chemical engineering will establish a whole new toolbox for chemistry. Recent developments are critically reviewed., BT/Biocatalysis

Oleate hydratase catalyses the addition of water to the CC double bond of oleic acid to produce (R)-10-hydroxystearic acid. The enzyme requires an FAD cofactor that functions to optimise the active site structure. A wide range of unsaturated fatty acids can be hydrated at the C10 and in some cases the C13 position. The substrate scope can be expanded using ‘decoy’ small carboxylic acids to convert small chain alkenes to secondary alcohols, albeit at low conversion rates. Systematic protein engineering and directed evolution to widen the substrate scope and increase the conversion rate is possible, supported by new high throughput screening assays that have been developed. Multi-enzyme cascades allow the formation of a wide range of products including keto-fatty acids, secondary alcohols, secondary amines and α,ω-dicarboxylic acids. Key points: • Phylogenetically distinct oleate hydratases may exhibit mechanistic differences. • Protein engineering to improve productivity and substrate scope is possible. • Multi-enzymatic cascades greatly widen the product portfolio., BT/Biocatalysis

An aqueous chemoenzymatic cascade reaction combining Pd-catalyzed C-C formation and enzymatic C=C asymmetric hydrogenation (AH) was developed for enantioselective synthesis of tertiary α-aryl cycloketones in good yields and excellent enantioselectivities. The stereopreference of the enzyme in AH of α-aryl cyclohexenones was studied. An enantiocomplementary enzyme was obtained by site-directed mutation., 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/Biocatalysis

Enantioenriched azido alcohols are precursors for valuable chiral aziridines and 1,2-amino alcohols, however their chiral substituted analogues are difficult to access. We established a cascade for the asymmetric azidohydroxylation of styrene derivatives leading to chiral substituted 1,2-azido alcohols via enzymatic asymmetric epoxidation, followed by regioselective azidolysis, affording the azido alcohols with up to two contiguous stereogenic centers. A newly isolated two-component flavoprotein styrene monooxygenase StyA proved to be highly selective for epoxidation with a nicotinamide coenzyme biomimetic as a practical reductant. Coupled with azide as a nucleophile for regioselective ring opening, this chemo-enzymatic cascade produced highly enantioenriched aromatic α-azido alcohols with up to >99% conversion. A bi-enzymatic counterpart with halohydrin dehalogenase-catalyzed azidolysis afforded the alternative β-azido alcohol isomers with up to 94% diastereomeric excess. We anticipate our biocatalytic cascade to be a starting point for more practical production of these chiral compounds with two-component flavoprotein monooxygenases., BT/Biocatalysis

Bacterial outer membrane vesicles (OMVs) are small unilamellar proteoliposomes, involved in various functions including cell-to-cell signalling and protein excretion. We have engineered the OMVs of Escherichia coli to nano-scaled bioreactors for the biotransformation of fatty acids by targeting a fatty acid double bond hydratase of Stentrophomonas maltophilia (SmOhyA) and/or a photoactivated fatty acid decarboxylase from Chlorella variabilis NC64 A (CvFAP) into OMVs. Engineered OMVs containing both SmOhyA and CvFAP were able to catalyse the transformation of oleic acid ((Z)-octadec-9-enoic acid) into 9-hydroxyheptadecane via (R)-10-hydroxyoctadecanoic acid. The specific biotransformation rates of oleic acid reached 8.0×10−12 μmol/min per OMV., BT/Biocatalysis

This paper outlines the immobilization of the recombinant dimeric unspecific peroxygenase from Agrocybe aegerita (rAaeUPO). The enzyme was quite stable (remaining unaltered its activity after 35 h at 47◦C and pH 7.0). Phosphate destabilized the enzyme, while glycerol stabilized it. The enzyme was not immobilized on glyoxyl-agarose supports, while it was immobilized albeit in inactive form on vinyl-sulfone-activated supports. rAaeUPO immobilization on glutaraldehyde pre-activated supports gave almost quantitative immobilization yield and retained some activity, but the biocatalyst was very unstable. Its immobilization via anion exchange on PEI supports also produced good immobilization yields, but the rAaeUPO stability dropped. However, using aminated agarose, the enzyme retained stability and activity. The stability of the immobilized enzyme strongly depended on the immobilization pH, being much less stable when rAaeUPO was adsorbed at pH 9.0 than when it was immobilized at pH 7.0 or pH 5.0 (residual activity was almost 0 for the former and 80% for the other preparations), presenting stability very similar to that of the free enzyme. This is a very clear example of how the immobilization pH greatly affects the final biocatalyst performance., BT/Biocatalysis

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., BT/Biocatalysis

Fatty acid photodecarboxylase (FAP) is one of the few photoenzymes in nature. The ability of FAP to convert fatty acids into alka(e)nes without the need for reducing equivalents put this enzyme into spotlight for biocatalytic applications. Although it has been discovered only a few years ago, many studies already emerged demonstrating its potential in areas from biofuel production and enzymatic kinetic resolution to being a critical component of multi-enzyme cascades. While there have been few protein engineering studies for modulating activity of FAP towards very short chain fatty acids, no study has yet addressed substrate selectivity within the medium to long chain fatty acid range, where FAP shows great promise for the synthesis of drop-in biofuels from ubiquitous fatty acids with chain lengths from C12 to C18. Here, after determining optimum expression and assay conditions for FAP, we screened 22 rationally designed mutant enzymes towards four naturally abundant fatty acid substrates; C12 : 0, C16 : 0, C18 : 0 and C18 : 1. Depending on the type of the exchanged amino acid, we observed selectivity shifts towards shorter or longer chains, compared to wild type enzyme. Notably, we obtained two groups of mutants; one group with high selectivity towards only C18 : 0, and another group that is selective towards C12 : 0 substrate. Moreover, we measured light and thermal stability of the wild type enzyme as well as the light stability of a mutant engineered for selectivity., 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/Biocatalysis

Versatile bio-derived catalysts have been under dynamic investigation as potential substitutes to conventional chemical catalysts for sustainable biodiesel production. This is because of their unique, low-cost benefits and production processes that are environmentally and economically acceptable. This critical review aspires to present a viable approach to the synthesis of environmentally benign and cost-effective heterogeneous solid-base catalysts from a wide range of biological and industrial waste materials for sustainable biodiesel production. Most of these waste materials include an abundance of metallic minerals like potassium and calcium. The different approaches proposed by researchers to derive highly active catalysts from large-scale waste materials of a re-usable nature are described briefly. Finally, this report extends to present an overview of techno-economic feasibility of biodiesel production, its environmental impacts, commercial aspects of community-based biodiesel production and potential for large-scale expansion., 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/Biocatalysis

The oxidation of alkanes into valuable chemical products is a vital reaction in organic synthesis. This reaction, however, is challenging, owing to the inertness of C−H bonds. Transition metal catalysts for C−H functionalization are frequently explored. Despite chemical alternatives, nature has also evolved powerful oxidative enzymes (e. g., methane monooxygenases, cytochrome P450 oxygenases, peroxygenases) that are capable of transforming C−H bonds under very mild conditions, with only the use of molecular oxygen or hydrogen peroxide as electron acceptors. Although progress in alkane oxidation has been reviewed extensively, little attention has been paid to small alkane oxidation. The latter holds great potential for the manufacture of chemicals. This Minireview provides a concise overview of the most relevant enzyme classes capable of small alkanes (C<6) oxyfunctionalization, describes the essentials of the catalytic mechanisms, and critically outlines the current state-of-the-art in preparative applications., 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/Biocatalysis

Multi-step cascade reactions have gained increasing attention in the biocatalysis field in recent years. In particular, multi-enzymatic cascades can achieve high molecular complexity without workup of reaction intermediates thanks to the enzymes’ intrinsic selectivity; and where enzymes fall short, organo-or metal catalysts can further expand the range of possible synthetic routes. Here, we present two enantiocomplementary (chemo)-enzymatic cascades composed of either a styrene monooxygenase (StyAB) or the Shi epoxidation catalyst for enantioselective alkene epoxidation in the first step, coupled with a halohydrin dehalogenase (HHDH)-catalysed regioselective epoxide ring opening in the second step for the synthesis of chiral aliphatic non-terminal azidoalcohols. Through the controlled formation of two new stereocenters, corresponding azidoalcohol products could be obtained with high regioselectivity and excellent enantioselectivity (99% ee) in the StyAB-HHDH cascade, while product enantiomeric excesses in the Shi-HHDH cascade ranged between 56 and 61%., BT/Biocatalysis

Bioelectrochemical technologies such as electro-fermentation and microbial CO2 electrosynthesis are emerging interdisciplinary technologies that can produce renewable fuels and chemicals (such as carboxylic acids). The benefits of electrically driven bioprocesses include improved production rate, selectivity, and carbon conversion efficiency. However, the accumulation of products can lead to inhibition of biocatalysts, necessitating further effort in separating products. The recent discovery of a new photoenzyme, capable of converting carboxylic acids to bio-alkanes, has offered an opportunity for system integration, providing a promising approach for simultaneous product separation and valorisation. Combining the strengths of photo/bio/electrochemical catalysis, we discuss an innovative circular cascading system that converts biomass and CO2 to value-added bio-alkanes (CnH2n+2, n = 2 to 5) whilst achieving carbon circularity., 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/Biocatalysis

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 to nonoptimized downstream processing, only 170 g of the enzyme has been isolated. The preparative usefulness of the so-obtained enzyme preparation has been demonstrated at a semipreparative scale (100 mL) as an example of the stereoselective hydroxylation of ethyl benzene. Using an adjusted H2O2 feed rate, linear product formation was observed for 7 days, producing more than 5 g L-1 (R)-1-phenyl ethanol. The biocatalyst performed more than 340.000 catalytic turnovers (942 g of the product per gram of rAaeUPO)., BT/Biocatalysis

DES are mixtures of two or more compounds, able to form liquids upon mixing, with lower freezing points when compared to the individual constituents (eutectic mixtures). This attitude is due to the specific hydrogen-bond interactions network between the components of the mixture. A notable characteristic of DES is the possibility to develop tailor-made mixtures by changing the components ratios or a limited water dilution, for special applications, making them attractive for pharmaceutical purposes. In this review, we focused our attention on application of ChCl-based DES in the synthesis of pharmaceutical compounds. In this context, these eutectic mixtures can be used as solvents, solvents/catalysts, or as chemical donors and we explored some representative examples in recent literature of such applications., BT/Biocatalysis

Following ionic liquids, (natural) deep eutectic solvents ((NA)DES) are receiving significant attention as performance additives for biocatalytic reactions. (NA)DES are increasingly evaluated as solvents to replace water in hydrolase-catalyzed esterification reactions thereby shifting the reaction equilibrium. They also frequently outperform water in terms of solubility properties of hydrophobic reagents and thereby enable higher space-time yields. Furthermore, (NA)DES frequently exceed stabilizing effects on enzymes and thereby enable more robust (and therefore economically more attractive) biocatalytic syntheses. In this contribution, we will summarize and critically evaluate the recent literature on (NA)DES-supported biocatalysis., 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/Biocatalysis

In this paper, we provide an overview of how Safe-by-Design is conceived and applied in practice in a large number of engineering disciplines. We discuss the differences, commonalities, and possibilities for mutual learning found in those practices and identify several ways of putting those disciplinary outlooks in perspective. The considered engineering disciplines in the order of historically grown technologies are construction engineering, chemical engineering, aerospace engineering, urban engineering, software engineering, bio-engineering, nano-engineering, and finally cyber space engineering. Each discipline is briefly introduced, the technology at issue is described, the relevant or dominant hazards are examined, the social challenge(s) are observed, and the relevant developments in the field are described. Within each discipline the risk management strategies, the design principles promoting safety or safety awareness, and associated methods or tools are discussed. Possible dilemmas that the designers in the discipline face are highlighted. Each discipline is concluded by discussing the opportunities and bottlenecks in addressing safety. Commonalities and differences between the engineering disciplines are investigated, specifically on the design strategies for which empirical data have been collected. We argue that Safe-by-Design is best considered as a specific elaboration of Responsible Research and Innovation, with an explicit focus on safety in relation to other important values in engineering such as well-being, sustainability, equity, and affordability. Safe-by-Design provides for an intellectual venue where social science and the humanities (SSH) collaborate on technological developments and innovation by helping to proactively incorporate safety considerations into engineering practices, while navigating between the extremes of technological optimism and disproportionate precaution. As such, Safe-by-Design is also a practical tool for policymakers, Safety and Security Science, Ethics & Philosophy of Technology, ChemE/Product and Process Engineering, BT/Biotechnology and Society, Circular Product Design, BT/Biocatalysis, Control & Simulation, Programming Languages, Quantum Communications Lab, Applied Mechanics

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’., BT/Biocatalysis

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 mutant from Agrocybe aegerita (PaDa-I variant), in the current study we have improved its activity in organic solvents by directed evolution. Mutant libraries constructed by random mutagenesis and in vivo DNA shuffling were screened in the presence of increasing concentrations of organic solvents that differed both in regard to their chemical nature and polarity. In addition, a palette of neutral mutations generated by genetic drift that improved activity in organic solvents was evaluated by site directed recombination in vivo. The final UPO variant of this evolutionary campaign carried nine mutations that enhanced its activity in the presence of 30% acetonitrile (vol/vol) up to 23-fold over PaDa-I parental type, and it was also active and stable in aqueous acetone, methanol and dimethyl sulfoxide mixtures. These mutations, which are located at the surface of the protein and in the heme channel, seemingly helped to protect UPO from harmful effects of cosolvents by modifying interactions with surrounding residues and influencing critical loops., BT/Biocatalysis

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 be subjected to nucleophilic ring opening, yielding non-racemic trans-disubstituted cyclohexadiene derivates, which in turn can be used for further chemical transformations. This approach may represent a promising shortcut for the synthesis of natural products and APIs., BT/Biocatalysis, BT/Environmental Biotechnology

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., BT/Biocatalysis

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., BT/Biocatalysis

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., BT/Biocatalysis

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/Biocatalysis

The oxidation of alkanes into valuable chemical products is a vital reaction in organic synthesis. This reaction, however, is challenging, owing to the inertness of C−H bonds. Transition metal catalysts for C−H functionalization are frequently explored. Despite chemical alternatives, nature has also evolved powerful oxidative enzymes (e. g., methane monooxygenases, cytochrome P450 oxygenases, peroxygenases) that are capable of transforming C−H bonds under very mild conditions, with only the use of molecular oxygen or hydrogen peroxide as electron acceptors. Although progress in alkane oxidation has been reviewed extensively, little attention has been paid to small alkane oxidation. The latter holds great potential for the manufacture of chemicals. This Minireview provides a concise overview of the most relevant enzyme classes capable of small alkanes (C<6) oxyfunctionalization, describes the essentials of the catalytic mechanisms, and critically outlines the current state-of-the-art in preparative applications., 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/Biocatalysis

DES are mixtures of two or more compounds, able to form liquids upon mixing, with lower freezing points when compared to the individual constituents (eutectic mixtures). This attitude is due to the specific hydrogen-bond interactions network between the components of the mixture. A notable characteristic of DES is the possibility to develop tailor-made mixtures by changing the components ratios or a limited water dilution, for special applications, making them attractive for pharmaceutical purposes. In this review, we focused our attention on application of ChCl-based DES in the synthesis of pharmaceutical compounds. In this context, these eutectic mixtures can be used as solvents, solvents/catalysts, or as chemical donors and we explored some representative examples in recent literature of such applications., BT/Biocatalysis

Versatile bio-derived catalysts have been under dynamic investigation as potential substitutes to conventional chemical catalysts for sustainable biodiesel production. This is because of their unique, low-cost benefits and production processes that are environmentally and economically acceptable. This critical review aspires to present a viable approach to the synthesis of environmentally benign and cost-effective heterogeneous solid-base catalysts from a wide range of biological and industrial waste materials for sustainable biodiesel production. Most of these waste materials include an abundance of metallic minerals like potassium and calcium. The different approaches proposed by researchers to derive highly active catalysts from large-scale waste materials of a re-usable nature are described briefly. Finally, this report extends to present an overview of techno-economic feasibility of biodiesel production, its environmental impacts, commercial aspects of community-based biodiesel production and potential for large-scale expansion., 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/Biocatalysis

Fatty acid photodecarboxylase (FAP) is one of the few photoenzymes in nature. The ability of FAP to convert fatty acids into alka(e)nes without the need for reducing equivalents put this enzyme into spotlight for biocatalytic applications. Although it has been discovered only a few years ago, many studies already emerged demonstrating its potential in areas from biofuel production and enzymatic kinetic resolution to being a critical component of multi-enzyme cascades. While there have been few protein engineering studies for modulating activity of FAP towards very short chain fatty acids, no study has yet addressed substrate selectivity within the medium to long chain fatty acid range, where FAP shows great promise for the synthesis of drop-in biofuels from ubiquitous fatty acids with chain lengths from C12 to C18. Here, after determining optimum expression and assay conditions for FAP, we screened 22 rationally designed mutant enzymes towards four naturally abundant fatty acid substrates; C12 : 0, C16 : 0, C18 : 0 and C18 : 1. Depending on the type of the exchanged amino acid, we observed selectivity shifts towards shorter or longer chains, compared to wild type enzyme. Notably, we obtained two groups of mutants; one group with high selectivity towards only C18 : 0, and another group that is selective towards C12 : 0 substrate. Moreover, we measured light and thermal stability of the wild type enzyme as well as the light stability of a mutant engineered for selectivity., 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/Biocatalysis

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 to nonoptimized downstream processing, only 170 g of the enzyme has been isolated. The preparative usefulness of the so-obtained enzyme preparation has been demonstrated at a semipreparative scale (100 mL) as an example of the stereoselective hydroxylation of ethyl benzene. Using an adjusted H2O2 feed rate, linear product formation was observed for 7 days, producing more than 5 g L-1 (R)-1-phenyl ethanol. The biocatalyst performed more than 340.000 catalytic turnovers (942 g of the product per gram of rAaeUPO)., BT/Biocatalysis

Multi-step cascade reactions have gained increasing attention in the biocatalysis field in recent years. In particular, multi-enzymatic cascades can achieve high molecular complexity without workup of reaction intermediates thanks to the enzymes’ intrinsic selectivity; and where enzymes fall short, organo-or metal catalysts can further expand the range of possible synthetic routes. Here, we present two enantiocomplementary (chemo)-enzymatic cascades composed of either a styrene monooxygenase (StyAB) or the Shi epoxidation catalyst for enantioselective alkene epoxidation in the first step, coupled with a halohydrin dehalogenase (HHDH)-catalysed regioselective epoxide ring opening in the second step for the synthesis of chiral aliphatic non-terminal azidoalcohols. Through the controlled formation of two new stereocenters, corresponding azidoalcohol products could be obtained with high regioselectivity and excellent enantioselectivity (99% ee) in the StyAB-HHDH cascade, while product enantiomeric excesses in the Shi-HHDH cascade ranged between 56 and 61%., BT/Biocatalysis

In this paper, we provide an overview of how Safe-by-Design is conceived and applied in practice in a large number of engineering disciplines. We discuss the differences, commonalities, and possibilities for mutual learning found in those practices and identify several ways of putting those disciplinary outlooks in perspective. The considered engineering disciplines in the order of historically grown technologies are construction engineering, chemical engineering, aerospace engineering, urban engineering, software engineering, bio-engineering, nano-engineering, and finally cyber space engineering. Each discipline is briefly introduced, the technology at issue is described, the relevant or dominant hazards are examined, the social challenge(s) are observed, and the relevant developments in the field are described. Within each discipline the risk management strategies, the design principles promoting safety or safety awareness, and associated methods or tools are discussed. Possible dilemmas that the designers in the discipline face are highlighted. Each discipline is concluded by discussing the opportunities and bottlenecks in addressing safety. Commonalities and differences between the engineering disciplines are investigated, specifically on the design strategies for which empirical data have been collected. We argue that Safe-by-Design is best considered as a specific elaboration of Responsible Research and Innovation, with an explicit focus on safety in relation to other important values in engineering such as well-being, sustainability, equity, and affordability. Safe-by-Design provides for an intellectual venue where social science and the humanities (SSH) collaborate on technological developments and innovation by helping to proactively incorporate safety considerations into engineering practices, while navigating between the extremes of technological optimism and disproportionate precaution. As such, Safe-by-Design is also a practical tool for policymakers, Safety and Security Science, Ethics & Philosophy of Technology, ChemE/Product and Process Engineering, BT/Biotechnology and Society, Circular Product Design, BT/Biocatalysis, Control & Simulation, Programming Languages, Quantum Communications Lab, Applied Mechanics

Following ionic liquids, (natural) deep eutectic solvents ((NA)DES) are receiving significant attention as performance additives for biocatalytic reactions. (NA)DES are increasingly evaluated as solvents to replace water in hydrolase-catalyzed esterification reactions thereby shifting the reaction equilibrium. They also frequently outperform water in terms of solubility properties of hydrophobic reagents and thereby enable higher space-time yields. Furthermore, (NA)DES frequently exceed stabilizing effects on enzymes and thereby enable more robust (and therefore economically more attractive) biocatalytic syntheses. In this contribution, we will summarize and critically evaluate the recent literature on (NA)DES-supported biocatalysis., 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/Biocatalysis

Bioelectrochemical technologies such as electro-fermentation and microbial CO2 electrosynthesis are emerging interdisciplinary technologies that can produce renewable fuels and chemicals (such as carboxylic acids). The benefits of electrically driven bioprocesses include improved production rate, selectivity, and carbon conversion efficiency. However, the accumulation of products can lead to inhibition of biocatalysts, necessitating further effort in separating products. The recent discovery of a new photoenzyme, capable of converting carboxylic acids to bio-alkanes, has offered an opportunity for system integration, providing a promising approach for simultaneous product separation and valorisation. Combining the strengths of photo/bio/electrochemical catalysis, we discuss an innovative circular cascading system that converts biomass and CO2 to value-added bio-alkanes (CnH2n+2, n = 2 to 5) whilst achieving carbon circularity., 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/Biocatalysis

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’., BT/Biocatalysis

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 mutant from Agrocybe aegerita (PaDa-I variant), in the current study we have improved its activity in organic solvents by directed evolution. Mutant libraries constructed by random mutagenesis and in vivo DNA shuffling were screened in the presence of increasing concentrations of organic solvents that differed both in regard to their chemical nature and polarity. In addition, a palette of neutral mutations generated by genetic drift that improved activity in organic solvents was evaluated by site directed recombination in vivo. The final UPO variant of this evolutionary campaign carried nine mutations that enhanced its activity in the presence of 30% acetonitrile (vol/vol) up to 23-fold over PaDa-I parental type, and it was also active and stable in aqueous acetone, methanol and dimethyl sulfoxide mixtures. These mutations, which are located at the surface of the protein and in the heme channel, seemingly helped to protect UPO from harmful effects of cosolvents by modifying interactions with surrounding residues and influencing critical loops., BT/Biocatalysis

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., BT/Biocatalysis

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., BT/Biocatalysis

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 be subjected to nucleophilic ring opening, yielding non-racemic trans-disubstituted cyclohexadiene derivates, which in turn can be used for further chemical transformations. This approach may represent a promising shortcut for the synthesis of natural products and APIs., BT/Biocatalysis, BT/Environmental Biotechnology

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., BT/Biocatalysis

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., BT/Biocatalysis

The use of a microscale atmospheric pressure plasma jet (μAPPJ) was investigated for its potential to supply hydrogen peroxide in biocatalysis. Compared to a previously employed dielectric barrier discharge (DBD), the μAPPJ offered significantly higher H2O2 production rates and better handling of larger reaction volumes. The performance of the μAPPJ was evaluated with recombinant unspecific peroxygenase from Agrocybe aegerita (rAaeUPO). Using plasma-treated buffer, no side reactions with other plasma-generated species were detected. For long-term treatment, rAaeUPO was immobilized, transferred to a rotating bed reactor, and reactions performed using the μAPPJ. The enzyme had a turnover of 36,415 mol mol−1 and retained almost full activity even after prolonged plasma treatment. Overall, the μAPPJ presents a promising plasma source for plasma-driven biocatalysis., BT/Biocatalysis

Peroxygenases have long inspired the selective oxyfunctionalization of various aliphatic and aromatic compounds, because of their broad substrate spectrum and simplicity of catalytic mechanism. This study provides a proof-of-concept of piezobiocatalysis by demonstrating peroxygenase-catalyzed oxyfunctionalization reactions fueled by piezocatalytically generated H2O2. Bismuth oxychloride (BiOCl) generated H2O2 in situ via an oxygen reduction reaction under ultrasonic wave conditions. Through the simple combination of water, ultrasound, recombinant, evolved unspecific peroxygenase from Agrocybe aegerita (rAaeUPO), and BiOCl, the piezobiocatalytic platform accelerated selective hydroxylation of ethylbenzene to enantiopure (R)-1-phenylethanol [total turnover number of rAaeUPO (TTNrAaeUPO), 2002; turnover frequency, 77.7 min-1 >99% enantiomeric excess (ee)]. The BiOCl-rAaeUPO couple also catalyzed other representative substrates (e.g., propylbenzene, 1-chloro-4-ethylbenzene, cyclohexane, and cis-β-methylstyrene) with high turnover frequency and selectivity. We alleviated the oxidative stress of piezocatalytically generated OH- on rAaeUPO by spatial separation of rAaeUPO and BiOCl, which resulted in greatly enhanced TTNrAaeUPO of >3900 and the notable prolongation of reaction time. Overall, the BiOCl-rAaeUPO couple serves as a mechanical-to-chemical energy conversion platform for driving peroxygenase-catalyzed reactions under ultrasonic conditions., 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/Biocatalysis

A biocatalytic cascade transforming castor oil into (R,Z)-octadec-9-en-7-ol is presented by combining a lipase catalysed hydrolysis of castor oil into ricinoleic acid followed by a photoenzymatic decarboxylation into (R,Z)-octadec-9-en-7-ol. Conversion of up to 41.7% and overall product concentrations of up to 60 mM, this new bienzymatic and photocatalytic cascade exhibits significant potential for the valorisation of non-edible castor oil. The scope and limitations of the current system are described and discussed., BT/Biocatalysis

In this chapter the catalytic and structural properties of the vanadium chloroperoxidases will be discussed with an emphasis on their superb activity and stability under operational conditions. These properties make these enzymes attractive catalysts in organic synthesis and allow a number of applications. Some of the more recent findings are highlighted, e.g., the use of vanadium chloroperoxidase (VCPO) in the formation of singlet oxygen, halogenation of phenols, alkenes, halocyclisation of ϵ,γ-unsaturated alcohols and the aza-Achmatowicz reaction., 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/Biocatalysis

A biocatalytic cascade transforming castor oil into (R,Z)-octadec-9-en-7-ol is presented by combining a lipase catalysed hydrolysis of castor oil into ricinoleic acid followed by a photoenzymatic decarboxylation into (R,Z)-octadec-9-en-7-ol. Conversion of up to 41.7% and overall product concentrations of up to 60 mM, this new bienzymatic and photocatalytic cascade exhibits significant potential for the valorisation of non-edible castor oil. The scope and limitations of the current system are described and discussed., BT/Biocatalysis

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 aryl-alcohol oxidase that supplies H2O2 in situ. Enzyme fusion libraries with peptide linkers of different sizes and amino acid compositions were designed, while attached leader sequences favored secretion in yeast. The most promising functional enzyme fusions were characterized biochemically and further tested for the synthesis of dextrorphan, a metabolite of the antitussive drug dextromethorphan. This reaction system was optimized to control the aromatic alcohol transformation rate, and therefore the H2O2 supply, to achieve total turnover numbers of 62,000, the highest value reported for the biocatalytic synthesis of dextrorphan to date. Accordingly, our study opens an avenue for the use of peroxygenase-aryl alcohol oxidase fusions in the pharmaceutical and chemical sectors., 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/Biocatalysis

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 strategies were investigated to prevent the dielectric barrier discharge plasma-induced inactivation of biocatalysts, using recombinant unspecific peroxygenase from Agrocybe aegerita (rAaeUPO), one of the most promising enzymes for plasma-driven biocatalysis. Treatment in oxygen-free atmospheres did not provide any advantage over treatment in synthetic air, indicating that the detrimental reactive species did not originate from oxygen in the plasma phase. Chemical scavengers were employed to eliminate undesired reactive species, without any long-term effect on enzyme lifetime. Similarly, chaperones, including the known stress response proteins Hsp33, CnoX, and RidA did not increase the lifetime of rAaeUPO. Immobilization of the biocatalyst proved effective in preserving enzyme activity. The residual activity of rAaeUPO after plasma treatment strongly depended on the specific immobilization support. Essentially complete protection for at least 15 min of plasma exposure was achieved with an epoxy-butyl-functionalized carrier. This study presents new insights into plasma-protein interactions and plots a path forward for protecting biocatalytic proteins from plasma-mediated inactivation., 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/Biocatalysis

In this invited Account, we highlight the enormous scientific breadth of our mentor Professor Manfred T. Reetz. It stretches from the development of organometallic reagents and transition metal catalysts to the adventurous idea of directed evolution of chemo-, stereo-, and regioselective enzymes, which he considered to be most important. We hope to show that Reetz did not consider these research areas to be totally unrelated realms, and attempt to reveal his transdisciplinary way of thinking about methodology development. Since biocatalysis has become crucial for chemical synthesis, we mainly focus on Reetz's contributions in this area. Some personal reflections from some of his former co-workers are also included, which reveal the stimulating atmosphere in the Reetz group in terms of science, career advice, and the importance of ethical considerations., BT/Biocatalysis

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., BT/Biocatalysis, RST/Applied Radiation & Isotopes, RST/Radiation, Science and Technology

Reductions play a key role in organic synthesis, producing chiral products with new functionalities. Enzymes can catalyse such reactions with exquisite stereo-, regio- and chemoselectivity, leading the way to alternative shorter classical synthetic routes towards not only high-added-value compounds but also bulk chemicals. In this review we describe the synthetic state-of-the-art and potential of enzymes that catalyse reductions, ranging from carbonyl, enone and aromatic reductions to reductive aminations., BT/Biocatalysis