Biocatalysis in Water or in Non-Conventional Media? Adding the CO2 Production for the Debate.

Biocatalysis can be applied in aqueous media and in different non-aqueous solutions (non-conventional media). Water is a safe solvent, yet many synthesis-wise interesting substrates cannot be dissolved in aqueous solutions, and thus low concentrations are often applied. Conversely, non-conventional media may enable higher substrate loadings but at the cost of using (fossil-based) organic solvents. This paper determines the CO2 production-expressed as kg CO2·kg product-1-of generic biotransformations in water and non-conventional media, assessing both the upstream and the downstream. The key to reaching a diminished environmental footprint is the type of wastewater treatment to be implemented. If the used chemicals enable a conventional (mild) wastewater treatment, the production of CO2 is limited. If other (pre)treatments for the wastewater are needed to eliminate hazardous chemicals and solvents, higher environmental impacts can be expected (based on CO2 production). Water media for biocatalysis are more sustainable during the upstream unit-the biocatalytic step-than non-conventional systems. However, processes with aqueous media often need to incorporate extractive solvents during the downstream processing. Both strategies result in comparable CO2 production if extractive solvents are recycled at least 1-2 times. Under these conditions, a generic industrial biotransformation at 100 g L-1 loading would produce 15-25 kg CO2·kg product-1 regardless of the applied media.

Nitrile Synthesis with Aldoxime Dehydratases: A Biocatalytic Platform with Applications in Asymmetric Synthesis, Bulk Chemicals, and Biorefineries.

Nitriles comprise a broad group of chemicals that are currently being industrially produced and used in fine chemicals and pharmaceuticals, as well as in bulk applications, polymer chemistry, solvents, etc. Aldoxime dehydratases catalyze the cyanide-free synthesis of nitriles starting from aldoximes under mild conditions, holding potential to become sustainable alternatives for industrial processes. Different aldoxime dehydratases accept a broad range of aldoximes with impressive high substrate loadings of up to >1 Kg L-1 and can efficiently catalyze the reaction in aqueous media as well as in non-aqueous systems, such as organic solvents and solvent-free (neat substrates). This paper provides an overview of the recent developments in this field with emphasis on strategies that may be of relevance for industry and sustainability. When possible, potential links to biorefineries and to the use of biogenic raw materials are discussed.

Production of Bulk Chemicals with Biocatalysis: Drivers and Challenges Reflected in Recent Industrial Granted Patents (2015-2020).

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.

Modeling Alcohol Dehydrogenase Catalysis in Deep Eutectic Solvent/Water Mixtures.

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 (aW ) in non-conventional media. At low aW values (<0.2), ADH does not show any activity; at higher aW values, the activity was still lower than that in pure water due to the high viscosities of the DES. These findings could be further explained by increased enzyme flexibility with increasing water content.

Microbial ß-etherases and glutathione lyases for lignin valorisation in biorefineries: current state and future perspectives.

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, ß-etherases offer the possibility for a selective cleavage of the ß-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. ß-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 ß-etherases and glutathione lyases, their biochemical and structural features, and critically discusses their potential for application in biorefineries.

Multi-step biocatalytic depolymerization of lignin.

Lignin is a biomass-derived aromatic polymer that has been identified as a potential renewable source of aromatic chemicals and other valuable compounds. The valorization of lignin, however, represents a great challenge due to its high inherent functionalization, what compromises the identification of chemical routes for its selective depolymerization. In this work, an in vitro biocatalytic depolymerization process is presented, that was applied to lignin samples obtained from beech wood through OrganoCat pretreatment, resulting in a mixture of lignin-derived aromatic monomers. The reported biocracking route comprises first a laccase-mediator system to specifically oxidize the Cα hydroxyl group in the ß-O-4 structure of lignin. Subsequently, selective ß-O-4 ether cleavage of the oxidized ß-O-4 linkages is achieved with ß-etherases and a glutathione lyase. The combined enzymatic approach yielded an oily fraction of low-molecular-mass aromatic compounds, comprising coniferylaldehyde and other guaiacyl and syringyl units, as well as some larger (soluble) fractions. Upon further optimization, the reported biocatalytic route may open a valuable approach for lignin processing and valorization under mild reaction conditions.

Enantioselective imine reduction catalyzed by imine reductases and artificial metalloenzymes.

Adding value to organic synthesis. Novel imine reductases enable the enantioselective reduction of imines to afford optically active amines. Likewise, novel bioinspired artificial metalloenzymes can perform the same reaction as well. Emerging proof-of-concepts are herein discussed.

Intensified, Kilogram-Scaled, and Environment-Friendly: Chemoenzymatic Synthesis of Bio-Based Acylated Hydroxystyrenes.

Lignin-derived styrene derivatives are versatile building blocks for the manufacture of biobased polymers. As shown previously, phenol-protected hydroxystyrenes are accessible under industrially sound conditions (>100 g L-1, >95% yield) by subjecting biogenic phenolic acids to enzymatic decarboxylation and base-catalyzed acylation in nonaqueous media (wet cyclopentyl methyl ether, CPME). Herein, we demonstrate the production of 1 kg of 4-acetoxy-3-methoxy-styrene in a 10 L reactor and present practical adjustments to the up- and downstream processing that warrant a straightforward process and high isolated yields. Additionally, an environmental assessment is conducted, starting with a thorough E factor analysis to identify the sources that contribute most to the environmental burden (solvent and downstream processing). Also, the total CO2 production of the process is studied, including contributions from energy use and the treatment of generated wastes. The energy impact is evaluated through thermodynamic analysis, and the environmental footprint contributions by wastes-organic and aqueous fractions-are assessed based on CO2 emissions from solvent incineration and wastewater treatment, respectively. Overall, the holistic assessment of the process, its optimization, scale-up, product isolation, and environmental analysis indicate the feasibility of multistep chemoenzymatic reactions to deliver high-volume, low-value chemicals from biorefineries.

Integrated preservation of water activity as key to intensified chemoenzymatic synthesis of bio-based styrene derivatives.

The valorization of lignin-derived feedstocks by catalytic means enables their defunctionalization and upgrading to valuable products. However, the development of productive, safe, and low-waste processes remains challenging. This paper explores the industrial potential of a chemoenzymatic reaction performing the decarboxylation of bio-based phenolic acids in wet cyclopentyl methyl ether (CPME) by immobilized phenolic acid decarboxylase from Bacillus subtilis, followed by a base-catalyzed acylation. Key-to-success is the continuous control of water activity, which fluctuates along the reaction progress, particularly at high substrate loadings (triggered by different hydrophilicities of substrate and product). A combination of experimentation, thermodynamic equilibrium calculations, and MD simulations revealed the change in water activity which guided the integration of water reservoirs and allowed process intensification of the previously limiting enzymatic step. With this, the highly concentrated sequential two-step cascade (400 g·L-1) achieves full conversions and affords products in less than 3 h. The chemical step is versatile, accepting different acyl donors, leading to a range of industrially sound products. Importantly, the finding that water activity changes in intensified processes is an academic insight that might explain other deactivations of enzymes when used in non-conventional media.

Benzaldehyde lyase-catalyzed diastereoselective C-C bond formation by simultaneous carboligation and kinetic resolution.

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.

Biomass pretreatment affects Ustilago maydis in producing itaconic acid.

BACKGROUND: In the last years, the biotechnological production of platform chemicals for fuel components has become a major focus of interest. Although ligno-cellulosic material is considered as suitable feedstock, the almost inevitable pretreatment of this recalcitrant material may interfere with the subsequent fermentation steps. In this study, the fungus Ustilago maydis was used to produce itaconic acid as platform chemical for the synthesis of potential biofuels such as 3-methyltetrahydrofuran. No studies, however, have investigated how pretreatment of ligno-cellulosic biomass precisely influences the subsequent fermentation by U. maydis. Thus, this current study aims to first characterize U. maydis in shake flasks and then to evaluate the influence of three exemplary pretreatment methods on the cultivation and itaconic acid production of this fungus. Cellulose enzymatically hydrolysed in seawater and salt-assisted organic-acid catalysed cellulose were investigated as substrates. Lastly, hydrolysed hemicellulose from fractionated beech wood was applied as substrate. RESULTS: U. maydis was characterized on shake flask level regarding its itaconic acid production on glucose. Nitrogen limitation was shown to be a crucial condition for the production of itaconic acid. For itaconic acid concentrations above 25 g/L, a significant product inhibition was observed. Performing experiments that simulated influences of possible pretreatment methods, U. maydis was only slightly affected by high osmolarities up to 3.5 osmol/L as well as of 0.1 M oxalic acid. The production of itaconic acid was achieved on pretreated cellulose in seawater and on the hydrolysed hemicellulosic fraction of pretreated beech wood. CONCLUSION: The fungus U. maydis is a promising producer of itaconic acid, since it grows as single cells (yeast-like) in submerged cultivations and it is extremely robust in high osmotic media and real seawater. Moreover, U. maydis can grow on the hemicellulosic fraction of pretreated beech wood. Thereby, this fungus combines important advantages of yeasts and filamentous fungi. Nevertheless, the biomass pretreatment does indeed affect the subsequent itaconic acid production. Although U. maydis is insusceptible to most possible impurities from pretreatment, high amounts of salts or residues of organic acids can slow microbial growth and decrease the production. Consequently, the pretreatment step needs to fit the prerequisites defined by the actual microorganisms applied for fermentation.

Robust and Continuous Lipase-Catalyzed Reactions in Deep Eutectic Solvents: Low Viscosity and Double CLEA-LentiKats Immobilization (CLEA-LK-Lipases).

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®. The low viscosity and high stability of the CLEA-LK-lipase enable the use of DES under flow conditions.

Integrating Biocatalysis with Viscous Deep Eutectic Solvents in Lab-On-A-Chip Microreactors.

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-1 at 45 °C while keeping their non-aqueous nature. This enables the setup of efficient enzymatic esterifications, which can also be applied in different continuous systems. Following those premises, the use of microreactors in biocatalytic reactions was explored using (low-viscous) DES-buffer media, showing that reactions could be performed efficiently. Under non-optimized conditions, the microreactor devices led to specific productivities considerably higher than those observed in the batch reactor (14 vs. 0.24 mgproduct min-1 mgbiocat -1 ) at the same enzyme loadings and conversion of 6 % (to assure a fair comparison). Looking beyond, the combination of several microchannels (e. g., in scale-out fashion) with DES-water media may lead to powerful, sustainable, and efficient tools for industrial synthesis.

Enzymatic Cascade for the Synthesis of 2,5-Furandicarboxylic Acid in Biphasic and Microaqueous Conditions: 'Media-Agnostic' Biocatalysts for Biorefineries.

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.

Biocatalysis for the asymmetric synthesis of Active Pharmaceutical Ingredients (APIs): this time is for real.

INTRODUCTION: 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. AREAS COVERED: 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. EXPERT OPINION: The reported enzymatic examples are performed with high substrate loadings (>100 g L-1) and with excellent selectivity, making them inspiring strategies for present and future industrial applications. The combination of powerful molecular biology techniques with the needs of the pharmaceutical industry can be aligned, creating promising platforms for synthesis under more sustainable conditions.

Biocatalysis as Key to Sustainable Industrial Chemistry.

Invited for this month's cover is the Working Group Sustainable Chemistry of the European Society of Applied Biocatalysis (ESAB). The image shows the significant contributions of Biocatalysis to science, industry, society, and environment as a technology of first choice for Sustainable Chemistry in the 21st century. The Perspective itself is available at 10.1002/cssc.202102709.

Preface to Special Issue on Biocatalysis as Key to Sustainable Industrial Chemistry.

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.

Biocatalysis as Key to Sustainable Industrial Chemistry.

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.

Biocatalytic strategies for the asymmetric synthesis of alpha-hydroxy ketones.

The development of efficient syntheses for enantiomerically enriched alpha-hydroxy ketones is an important research focus in the pharmaceutical industry. For example, alpha-hydroxy ketones are found in antidepressants, in selective inhibitors of amyloid-beta protein production (used in the treatment of Alzheimer's), in farnesyl transferase inhibitors (Kurasoin A and B), and in antitumor antibiotics (Olivomycin A and Chromomycin A3). Moreover, alpha-hydroxy ketones are of particular value as fine chemicals because of their utility as building blocks for the production of larger molecules. They can also be used in preparing many other important structures, such as amino alcohols, diols, and so forth. Several purely chemical synthetic approaches have been proposed to afford these compounds, together with some organocatalytic strategies (thiazolium-based carboligations, proline alpha-hydroxylations, and so forth). However, many of these chemical approaches are not straightforward, lack selectivity, or are economically unattractive because of the large number of chemical steps required (usually combined with low enantioselectivities). In this Account, we describe three different biocatalytic approaches that have been developed to efficiently produce alpha-hydroxy ketones: (i) The use of thiamine diphosphate-dependent lyases (ThDP-lyases) to catalyze the umpolung carboligation of aldehydes. Enantiopure alpha-hydroxy ketones are formed from inexpensive aldehydes with this method. Some lyases with a broad substrate spectrum have been successfully characterized. Furthermore, the use of biphasic media with recombinant whole cells overexpressing lyases leads to productivities of approximately 80-100 g/L with high enantiomeric excesses (up to >99%). (ii) The use of hydrolases to produce alpha-hydroxy ketones by means of (in situ) dynamic kinetic resolutions (DKRs). Lipases are able to successfully resolve racemates, and many outstanding examples have been reported. However, this approach leads to a maximum theoretical yield of 50%. As a means of overcoming this problem, these traditional lipase-catalyzed kinetic resolutions are combined with racemization of remnant substrate, which can be done in situ or in separate compartments. Examples showing high conversions (>90%) and enantiomeric excesses (>99%) are described. (iii) Whole-cell redox processes, catalyzed by several microorganisms, either by means of free enzymes (applying a cofactor regeneration system) or by whole cells. Through the use of redox machineries, different strategies can lead to high yields and enantiomeric excesses. Some enantiopure alpha-hydroxy ketones can be formed by reductions of diketones and by selective oxidations of vicinal diols. Likewise, some redox processes involving sugar chemistry (involving alpha-hydroxy ketones) have been developed on the industrial scale. Finally, the redox whole-cell concept allows racemizations (and deracemizations) as well. These three strategies provide a useful and environmentally friendly synthetic toolbox. Likewise, the field represents an illustrative example of how biocatalysis can assist practical synthetic processes, and how problems derived from the integration of natural tools in synthetic pathways can be efficiently tackled to afford high yields and enantioselectivities.