Search

Enantiopure tertiary alcohols are very valuable building blocks for the synthesis of many different natural products and pharmaceuticals. As a consequence, several chemical and enzymatic strategies to afford such chiral structures have been described. Promising enzymatic approaches with agents such as epoxide hydrolases, dehalogenases and hydroxynitrile lyases have been reported, as well as dihydroxylation by microorganisms. Apart from those valuable options, the hydrolase-based kinetic resolution of tertiary alcohols has been known for the last three decades, as several wild-type enzymes have been reported to be able to accept these sterically hindered molecules. More recently, the existence of an amino acid motif within an enzyme's active site has been identified as highly relevant for the acceptance of such bulky structures. This discovery clearly facilitates the identification of novel biocatalysts for this application. Although several tertiary alcohols have been successfully resolved with wild-type biocatalysts, enantioselectivities have often been too low for synthetic purposes. These limitations have recently been overcome by accessing enzymes from the metagenome through directed evolution or by rational protein design. This minireview describes the state of the art in this area, highlighting aspects of basic academic research into the practical application of biocatalysts for the synthesis of optically active tertiary alcohols.

The oxidation of 5‐hydroxymethylfurfural (HMF) to 2,5‐diformylfuran (DFF) is a key reaction in valorizing biomass. DFF is hardly soluble in water, while HMF is often obtained from biorefineries in crude wet organic fractions. Thus, the reaction is challenging for both biocatalysis performed in aqueous media, and for chemocatalysis where the presence of water often results in catalyst poisoning. Galactose oxidase (GalOx) can selectively oxidize HMF to DFF and displays promising activity in aqueous‐organic media. In this study, GalOx was immobilized on ten carriers, assessing the immobilization yield, activity, and stability. Covalently immobilized GalOx catalyzed the oxidation of HMF to DFF in neat and water‐saturated EtOAc, and in biphasic systems of various water contents. At 50 % v/v H2O, the reaction was conducted at a semi‐preparative scale (50 mL) with no adverse effect on DFF yield. Some limitations arise, such as enzyme deactivation, and adsorption of DFF to the support, particularly in the aqueous fraction. Future options to upgrade the route may include designed stable enzymes under the presence of HMF/DFF, and the setup of microaqueous systems where DFF adsorption is minimized. The use of wet EtOAc media would be a promising approach in future biorefineries employing inexpensive crude wet organic fractions. [ABSTRACT FROM AUTHOR]

Biocatalysis holds immense potential for pharmaceutical development as it enables synthetic routes to various chiral building blocks with unparalleled selectivity. Therein, solvent and water use account for a large contribution to the environmental impact of the reactions. In the spirit of Green Chemistry, a transition from traditional highly diluted aqueous systems to intensified non-aqueous media to overcome limitations (e.g., water shortages, recalcitrant wastewater treatments, and low substrate loadings) has been observed. Benefiting from the spectacular advances in various enzyme stabilization techniques, a plethora of biotransformations in non-conventional media have been established. Deep eutectic solvents (DESs) emerge as a sort of (potentially) greener non-aqueous medium with increasing use in biocatalysis. This review discusses the state-of-the-art of biotransformations in DESs with a focus on biocatalytic pathways for the synthesis of active pharmaceutical ingredients (APIs). Representative examples of different enzyme classes are discussed, together with a critical vision of the limitations and discussing prospects of using DESs for biocatalysis. [ABSTRACT FROM AUTHOR]

5‐hydroxymethylfurfural (HMF) is produced upon dehydration of C6 sugars in biorefineries. As the product, it remains either in aqueous solutions, or is in situ extracted to an organic medium (biphasic system). For the subsequent oxidation of HMF to 2,5‐furandicarboxylic acid (FDCA), 'media‐agnostic' catalysts that can be efficiently used in different conditions, from aqueous to biphasic, and to organic (microaqueous) media, are of interest. Here, the concept of a one‐pot biocatalytic cascade for production of FDCA from HMF was reported, using galactose oxidase (GalOx) for the formation of 2,5‐diformylfuran (DFF), followed by the lipase‐mediated peracid oxidation of DFF to FDCA. GalOx maintained its catalytic activity upon exposure to a range of organic solvents with only 1 % (v/v) of water. The oxidation of HMF to 2,5‐diformylfuran (DFF) was successfully established in ethyl acetate‐based biphasic or microaqueous systems. To validate the concept, the reaction was conducted at 5 % (v/v) water, and integrated in a cascade where DFF was subsequently oxidized to FDCA in a reaction catalyzed by Candida antarctica lipase B. [ABSTRACT FROM AUTHOR]

In nature, enzymes conventionally operate under aqueous conditions. Because of this, aqueous buffers are often the choice for reaction media when enzymes are applied in chemical synthesis. However, to meet the demands of an industrial application, due to the poor water solubility of many industrially relevant compounds, an aqueous reaction system will often not be able to provide sufficient substrate loadings. A switch to a non-aqueous solvent system can provide a solution, which is already common for lipases, but more challenging for biocatalysts from other enzyme classes. The choices in solvent types and systems, however, can be overwhelming. Furthermore, some engineering of the protein structure of biocatalyst formulation is required. In this review, a guide for those working with biocatalysts, who look for a way to increase their reaction productivity, is presented. Examples reported clearly show that bulk water is not necessarily required for biocatalytic reactions and that clever solvent systems design can support increased product concentrations thereby decreasing waste formation. Additionally, under these conditions, enzymes can also be combined in cascades with other, water-sensitive, chemical catalysts. Finally, we show that the application of non-aqueous solvents in biocatalysis can actually lead to more sustainable processes. At the hand of flowcharts, following simple questions, one can quickly find what solvent systems are viable. [ABSTRACT FROM AUTHOR]

Enzyme catalysis (biocatalysis) is a mature strategy to perform organic synthesis with high selectivity and under mild reaction conditions. However, despite their origin and biodegradability, the simple use of enzymes is not sufficient to validate a process as green or sustainable. To reinforce the value that biocatalysis may offer, enzymatic processes can be established in environmentally‐friendly media, such as water, neoteric solvents (e. g. deep‐eutectic‐solvents, DES), biogenic solvents (e. g. 2‐MeTHF or terpenes), or even solvent‐free systems (if substrates allow that). Furthermore, these options can be conducted in continuous biocatalytic processes, which are more efficient and sustainable. Hence, a triple synergy is generated, and very promising environmentally‐friendly options can be envisaged. This conceptual paper discusses these alternatives, providing relevant, recently reported, successful examples. [ABSTRACT FROM AUTHOR]

Biogenic furans (furfural and 5-hydroxymethylfurfural) are expected to become relevant building blocks based on their high degree of functionality and versatility. However, the inherent instability of furans poses considerable challenges for their synthetic modifications. Valorization routes of furans typically generate byproducts, impurities, wastes, and a cumbersome downstream processing, compromising their ecological footprint. Biocatalysis may become an alternative, given the high selectivity of enzymes, together with the mild reaction conditions applied. This Review critically discusses the options for enzymes in the upgrading of furans. Based on previous reports, a variety of biocatalytic transformations have been applied to furans, with successful cases both in aqueous and in water-free media. Options comprise the biodetoxification of toxic furans in hydrolysates, selective syntheses based on oxidation-reduction processes, solvent-free esterifications, or carboligations to afford C12 derivatives. Reported strategies show in general promising but still modest productivities (2-30 gproduct L−1 d−1, depending on the example). There are opportunities with high potential and deserving of further development, scale-up, and technoeconomic assessment, to entirely validate them as realistic alternatives. [ABSTRACT FROM AUTHOR]

The use of deep eutectic solvent (DES)/water mixtures were explored for the selective enzymatic synthesis of α-monobenzoate glycerol (α-MBG) from glycerol and benzoic acid as substrates. Experiments were performed with four DES, three of them containing choline chloride (ChCl), combined with urea (URA), glycerol (GLY), and ethylene glycol (ETA) (in all cases ChCl/HBD 1:2 mol ratio), and another one formed with methylammonium chloride and glycerol (MA/GLY 1:3 mol ratio). The best conversions (99 %) were achieved with immobilized lipase B from Candida antarctica (CAL-B) when ChCl/GLY was used as the solvent and the substrate at the same time. The use of water as a cosolvent (8 % v/ v) led to a significant decrease in the viscosity of the DES, and full conversions were then reached. Reusability studies of the biocatalyst revealed a 37 % decrease in activity after the first batch, but the activity remained mostly constant for the rest of the cycles. [ABSTRACT FROM AUTHOR]

In their Editorial for the Special Issue on Biocatalysis as Key to Sustainable Industrial Chemistry, Guest Editors Andrés Alcántara, Pablo Domínguez de María, Jennifer Littlechild, and Roland Wohlgemuth and their co‐workers on the European Society of Applied Biocatalysis' (ESAB) Working Group on Sustainable Chemistry Martin Schürmann and Roger Sheldon discuss the Special Issue and the importance of biocatalysis in carrying out cutting‐edge industrial chemistry in a sustainable way, as well as the future prospects for the field. [ABSTRACT FROM AUTHOR]

Glutathione-dependent β-etherases and glutathione lyases are key-enzymes for the biocatalytic depolymerization of lignin. In the first step, the nucleophilic attack of glutathione to the common β-O-4-aryl-ether motif in lignin is catalyzed by β-etherases and afterwards the glutathione is removed again by the action of glutathione lyases. Given their potential impact for lignin valorization, in this paper novel glutathione lyases are reported and biocatalytically characterized based on lignin model compounds. As a result, an enzyme exhibiting increased thermostability and lowered enantioselectivity – key features for implementation of glutathione lyases in enzymatic lignin depolymerization processes – was identified. Furthermore, first mutational studies of these enzymes revealed the possibility to further alter the activity as well as enantioselectivity of glutathione lyases by means of protein engineering. From a practical perspective, one-pot multi-step processes combining β-etherases and glutathione lyases are successfully set-up, giving hints on the potential that the implementation of these biocatalysts may bring for biorefinery purposes. [ABSTRACT FROM AUTHOR]

Microbial β-etherases, which selectively cleave the β- O-4 aryl ether linkage present in lignin, hold great promise for future applications in lignin valorization. However, very few members have been reported so far and little is known about these enzymes. By using a database mining approach, four novel bacterial β-etherases were identified, recombinantly produced in Escherichia coli, and investigated together with known β-etherases in the conversion of various lignin and non-lignin-type model compounds. The resulting activities revealed the significant influence of the substituents at the phenyl ring adjacent to the ether bond. Finally, β-etherase activity on polymeric substrates, measured by using a fluorescently labeled synthetic lignin, was also proven; this underlined the applicability of the enzymes for the conversion of lignin into renewable chemicals. [ABSTRACT FROM AUTHOR]

Hydrolases catalyze synthetic reactions in nonaqueous media, whereas they perform hydrolysis under aqueous solutions. An acyl transferase from Mycobacterium smegmatis (MsAcT) is able to catalyze synthetic reactions in buffer because of its highly hydrophobic active site, which enables efficient transesterification reactions even at 99.9 % v/v buffer solution. This unique feature of MsAcT among hydrolases may open new opportunities to conduct synthetic (bio)catalysis in aqueous media. With these goals in mind, this paper explores some evidence of such potential: MsAcT can perform enantioselective transesterifications (e.g., ( S)-2-octanol), which could be combined with other aqueous multistep (asymmetric) reactions; 5-hydroxymethylfurfural (HMF) can be esterified to produce more hydrophobic and easily extractable HMF esters (e.g., for downstream processing or wastewater treatment); and upon addition of dilute H2O2, MsAcT works efficiently as a perhydrolase to form in situ peracids-in bulk water-that can be used for oxidations (e.g., furfural to furoic acid oxidation). Overall, these and many other new applications can be envisaged by using MsAcT in aqueous solutions. [ABSTRACT FROM AUTHOR]

Deep eutectic solvents (DESs) are formed by mixing quaternary ammonium salts (e.g., choline chloride) and hydrogen-bond donors (e.g., glycerol or urea), which leads to biodegradable and readily available ionic solvents at room temperature. Analogous to other ionic liquids, DESs represent a promising reaction media if hydrophobic and hydrophilic substrates need to be combined. This paper assesses DESs as reaction media for chymotrypsin-catalyzed peptide synthesis. After careful determination of the reaction conditions (e.g., water content, enzyme loading), α-chymotrypsin displayed high activity for peptide synthesis in choline chloride/glycerol mixtures to afford productivities of ca. 20 g L-1 h-1 and with complete selectivity for the peptide, which is in contrast to the detrimental hydrolysis pathway observed in aqueous media. The nonimmobilized suspended enzyme could be reused several times by simple filtration with excellent to moderate activities. Overall, the results reported suggest that choline chloride based DESs may become promising neoteric solvents for peptide synthesis through biocatalysis. [ABSTRACT FROM AUTHOR]

Furfural and 5-hydroxymethylfurfural (HMF) are important biomass-derived platform chemicals that can be obtained from the dehydration of lignocellulosic sugars. A possible route for the derivatization of furanics is their oxidation to afford a broad range of chemicals with promising applications (e.g., diacids, hydroxyl acids, aldehyde acids, monomers for novel polymers). Herein we explore the organic peracid-assisted oxidation of furanics under mild reaction conditions. Using lipases as biocatalysts, alkyl esters as acyl donors, and aqueous solutions of hydrogen peroxide (30 % v/ v) added stepwise, peracids are formed in situ, which subsequently oxidize the aldehyde groups to afford carboxylic acids with high yields and excellent selectivities. Furthermore, the use of an immobilized silica-based 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) affords the selective oxidation of the hydroxymethyl group of HMF to afford 2,5-diformylfuran. That product can be subsequently oxidized using again lipases for the in situ peracid formation to yield 2,5-furandicarboxylic acid, which is considered to be a key building block for biorefineries. These lipase-mediated reactions proceeded efficiently even with high substrate loadings under still non-optimized conditions. Overall, a proof-of-concept for the oxidation of furanics (based on in situ formed organic peracids as oxidants) is provided. [ABSTRACT FROM AUTHOR]

Pulp & Oil: We report the unprecedENted formation of de ‐ aromatized lignin oil together with a white polysaccharide fraction whEN lignocellulose is treated with peracids, which are formed in situ by ENzymes. A preliminary characterization of the lignin oil is provided, together with the evidENce that the delignified lignocellulose is accessible to cellulases to afford fermENtable sugars. As a first proof ‐ of ‐ concept, the reported approach may bring promising new research lines in the future. [ABSTRACT FROM AUTHOR]

The profen family belongs to the most important non-steroidal anti-inflammatory drugs (NSAIDs). A considerable number of biocatalytic processes for the synthesis of optically pure (S)-profens have been proposed. Despite of the excellent enantioselectivity and the large advantages that enzyme catalysis offers in terms of sustainability, biocatalytic processes have failed so far to meet the technical and economic challenges of commercialization. This critical review outlines recent trends and developments of novel applications that appear very promising in terms of enantioselectivity, efficiency, sustainability and yield. Special emphasis is placed on the contribution of protein engineering in overcoming the limitations of these enzymes for technical applications, and thus providing promising biocatalysts for the preparation of pharmaceutical products. The natural catalytic diversity, assisted by modern methods of protein engineering, provides novel concepts and leads for the environmentally friendly synthesis of pharmacologically important drugs. Considerable progress can be expected in the coming decades. Furthermore, aspects regarding ecological footprints and the impact of each biocatalytic route are critically addressed, considering aspects like the type of solvent, waste produced, availability of substrate, etc. When possible, suggestions for combining efficiency with more sustainable synthetic approaches are also given. [ABSTRACT FROM AUTHOR]

Abstract: The use of oxidoreductases as biocatalysts for the production of a wide number of chiral building blocks is presently a mature (bio-)technology. In this context some industrial applications are currently performed by means of those enzymatic approaches, and new examples are expected to be realized. Moreover, oxidoreductases provide an interesting academic platform to undertake fundamental research in enzymology, to acquire a better understanding on catalytic mechanisms, and to facilitate the development of new biocatalytic applications. Within this area, a wide number of oxidoreductases from genus Candida spp. have been characterized and used as biocatalysts. These enzymes are rather diverse, and are able to carry out many useful reactions, like highly (enantio)selective keto-reductions, (de)racemizations and stereoinversions, and promiscuous catalytic imine reductions. In addition, some Candida spp. dehydrogenases are very useful for regenerating the cofactors, with the aid of sacrificial substrates. Addressing those features, the present paper aims to give an overview of these enzymes, by focusing on practical applications that these biocatalysts can provide. Furthermore, when possible, academic insights on the enzymatic performances will be discussed as well. [Copyright &y& Elsevier]

Abstract: The enzymatic promiscuity concept involves the possibility that one active site of an enzyme can catalyse several different chemical transformations. A rational understanding of the mechanistic reasons for this catalytic performance could lead to new practical applications. The capability of certain hydrolases to perform the perhydrolysis was described more than a decade ago, and recently its molecular basis has been elucidated. Remarkably, a similarity between perhydrolases (cofactor-free haloperoxidases) and serine hydrolases was found, with both groups of enzymes sharing a common catalytic triad, which suggests an evolution from a common ancestor. On the other hand, several biotechnological applications derived from the capability of hydrolases to catalyse the synthesis of peracids have been reported: the use of hydrolases as bleaching agents via in situ generation of peracids; (self)-epoxidation of unsaturated fatty acids, olefins, or plant oils, via Prileshajev epoxidation; Baeyer-Villiger reactions. In the present review, the molecular basis for this promiscuous hydrolase capability, as well as identified applications are reviewed and described in detail. [Copyright &y& Elsevier]

Abstract: In recent years, the Carica papaya lipase (CPL) is attracting more and more interest. This hydrolase, being tightly bonded to the water-insoluble fraction of crude papain, is thus considered as a “naturally immobilized” biocatalyst. To date, several CPL applications have already been described: (i) fats and oils modification, derived from the sn-3 selectivity of CPL as well as from its preference for short-chain fatty acids; (ii) esterification and inter-esterification reactions in organic media, accepting a wide range of acids and alcohols as substrates; (iii) more recently, the asymmetric resolution of different non-steroidal anti-inflammatory drugs (NSAIDs), 2-(chlorophenoxy)propionic acids, and non-natural amino acids. Taking into account the novelty and the current interest of the topic, this review aims to highlight the origin, features, and applications of the C. papaya lipase, with the objective to prompt research groups to further investigate the spectra of applications that this emerging and versatile CPL could have in the future. [Copyright &y& Elsevier]

Abstract: Candida rugosa lipase (CRL) is one of the enzymes most frequently used in biotransformations. However, there are some irreproducibility problems inherent to this biocatalyst, attributed either to differences in lipase loading and isoenzymatic profile or to other medium-engineering effects (temperature, a w, choice of solvent, etc.). In addition, some other properties (influence of substrate and reaction conditions on the lid movement, differences in the glycosylation degree, post-translational modifications) should not be ruled out. In the present paper the recent developments published in the CRL field are overviewed, focusing on: (a) comparison of structural and biochemical data among isoenzymes (Lip1–Lip5), and their influence in the biocatalytical performance; (b) developments in fermentation technology to achieve new crude C. rugosa lipases; (c) biocatalytical reactivity of each isoenzyme, and methods for characterising them in crude CRL; (d) state-of-the-art of new applications performed with recombinant CRLs, both in CRL-second generation (wild-type recombinant enzymes), as well as in CRL-third generation, (mutants of the wt-CRL). [Copyright &y& Elsevier]

Whole-cell biocatalysis with the use of baker's yeast is demonstrated in different mixtures of water with deep eutectic solvents (DESs; choline chloride/glycerol, 1:2 mol/mol). Enantioselective ketone reduction is observed for long reaction times (>200 h), which suggests that the whole cells remain stable in these neoteric solvents. By changing the proportion of the DES added, a complete inversion of enantioselectivity is observed, from approximately 95 % enantiomeric excess ( ee) ( S) in pure water to approximately 95 % ee ( R) in the pure DES. Presumably, some ( S)-oxidoreductases present in baker's yeast are inhibited by DESs. [ABSTRACT FROM AUTHOR]

The article discusses a study on a novel type of enzymes, the nitrile reductases or nitrile oxidoreductases which catalyze the four-electron reduction to afford primary amines from nitriles. It cites efforts for the elucidation of the nitrile reduction mechanism proposing a reaction pathway that involve two molecules of cofactor in each catalytic turnover. It suggests the these enzymes may be widely spread in nature and opens the lead for new biocatalytic applications in the nitrile chemistry.

• Lipase-CLEA aggregates can be efficiently immobilized in Lentikats®, leading to a highly robust biocatalyst. • The new biocatalyst can be used in low viscous non-conventional Deep Eutectic Solvent – Buffer systems. • The new biocatalyst displays outstanding stability and straightforward recovery. The immobilization of lipases in cross linked aggregates (CLEA) leads to a robust biocatalyst that remains very stable in low viscous non-conventional Deep Eutectic Solvents – Buffer mixtures. To reinforce that stability, and to facilitate the biocatalyst recovery, this paper explores the immobilization of Lipase-CLEA derivatives in Lentikats®. This double immobilization can be successfully used in esterifications in DES-buffer media, using substrates of unpaired solubilities (e.g. benzoic acid and glycerol), in batch and continuous processes, and reaching full conversion. Under these conditions, the derivatives display an improved stability (compared to the Lipase-CLEA derivatives) and enable the reuse of the reaction media in continuous devices for at least 6 cycles under non-optimized conditions, accumulating 10 g product L−1, enhancing the productivity, and opening exciting future options for sustainable chemistry. [ABSTRACT FROM AUTHOR]