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The combination of ball milling (BM), microwave irradiation (MI), and deep eutectic solvents (DES) results synergistic for an efficient, selective, and very rapid (10 min) delignification of materials with high lignin content (ca. 50 wt%) such as walnut shells (WS). Lignin is dissolved in the DES, whereas the polysaccharide fractions remain suspended with limited degradation, due to the rapid pretreatment. After ball milling procedure (3 h), biomass loadings in the range of 100–200 g L−1 are selectively delignified in 10 min at 150 °C by using choline chloride:formic acid DES (1:2 molar ratio), rendering lignin yields of 60–80% (ca. ~ 40–60 g lignin L−1). Ball milling, microwave irradiation, and DES systems are much more efficient than ball milling, conventional heating, and DES system. The obtained lignins exhibited similar Fourier-transform infrared spectroscopy (FTIR) profile to that of milled wood lignin (MWL), indicating minimal functional group changes. Graphical abstract

Biocatalysis has emerged as a powerful and useful strategy for the synthesis of active pharmaceutical ingredients (APIs). The outstanding developments in molecular biology techniques allow nowadays the screening, large-scale production, and designing of biocatalysts, adapting them to desired reactions. Many enzymes can perform reactions both in aqueous and non-aqueous media, broadening even further the opportunities to integrate them in complex pharmaceutical multi-step syntheses.This paper showcases several examples of biocatalysis in the pharmaceutical industry, covering examples of different enzymes, such as lipases, oxidoreductases, and transaminases, to deliver active drugs through complex synthetic routes. Examples are critically discussed in terms of reaction conditions, motivation for using an enzyme, and how biocatalysts can be integrated in multi-step syntheses. When possible, biocatalytic routes are benchmarked with chemical reactions.The reported enzymatic examples are performed with high substrate loadings (100 g L

The use of deep eutectic solvents (DES) with buffer as cosolvent (up to 10 % v/v) leads to low-viscous media in which lipases can perform synthetic reactions, instead of hydrolysis. This paper explores the immobilization of Pseudomonas stutzeri lipase (TL) in cross-linking aggregates (CLEA) to deliver robust derivatives that are active in media like choline chloride – glycerol DES with buffer as cosolvent. While the free TL enzyme was markedly inactive in these media, TL-CLEA derivatives perform esterifications and can be reused several times. Overall, results are consistent with previous experiments reported for other lipases in these DES-water media and confirm that CLEA immobilization turns out a very useful and straightforward alternative for generating active (bio)catalysts for DES-aqueous media systems. Immobilized systems open the possibility of performing continuous processes in low-viscous DES-buffer media.

The use of oxidoreductases in organic-aqueous biphasic systems is advantageous (effective solvation of reactants, minimization of substrate/product-induced inhibition, improved volumetric productivity, and straightforward downstream processing). This paper explores the effects of organic solvents on horse liver alcohol dehydrogenase (HLADH) by combining experimental and computational studies. Various organic solvents displaying a broad range of hydrophobicity and functionalities are used, namely, ethyl acetate, 2-methyltetrahydrofuran, methyl tert-butyl ether, cyclopentyl methyl ether, toluene, cyclohexane, heptane, and dodecane. The catalytic performance of model enzyme horse liver alcohol dehydrogenase concerning its activity, stability, and selectivity is experimentally evaluated. The results are interpreted with molecular dynamics simulations by assessing the (i) protein location in biphasic media, (ii) organic solvent distribution, and (iii) enzyme conformation. Herein, the stability states the robustness of the enzyme while storing it in biphasic media without catalysis taking place. Overall, different toxicities of the solvent to the enzyme can be pinpointed: "molecular toxicity", related to the solvent functional groups, and "interfacial toxicity", related to the position of the enzyme at the interface. Likewise, some solvents are more prone to be located close to the active site of the enzyme, triggering other effects on the enzymatic performance. Thus, methyl tert-butyl ether resulted as an optimal option for the enzyme, whereas other solvents like toluene and 2-methyltetrahydrofuran were detrimental. The combined forces of experiments and simulations have been shown to be useful tools to study the effects of reaction media, thus guiding solvent selection.

Deep Eutectic Solvents (DES) are used as reaction media for lipase-catalyzed esterifications in continuous devices. In particular, DES may be useful for lipophilization-like reactions involving substrates with unpaired solubilities. Aspects to be considered are the viscosity of the solvent, as well as the stability of the enzyme in the non-conventional media. The viscosity can be decreased by adding buffer as cosolvent (up to 20% v/v) and keeping the non-conventional nature. Lipases can be stabilized by following a double immobilization pattern, comprising CLEA formation and entrapment in LentiKats

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.

The role and power of biocatalysis in sustainable chemistry has been continuously brought forward step by step to its present outstanding position. The problem-solving capabilities of biocatalysis have been realized by numerous substantial achievements in biology, chemistry and engineering. Advances and breakthroughs in the life sciences and interdisciplinary cooperation with chemistry have clearly accelerated the implementation of biocatalytic synthesis in modern chemistry. Resource-efficient biocatalytic manufacturing processes have already provided numerous benefits to sustainable chemistry as well as customer-centric value creation in the pharmaceutical, food, flavor, fragrance, vitamin, agrochemical, polymer, specialty, and fine chemical industries. Biocatalysis can make significant contributions not only to manufacturing processes, but also to the design of completely new value-creation chains. Biocatalysis can now be considered as a key enabling technology to implement sustainable chemistry.

The combination of deep eutectic solvents (DESs, ChCl/glycerol 1 : 2) with buffer (up to 15 % v/v) leads to solvent mixtures that exert viscosities below 25 mPa s

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. © 2022 The Authors. ChemSusChem published by Wiley-VCH GmbH.

The quest for sustainable solvents is currently a matter of intense research and development, as solvents significantly contribute heavily to the waste generated by chemical industries. Cyclopentyl methyl ether (CPME) is a promising eco-friendly solvent with valuable properties such as low peroxide formation rate, stability under basic and acidic conditions, and relatively high boiling point. This Review discusses the potential use of CPME for applications in biotechnology (e.g., biotransformations, as solvent or cosolvent), biorefineries, and bioeconomy (e.g., for furan synthesis or as an extractive agent in liquid-liquid separations), as well as for other purposes, such as chromatography or peptide synthesis. Although CPME is currently produced by petrochemical means with a remarkably high atom economy, its biogenic production can be envisaged from substrates such as cyclopentanol or cyclopentanone, which can be derived from furfural or from (bio-based) adipic acid, respectively. The combination of the promising properties of CPME as a (co)solvent with a future (economic) biogenic origin would be advantageous for setting strategies aligned with the sustainable chemistry principles.

Upon CO2 pressurization, 2-methyltetrahydrofuran (2-MTHF) expands. The expanded phase exerts different solvation properties, triggering fractional lignin precipitation. Depending on the actual CO2 pressure, fractions of lignin with a gradient of different sizes and chemical properties are selectively precipitated. A range of raw materials and applications may be thus reached.

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., It's not only lipases which can be applied in alternative solvent systems to meet industrial and environmental demands. At the hand of case studies and flowcharts this review quickly shows what solvent systems are viable.

The application of biocatalysis and White Biotechnology tools in chemical areas concerning the production of bulk compounds and other related low-added value products (with high volumes) has been gaining importance in recent years. The expected drivers of biocatalysis for these sectors are energy savings, regioselectivity (leading to cleaner products), the possibility of using thermolabile substrates, as well as the generation of less by-products and manageable wastes. This paper explores some recent industrial granted patents related to biocatalysis and bulk chemicals. Several patents have been identified in fields such as biodiesel and esterification reactions, and sugar or furan chemistry. Overall, innovative strategies involve the identification of novel enzymes, the set-up of improved immobilization methods, as well as novel reactor designs that can offer improved performances and economics. The reported examples indicate that biocatalysis can certainly offer opportunities for these areas as well, far from the typical pharmaceutical and fine chemical applications often reported in the literature.

In this series of articles, the board members of ChemSusChem discuss recent research articles that they consider of exceptional quality and importance for sustainability. This entry features Dr. P. Domínguez de María, who introduces the biocatalytic synthesis of esters and amides in aqueous media by means of some acyltransferases that seem to proceed against Le Chatelier's principle. Continuous processes with immobilized forms of these enzymes enable the efficient production of aroma esters and important amides (e. g., melatonin) in aqueous solutions and using natural substrates with limited environmental impact.

Alcohol dehydrogenase (ADH) catalyzed reductions in deep eutectic solvents (DESs) may become efficient and sustainable alternatives to afford alcohols. This paper successfully explores the ADH-catalyzed reduction of ketones and aldehydes in a DES composed of choline chloride and 1,4-butanediol, in combination with buffer (Tris-HCl, 20% v/v). 1,4-butanediol (a DES component), acts as a smart cosubstrate for the enzymatic cofactor regeneration, shifting the thermodynamic equilibrium to the product side. By means of the novel DES media, cyclohexanone reduction was optimized to yield maximum productivity with low enzyme amounts (in the range of 10 g L−1 d−1). Notably, with the herein developed reaction media, cinnamaldehyde was reduced to cinnamyl alcohol, an important compound for the fragrance industry, with promising high productivities of ~75 g L−1 d−1. Fil: Chanquia, Santiago Nahuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; Argentina Fil: Huang, Lei. University Aarhus; Dinamarca Fil: Garcia Liñares, Guadalupe Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; Argentina Fil: Domínguez de María, Pablo. Sustainable Momentum; España Fil: Kara, Selin. University Aarhus; Dinamarca

This work explores for the first time the use of Deep Eutectic Solvents (DES) with phosphate buffer 100 mM pH 7 as cosolvent (10% v/v) in biocatalytic reactions in fed-batch and packed-bed bioreactors. The lipase-catalyzed esterification of glycerol and benzoic acid is studied, as it involves two substrates with different polarities (for which DES are needed). In the fed-batch bioreactor, the highest conversion (90%) was obtained at a substrate flow rate of 0.01 mL/min. The fed-batch operation increased the conversion by 59% compared to the batch mode. Regarding productivity, semi-continuous and continuous bioreactors showed analogous results. Upon recirculation of the reaction media in the continuous bioreactor, a conversion of 67% was achieved in 7 cycles of operation. The stability of the biocatalyst in the packed-bed bioreactor decreased only 2% in 10 days, demonstrating the attractiveness that low viscous DES-water mixtures with continuous processes may have.

The use of oxidoreductases (EC1) in non‐conventional reaction media has been increasingly explored. In particular, deep eutectic solvents (DESs) have emerged as a novel class of solvents. Herein, an in‐depth study of bioreduction with an alcohol dehydrogenase (ADH) in the DES glyceline is presented. The activity and stability of ADH in mixtures of glyceline/water with varying water contents were measured. Furthermore, the thermodynamic water activity and viscosity of mixtures of glyceline/water have been determined. For a better understanding of the observations, molecular dynamics simulations were performed to quantify the molecular flexibility, hydration layer, and intraprotein hydrogen bonds of ADH. The behavior of the enzyme in DESs follows the classic dependence of water activity (a W) in non‐conventional media. At low a W values (, Enzyme solvation: The use of deep eutectic solvents (DESs) is examined experimentally and computationally for the alcohol dehydrogenase catalyzed reaction. The outcomes of molecular dynamics simulations complement experimental observations on catalytic performance of the enzyme for a greater understanding of influencing factors.

The combination of Deep-eutectic-solvents (DES) with water as “co-solvent” enables a low-viscous reaction medium that keeps its “non-conventional” nature and thus enables synthetic lyophilization reactions (e.g. esterification) catalyzed by hydrolases. Substrates with different polarity may be employed. This paper shows how the enzyme immobilization with cross-linking aggregates (CLEA) leads to highly stable and active immobilized catalysts in different DES. As a remarkable case, when choline chloride-glycerol DES is used, CLEA derivatives of Candida antarctica lipase B (CLEA-CALB) are stable for at least 14 days without any loss of activity. The immobilized biocatalysts are applied in non-viscous DES-water blends (8% v/v) to catalyze the esterification of benzoic acid and glycerol to furnish glyceryl monobenzoate (α-MBG) in productivities of ∼35 g α-MBG L−1d−1. Compared to other commercial immobilized CALB, the CLEA-CALB derivatives rendered more product (higher conversions by 30%). Moreover, CLEA derivatives were successfully reused for six times without any loss of activity. Given the ease of immobilization (CLEA), their excellent performance in DES and the low viscosity of the DES-water blends, the reported approach may be useful for many synthetic procedures and even for continuous processes with largely optimized outcomes.

To develop novel biorefinery concepts, the use of bio-based catalysts and solvents must be aligned with the principles of green chemistry. In this context, biogenic 2,5-furandicarboxylic acid (FDCA) is a very promising yet underused molecule with high potential for application as an acid catalyst, combining feasibility and sustainability with efficient and straightforward recovery. In this study, FDCA was evaluated as a catalyst in the recently developed OrganoCat pretreatment, a biphasic lignocellulose fractionation system. The catalyst was investigated for the efficient fractionation of the three main components-lignin, cellulose and noncellulosic sugars-with particular focus on the lignin quality, on the effect on enzymatic hydrolysis of the cellulosic residue, and on the noncellulosic sugar extraction. To address recovery of FDCA from the OrganoCat system, a method was developed, leading to the recovery of >97 % of FDCA with a spectroscopic purity of >99 %, maintaining full activity in consecutive runs.

Lignin is the major aromatic biopolymer in nature, and it is considered a valuable feedstock for the future supply of aromatics. Hence, its valorisation in biorefineries is of high importance, and various chemical and enzymatic approaches for lignin depolymerisation have been reported. Among the enzymes known to act on lignin, β-etherases offer the possibility for a selective cleavage of the β-O-4 aryl ether bonds present in lignin. These enzymes, together with glutathione lyases, catalyse a reductive, glutathione-dependent ether bond cleavage displaying high stereospecificity. β-Etherases and glutathione lyases both belong to the superfamily of glutathione transferases, and several structures have been solved recently. Additionally, different approaches for their application in lignin valorisation have been reported in the last years. This review gives an overview on the current knowledge on β-etherases and glutathione lyases, their biochemical and structural features, and critically discusses their potential for application in biorefineries.

Biocatalysis has been traditionally considered an environment-friendly and industrial toolbox for organic synthesis. However, in many cases these statements are not substantiated with real metrics (e.g. E-factor and productivities). This paper discusses recent developments that may contribute to generate greener biocatalytic applications with industrial interest. Namely, aqueous-based processes with extremely high substrate loadings; solvent-free reactions in which neither solvents nor water are used; and finally, biogenic solvents as well as deep eutectic solvents. From all of them, solvent-free systems are superior both in terms of greenness and industrial use (but they cannot be always implemented). The described systems can be combined in flow (continuous), and with solvent and water recycling, leading to powerful alternatives for future sustainable processes. Overall, providing metrics is key to honestly assess a biotransformation and to put forth further research to improve systems.

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.

The conversion of lignocellulose into its building blocks and their further transformation into valuable platform chemicals (e. g., furfural) are key technologies to move towards the use of renewable resources. This paper explored the disentanglement of lignocellulose into hemicellulose‐derived sugars, cellulose, and lignin in a biphasic solvent system (water/2‐methyltetrahydrofuran) using phosphoric acid as recyclable catalyst. Integrated with the biomass fractionation, in a second step hemicellulose‐derived sugars (mainly xylose) were converted to furfural, which was in situ extracted into 2‐methyltetrahydrofuran with high selectivity (70 %) and yield (56 wt %). To further increase the economic feasibility of the process, a downstream and recycling strategy enabled recovery of phosphoric acid without loss of process efficiency over four consecutive cycles. This outlines a more efficient and sustainable use of phosphoric acid as catalyst, as its inherent costs can be significantly lowered., Lignin, cellulose, and furfural: In a two‐step procedure, lignocellulose is first fractionated into lignin, cellulose, and monosaccharides to then synthesize furfural from the pentoses in the sugar hydrolyzate. Phosphoric acid is used as catalyst for the whole process and recycled over four cycles without loss in activity. The biphasic 2‐MTHF‐aqueous system allows for high selectivity, straightforward product separation, and downstream processing.

Amino acids are of paramount importance as chiral building blocks of life, for drug development in modern medicinal chemistry, and for the manufacture of industrial products. In this work, the stereoselective synthesis of (S)- and (R)-2-amino-4-hydroxybutanoic acid was accomplished using a systems biocatalysis approach comprising a biocatalytic one-pot cyclic cascade by coupling of an aldol reaction with an ensuing stereoselective transamination. A class II pyruvate aldolase from E. coli, expressed as a soluble fusion protein, in tandem with either an S- or R-selective, pyridoxal phosphate dependent transaminase was used as a catalyst to realize the conversion, with formaldehyde and alanine being the sole starting materials. Interestingly, the class II pyruvate aldolase was found to tolerate formaldehyde concentrations of up to 1.4 M. The cascade system was found to reach product concentrations for (S)- or (R)-2-amino-4-hydroxybutanoic acid of at least 0.4 M, rendering yields between 86% and >95%, respectively, productivities of >80 g L–1 d–1, and ee values of >99%., This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 635595 (CarbaZymes), the Ministerio de Economía y Competitividad (MINECO), the Fondo Europeo de Desarrollo Regional (FEDER) (grant no. CTQ2015-63563-R to P.C.), and COST action CM1303 Systems Biocatalysis., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

The low solubility of glycosylated flavonoids represents a hurdle to conduct efficient enzymatic deglycosylations in aqueous media. To overcome this drawback, environmentally-unfriendly dimethylsulfoxide (DMSO) is typically used as co-solvent. Using a specific diglycosidase from Acremonium sp. DSM24697 for the deglycosylation of the rutinosylated flavonoid (hesperidin) as model reaction, this communication explores the use of (non-hazardous and biodegradable) deep eutectic solvents (DESs) as co-solvents in flavonoid biocatalysis. The enzymatic deglycosylation was observed when DES composed of choline chloride and glycerol or ethylene-glycol was used at proportions of up to 40% (DES-Buffer, v/v), displaying a promising framework to combine enhanced flavonoid solubilities and high enzymatic activities. The deglycosylation activity significantly increased when the single DES components – glycerol and ethylene-glycol – were added (e.g. 140% of enzyme activity at glycerol at 40% v/v), whereas deleterious effects were observed when choline chloride was solely added, presumably due to its chaotropic effect. Future research opportunities may be envisaged in the genetic design to evolve more robust biocatalysts, and in tailoring DES to deliver more enzyme-compatible solvents. Fil: Weiz, Gisela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencias de la Tierra y Ambientales de La Pampa. Universidad Nacional de La Pampa. Facultad de Ciencias Exactas y Naturales. Instituto de Ciencias de la Tierra y Ambientales de La Pampa; Argentina Fil: Braun, Lucas Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencias de la Tierra y Ambientales de La Pampa. Universidad Nacional de La Pampa. Facultad de Ciencias Exactas y Naturales. Instituto de Ciencias de la Tierra y Ambientales de La Pampa; Argentina Fil: Lopez, Laura Rosana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencias de la Tierra y Ambientales de La Pampa. Universidad Nacional de La Pampa. Facultad de Ciencias Exactas y Naturales. Instituto de Ciencias de la Tierra y Ambientales de La Pampa; Argentina Fil: de María, Pablo Domínguez. Sustainable Momentum; España Fil: Breccia, Javier Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencias de la Tierra y Ambientales de La Pampa. Universidad Nacional de La Pampa. Facultad de Ciencias Exactas y Naturales. Instituto de Ciencias de la Tierra y Ambientales de La Pampa; Argentina

An emerging biogenic aprotic solvent is the cellulose-derived dihydrolevoglucosenone, or 6,8-dioxabicyclooctanone (Cyrene™). This paper explores the use of Cyrene in lipase-catalyzed biotransformations, both in aqueous solutions – as co-solvent – as well as non-conventional media for synthesis. Cyrene is useful as organic solvent for lipophilizations using benzoic acid and glycerol as a model system for substrates with unpaired solubilities. The immobilized lipase B from Candida antarctica is active in Cyrene, yielding up to ∼ 10 g product L−1. Interestingly, crosslinked aggregates immobilized lipases (CLEA) remain significantly stable in Cyrene, enabling its use along several catalytic cycles. Cyrene is highly hygroscopic and forms geminal-diol structures with water, leading to solvent mixtures with a (tailored) gradient of polarities, what may be promising for biocatalysis in aqueous solutions. CALB displays hydrolytic reactions with different proportions of Cyrene as cosolvent, and CLEA derivatives remain fully active over 6 cycles at 30 % v/v Cyrene.

Deep eutectic solvents (DES) may become important alternatives as versatile, biodegradable, and cost-effective solvents for biocatalysis. Especially for reactions where substrates and products of different polarities are combined, the design of a tailored solvent that may dissolve all compounds-while being enzyme-compatible at the same time-appears to be a strong ally in sustainable chemistry. Herein it is shown that the combination of DES with "water as cosolvent" (in a range from 5% to 20% water, v/v) leads to non-conventional solvents with significantly reduced viscosity. In these media, lipases and proteases can perform synthetic reactions efficiently, and hydrolytic side reactions remain suppressed (even at 20% water, v/v). The use of these less viscous non-conventional media could also provide options for hydrolase-catalyzed synthetic reactions even in continuous fashion.