Search

In this contribution, we report chemoenzymatic bromodecarboxylation (Hunsdiecker-type) of α,ß-unsaturated carboxylic acids. The extraordinarily robust chloroperoxidase from Curvularia inaequalis (CiVCPO) generated hypobromite from H2O2and bromide, which then spontaneously reacted with a broad range of unsaturated carboxylic acids and yielded the corresponding vinyl bromide products. Selectivity issues arising from the (here undesired) addition of water to the intermediate bromonium ion could be solved by reaction medium engineering. The vinyl bromides so obtained could be used as starting materials for a range of cross-coupling and pericyclic reactions.

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.

Oxidation reactions are at the heart of organic chemistry. In industry, the epoxidation of ethylene and propylene or the hydroxylation of benzene for example is conducted at a scale of several million tonnes per year. At the same time, many of the established oxidation processes require relatively harsh reaction conditions and suffer from selectivity issues. As a consequence, the environmental impact of industrial oxidations remains high.Biocatalysis has a lot to offer here. Enzymes not only operate efficiently under very mild reaction conditions but also excel in terms of selectivity making them attractive alternatives to currently widespread transition metal catalysts for oxidation chemistry.In this special issue, we have assembled contributions from some of the leaders in the academic research community working on biocatalytic oxidation chemistry. We highlight the current state-of-the-art in the field demonstrating its enormous potential for industrial scale application but also highlighting its current limitations.Alcohol dehydrogenases have been among the first enzymes investigated for biocatalytic oxidation reactions. The manifold applications of alcohol dehydrogenases for the stereo- and regio-selective oxidation of primary and secondary alcohols are summarised in the contribution by Milagre and coworkers.While alcohol dehydrogenases transfer the reducing equivalents liberated from the starting materials to nicotinamide cofactors (NAD(P)+), oxidases utilise molecular oxygen as a sacrificial electron acceptor. Hence, oxidases appear promising, more cost-efficient alternatives to NAD(P)+-dependent dehydrogenases. The recent rapid developments in oxidase catalysis are critically summarised and discussed in the contribution by Heath and Turner.Dehydrogenases and oxidases catalyse dehydrogenation (hydride abstraction) reactions and therefore require pre-existing functional groups such as alcohols or amines for activity. In contrast, (non-) heme iron oxygenases catalyse the insertion of O-atoms (and more) even into non-activated C–H bonds. Hence, these oxygenases often accomplish chemistries, which are not achievable by existing chemical technologies.The so-called P450 monooxygenases have been forerunners for biocatalytic oxyfunctionalisation chemistry resulting in an enormous portfolio of highly regio- and stereoselective oxyfunctionalisation reactions. This wealth of possibilities is nicely summarised in the contribution by Opperman and co-workers.More recently, P450 monooxygenases have been challenged as catalysts of choice by the so-called unspecific peroxygenases (UPOs) essentially catalysing the same reactions as P450 monooxygenases but mechanistically far simpler. The rapid development of peroxygenases as catalysts for oxyfunctionalisation chemistry is summarised by Alcalde and co-workers. Xu et al. critically compare both enzyme classes and importantly highlight some limitations en route to large-scale application.Halogenases represent a truly exciting emerging class of oxygenases as for the highly selective introduction of halogens into non-activated C–H bonds where no chemical equivalents are known. The contribution by Buller and coworkers provides a concise introduction to the various halogenases known today, mechanistic insights and provides an overview over synthetic possibilities.Generally, oxidation reactions yield more reactive products such as occurs with alcohol oxidation or alkene epoxidation. Hence, the products of biocatalytic oxidation reactions themselves serve as starting materials for further transformations. The cascade catalysis concept envisions performing the oxidation step and the subsequent derivatisation step integrated together, eliminating time- and resource-consuming isolation and purification of the intermediates. This fascinating research is introduced in the contribution by Zhi and co-workers.Despite the wealth of engineered biocatalysts available, oxidative biocatalysis still is underrepresented, especially in the synthesis of commodity and bulk chemicals. Several issues still require intensive R&D efforts before oxidative enzymes become competitive also for these lower value-added compounds. In two contributions by Sieber and co-workers and Bommarius the role of molecular oxygen in enzyme- and process instability is highlighted. Molecular and physico-chemical mechanisms of enzyme inactivation are discussed together with recent strategies to overcome such limitations.Water is often considered as an ideal solvent from a sustainability point-of-view. However, for a broad range of reactants of interest, water is a sub-optimal solvent limiting traditional biocatalysis to low product titres. Sheldon and co-workers discuss this issue and present some promising green alternatives to water as a process solvent.Along these lines, the contribution of Kara and co-workers emphasises the potential of process intensification to render biocatalytic oxidation reactions both environmentally and economically more attractive.We are proud to have assembled contributions from leading researchers in the field of biocatalytic oxidation chemistry. The different aspects highlighted in this special issue give an impressive, though certainly not comprehensive, overview of this dynamic and exciting research field. We hope to enthral students and researchers alike to join us in the quest for greener industrial oxidation processes using enzymes.

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 selective fatty acid oxyfunctionalisation. The ω-1 hydroxy fatty acid (esters) produced are further transformed into lactones, alcohols, esters and amines via multi-enzyme cascades thereby paving the way for new fatty acid valorisation pathways.

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., A one-pot enzymatic cascade for the asymmetric azidohydroxylation of styrenes leads to chiral 1,2-azido alcohols with up to two stereocenters.

Propargylic alcohols and amines are versatile building blocks in organic synthesis. We demonstrate a straightforward enzymatic cascade to synthesize enantiomerically pure propargylic alcohols and amines from readily available racemic starting materials. In the first step, the peroxygenase from Agrocybe aegerita converted the racemic propargylic alcohols into the corresponding ketones, which then were converted into the enantiomerically pure alcohols using the (R)-selective alcohol dehydrogenase from Lactobacillus kefir or the (S)-selective alcohol dehydrogenase from Thermoanaerobacter brokii. Moreover, an enzymatic Mitsunobu-type conversion of the racemic alcohols into enantiomerically enriched propargylic amines using (R)-selective amine transaminase from Aspergillus terreus or (S)-selective amine transaminase from Chromobacterium violaceum was established. The one-pot two-step cascade reaction yielded a broad range of enantioenriched alcohol and amine products in 70-99% yield.

Chemical communications: ChemComm 58, 10540-10543 (2022). doi:10.1039/D2CC02411J, Published by The Royal Society of Chemistry, Cambridge

The fatty acid photodecarboxylase from Chlorella variabilis NC64A (CvFAP) promotes the elimination of CO2 from fatty acids (Cn) producing the corresponding hydrocarbon (Cn-1). Therefore, this enzyme is of great biotechnological interest since it can be used in alternative biofuel production routes matching the concept of green chemistry. However, due to its recent discovery, this reaction still requires optimizations, which was the focus of the present work together with the application of continuous flow system. The results in batch reactors showed the importance of using high power LED lamps (300 W) to reduce the reaction time for full conversion (30 min, >99%). In another approach, a continuous flow system demonstrated high potential, as it enabled full conversion with a half concentration of enzyme extract in a very short residence time of 15 min. Furthermore, the use of less expensive and sustainable light sources, not previously reported for reactions with CvFAP, were evaluated with full conversion (>99%) after 1 h for continuous flow reactions using 300 W common white LED lamp and based preliminary batch reactions investigations using direct sunlight. Thus, important advances and new perspectives for CvFAP photodecarboxylation reactions could be achieved with the present report.

Phthalic acid esters (PAEs) are widely used as plastic additives to increase the flexibility and durability of plastics. Constantly leaching out from plastics, PAEs are ubiquitously found in the environment. As PAEs exhibit biological activities such as being endocrine disruptive, the quest for efficient degradation strategies continues. Here, we report a bienzymatic degradation system for PAEs to phthalic acid (PA) using a cascade comprising two hydrolases, EstJ6 and P8219. The reaction conditions were optimized with respect to concentrations of both enzymes, temperature, and initial pH. Finally, the substrate scope of the new cascade was investigated, revealing that particularly PAEs with relatively small alcohols were degraded to more than 90 %. This present study provides a potential doable biocatalytic strategy for the complete hydrolysis of PAEs.

A photochemoenzymatic halodecarboxylation of ferulic acid was achieved using vanadate-dependent chloroperoxidase as (bio)catalyst and oxygen and organic solvent as sole stoichiometric reagents in a biphasic system. Performance and selectivity were improved through a phase transfer catalyst, reaching a turnover number of 660.000 for the enzyme.

Halide assays are important for the study of enzymatic dehalogenation, a topic of great industrial and scientific importance. Here we describe the development of a very sensitive halide assay that can detect less than a picomole of bromide ions, making it very useful for quantifying enzymatic dehalogenation products. Halides are oxidised under mild conditions using the vanadium‐dependent chloroperoxidase from Curvularia inaequalis, forming hypohalous acids that are detected using aminophenyl fluorescein. The assay is up to three orders of magnitude more sensitive than currently available alternatives, with detection limits of 20 nM for bromide and 1 μM for chloride and iodide. We demonstrate that the assay can be used to determine specific activities of dehalogenases and validate this by comparison to a well‐established GC‐MS method. This new assay will facilitate the identification and characterisation of novel dehalogenases and may also be of interest to those studying other halide‐producing enzymes., How sensitive! A simple, safe, and very sensitive assay for enzymatic dehalogenation is presented. The halides produced by dehalogenases are generally very unreactive and thus hard to detect. We use hydrogen peroxide and a haloperoxidase to activate halides to hypohalous acids, which are easily detected using the sensitive fluorogenic probe aminophenyl fluorescein.

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%.

Contains fulltext : 237091.pdf (Publisher’s version ) (Open Access) 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 and risk assessors that helps shape governance arrangements for accommodating and incentivizing safety, while fully acknowledging uncertainty. 28 p.

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.

The use of natural deep eutectic solvents (NADES) as multifunctional solvents for limonene bioprocessing was reported. NADES were used for the extraction of limonene from orange peel wastes, as solvent for the chemoenzymatic epoxidation of limonene, and as sacrificial electron donor for the in situ generation of H2O2 to promote the epoxidation reaction. The proof‐of‐concept for this multifunctional use was provided, and the scope and current limitations of the concept were outlined.

A chemoenzymatic method for the halocyclization of unsaturated alcohols and acids by using the robust V‐dependent chloroperoxidase from Curvularia inaequalis (CiVCPO) as catalyst has been developed for the in situ generation of hypohalites. A broad range of halolactones and cyclic haloethers are formed with excellent performance of the biocatalyst., You had me at “halo”: A chemoenzymatic method for the halocyclization of unsaturated alcohols and acids by using a robust V‐dependent chloroperoxidase as catalyst has been developed for the in situ generation of hypohalites. A broad range of halolactones and cyclic haloethers are formed with excellent biocatalyst performance.

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.

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’., Keep me busy: The photoactivated fatty acid decarboxylase from Chlorella variabilis NC64 A (CvFAP) is a promising catalyst to generate next generation biodiesel from (waste) fatty acids. CvFAP, however is also plagued by pronounced photoinactivation, which can be alleviated by keeping it busy, i. e. converting carboxylic acids such as caprylic acid.

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.

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.

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).