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Biocatalysis will play a central role in future chemical conversions, aiming at more sustainable strategies while keeping efficiency and economics. The mere use of enzymes does not ensure the greenness of a process, and quantitative environmental metrics must substantiate this. Simple but meaningful approaches are needed, particularly when reactions are still at a preliminary phase of research. This paper explores the use of the E-factor and a gate-to-gate strategy to assess environmental metrics for biocatalysis at the proof-of-concept stage. The upstream part – the actual biocatalytic reaction – and the downstream part – the purification of the product – are considered, providing the complete E-factor of the synthesis. As a case study, the hydration of oleic acid catalyzed by oleate hydratases (OHY) is used. Depending on the reaction conditions (particularly the substrate loadings), and the chosen downstream, total E-factors range from 15–20 to 160, mostly comprising wastewater (from the reaction media) and solvent (from the downstream part). The evaluation is completed with the estimation of the Total Carbon Dioxide Release (TCR), kg of CO2 per kg of product, which provides comparable data to benchmark synthetic strategies. The timely identification of hot spots (large contributors to the environmental impact) enables researchers and practitioners to propose experiments to show that biocatalysis can be performed under more sustainable conditions. [ABSTRACT FROM AUTHOR]

For a knowledge-based design of enzyme catalysis in deep eutectic solvents (DESs), the influence of the DES properties (e.g., water activity and viscosity) and the impact of DESs and their individual components must be assessed. This paper investigates three different DESs: choline chloride–glycerol (ChCl–Gly), choline chloride–ethylene glycol (ChCl–EG), and ethyl ammonium chloride–glycerol (EACl–Gly). The specific activity and half-life time of horse liver alcohol dehydrogenase (HLADH) were experimentally determined in these DESs with water contents ranging from 0%–100%. HLADH showed limited activity in neat DESs, which was enhanced by adding water. Experiments with individual DES components of ChCl–Gly were carried out to clarify their individual influence. Glycerol acts as a strong stabilizer for the enzyme, whereas choline chloride's results are deleterious. To understand the experimental findings, molecular dynamics (MD) simulations were carried out to quantify the solvation layer and calculate the spatial distribution of the solvent molecules around HLADH. The experimental and the in silico approach suggests that designing novel DESs with higher glycerol loadings would result in improved media for biocatalysis. This is demonstrated by performing the reduction of cinnamaldehyde to cinnamyl alcohol—a relevant compound for the food industry and cosmetics—in ChCl–Gly (1 : 9 molar ratio), with 20 vol% water to decrease the viscosity. HLADH is highly active and stable under these new conditions, giving promising productivity (15.3 g L−1 d−1). This paper demonstrates that DESs can be designed to be both substrate-solubilizers and enzyme-compatible, opening new research lines for green chemistry and biocatalysis. [ABSTRACT FROM AUTHOR]

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. [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]

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]

The fractionation of lignocellulose in its three main components, hemicellulose, lignin and cellulose pulp can be achieved in a biphasic system comprising water and bio-based 2-methyltetrahydrofuran (2-MeTHF) as solvents and oxalic acid as catalyst at mild temperatures (up to 140 °C). This so-called OrganoCat concept relies on selective hemicellulose depolymerization to form an aqueous stream of the corresponding carbohydrates, whereas solid cellulose pulp remains suspended and the disentangled lignin is to a large extent extracted in situ with the organic phase. In the present paper, it is demonstrated that biomass loadings of 100 g L−1 can be efficiently fractionated within 3 h whereby the mild conditions assure that no significant amounts of by-products (e.g. furans) are formed. Removing the solid pulp by filtration allows to re-use the water and organic phase without product separation in repetitive batch mode. In this way, (at least) 400 g L−1 biomass can be processed in 4 cycles, leading to greatly improved biomass-to-catalyst and biomass-to-solvent ratios. Economic analysis of the process reveals that the improved biomass loading significantly reduces capital and energy costs in the solvent recycle, indicating the importance of process integration for potential implementation. The procedure was successfully scaled-up from the screening on bench scale to 3 L reactor. The feedstock flexibility was assessed for biomasses containing moderate-to-high hemicellulose content. [ABSTRACT FROM AUTHOR]

Benzaldehyde lyase (BAL) results in an efficient biocatalyst for the umpolung carboligation of furfural, HMF, and mixtures of them, leading to blended C10–C12 platform chemicals. Subsequently, the mixing and gentle heating (＜100 °C) of the formed hydroxy-ketone with choline chloride leads to the formation of a novel biomass-derived deep-eutectic-solvent. [ABSTRACT FROM AUTHOR]

Cellulases are promising catalysts for the depolymerization of cellulose under mild conditions. Reengineered cellulases are required to match application demands in biorefineries and to avoid cost-intensive downstream processing. This manuscript provides a novel fluorescence-based high throughput screening method for directed evolution of cellulases, based on 4-methylumbelliferyl-β-d-cellobioside (4-MUC). The 4-MUC high throughput screening system was successfully employed to identify CelA2 variants with enhanced stability and activity in mixtures of water with deep eutectic solvents like choline chloride : glycerol (ChCl : Gly), and seawater. The cellulase variant 4D1 (L21P; L184Q; H288R; K299I; D330G; N442D) was isolated and showed, compared to wild type, an increase in specific activity in 30% (v/v) ChCl : Gly (7.5-fold; 0.4 to 3.0 U mg−1) and in concentrated seawater (1.6-fold; 5.5 to 9.3 U mg−1). In addition, the residual activity of 4D1 in the presence of 3-fold concentrated seawater is unaffected whereas CelA2 wild type loses ＞50% of its activity. Furthermore, the position H288 was identified as a key position for activity and resistance in 4D1. [ABSTRACT FROM AUTHOR]

A one-pot multi-step approach comprising enzymatic oxidation-hydroxymethylation-reduction enables the synthesis of optically active α-aryl vicinal diols with high yields and enantioselectivities. Formaldehyde required for the hydroxymethylation step is enzymatically produced in situ using less hazardous methanol as substrate. [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]

A concept for a highly integrated fractionation of lignocellulose in its main components (cellulose-pulp, soluble hemicellulose sugars and lignin) is described, based on the selective catalytic depolymerization of hemicellulose in a biphasic solvent system. This leads to an effective disentanglement of the compact lignocellulose structure, liberating and separating the main components in a single step. At mild temperatures (80–140 °C), oxalic acid catalyzes selectively the depolymerization of hemicellulose to soluble sugars in aqueous solution, whereas the more crystalline cellulose-pulp remains solid and inaccessible to the acid catalysis. In the presence of a second organic phase consisting of bio-based 2-methyltetrahydrofuran (2-MTHF), lignin is directly separated from the pulp and the soluble carbohydrates by in situextraction. The oxalic acid catalyst can be crystallized from the aqueous solution, recovered and re-used. The delignified cellulose-pulp obtained from this biphasic system can be directly subjected to enzymatic depolymerization, affording soluble oligomers and glucose at rates almost comparable to those observed for the hydrolysis of commercial microcrystalline Avicel®. Overall, the concept may offer a promising approach for an efficient and selective pre-treatment of lignocellulosic materials under mild and environmentally-friendly conditions. [ABSTRACT FROM AUTHOR]

Dicarboxylic acids (e.g.oxalic and maleic acid) are able to depolymerize cellulose, producing oligomers and glucose. However, to reach efficient organic-acid-catalyzed performances with crystalline celluloses, high temperatures (>160 °C) are needed. These energetically-demanding conditions lead to undesired sugar degradation as well. Herein it is shown that organic acid-catalyzed cellulose depolymerization can proceed efficiently in water under mild reaction conditions (100–125 °C) by the addition of inexpensive NaCl (30 wt%). The application of some pressure in the reactor (10–30 bar) also influences and improves the depolymerization outcome. It is believed that the salt solutions act in a mechanism similar to ionic liquids and disrupt the hydrogen-bond matrix among cellulose fibers. Depolymerization proceeds efficiently with amorphous cellulose, α-cellulose, as well as with microcrystalline cellulose (Avicel®). Importantly, catalysis can be easily controlled by temperature, catalyst loading and salt concentrations, as well as by the applied pressure in the reactor, and thus sugar degradation can be diminished. Furthermore, experiments conducted using concentrated seawater as solvent and maleic acid as catalyst showed positive results in the hydrolysis of Avicel®. [ABSTRACT FROM AUTHOR]

Correction for ‘Fractionation of lignocellulosic biomass using the OrganoCat process’ by Philipp M. Grande et al., Green Chem., 2015, 17, 3533–3539. [ABSTRACT FROM AUTHOR]

Enzymes create chiral microenvironments that may simultaneously generate several stereogenic centers in the same catalytic cycle, broadening the possibilities of biocatalysis. Benzaldehyde lyase (BAL) affords highly diastereoselective α-hydroxy-ketones by simultaneously performing ligation and kinetic resolution of a racemic aldehyde. Thus, to the well-known enantioselective BAL-carboligation of aldehydes (C-C bond formation), another property, namely diastereoselectivity, is added in this paper for the first time.

Ether bonds are found in a wide variety of natural products--mainly secondary metabolites--including lipids, oxiranes, terpenoids, flavonoids, polyketides, and carbohydrate derivatives, to name some representative examples. To furnish such a biodiversity of structures, a large number of different enzymes are involved in several different biosynthetic pathways. Depending on the compound and on the (micro) environment in which the reaction is performed, ethers are produced by very different (enzymatic) reactions, thus providing an impressive display of how Nature has combined evolution and thermodynamics to be able to produce a vast number of compounds. In addition, many of these compounds possess different biological activities of pharmacological interest. Moreover, some of these ethers (i.e., epoxides) have high chemical reactivity, and can be useful starting materials for further synthetic processes. This review aims to provide an overview of the different strategies that are found in Nature for the formation of these "bioethers". Both fundamental and practical insights of the biosynthetic processes will be discussed.

An ATR-FT-IR-based method was applied to measure the degree of substitution (DS) of esterified cellulose. The strategy makes use of the capability of certain ionic liquids (ILs) to dissolve cellulose derivatives. To avoid high viscosity that could hamper the correct measurement, a mixture of DMSO/IL (2 : 1, w/w) was applied. In ATR-FT-IR, ester absorption appeared at ca. 1740 cm(-1), whereas carboxylic groups (acids) did at ca. 1710 cm(-1), and carboxylates at ca. 1650 cm(-1). The overlap of absorbances (ester-carboxylic acid) was circumvented by adding base equivalents, i.e. monoethanolamine (MEA). The base converted all free carboxylic acids in MEA-carboxylates, thus shifting the ATR-FT-IR integrated absorbance of carboxylic acid from ca. 1710 cm(-1) to ca. 1650 cm(-1), whereas the ester absorbance remained at ca. 1740 cm(-1). Therefore a proper separation among peaks (esters vs. carboxylates) was achieved, and quantitative measurements of DS (ester) were obtained even when excesses of carboxylic acids were present (which can be analogous to actual reaction conditions for the esterification of cellulose). The high versatility of ILs suggests that the herein reported strategy might be rapidly extended to the study of other polymeric derivatives (i.e. containing carboxylic acids, etc.), by choosing the correct tailor-made IL, as well as to other analytic techniques.