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.

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.

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.

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.

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.

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.

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 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.

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.

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.

Immobilization of Pseudomonas stutzeri lipase through Cross-linking Aggregates (CLEA) for reactions in Deep Eutectic Solvents.

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.

Applied biocatalysis beyond just buffers - from aqueous to unconventional media. Options and guidelines.

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.

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.

Lignocellulose Fractionation Using Recyclable Phosphoric Acid: Lignin, Cellulose, and Furfural Production.

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.

Across the Board: Pablo Domínguez de María on the Biocatalytic Synthesis of Esters and Amides in Aqueous Media.

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.

Multiscale analysis of lignocellulose recalcitrance towards OrganoCat pretreatment and fractionation.

BACKGROUND: Biomass recalcitrance towards pretreatment and further processing can be related to the compositional and structural features of the biomass. However, the exact role and relative importance to those structural attributes has still to be further evaluated. Herein, ten different types of biomass currently considered to be important raw materials for biorefineries were chosen to be processed by the recently developed, acid-catalyzed OrganoCat pretreatment to produce cellulose-enriched pulp, sugars, and lignin with different amounts and qualities. Using wet chemistry analysis and NMR spectroscopy, the generic factors of lignocellulose recalcitrance towards OrganoCat were determined. RESULTS: The different materials were processed applying different conditions (e.g., type of acid catalyst and temperature), and fractions with different qualities were obtained. Raw materials and products were characterized in terms of their compositional and structural features. For the first time, generic correlation coefficients were calculated between the measured chemical and structural features and the different OrganoCat product yields and qualities. Especially lignin-related factors displayed a detrimental role for enzymatic pulp hydrolysis, as well as sugar and lignin yield exhibiting inverse correlation coefficients. Hemicellulose appeared to have less impact, not being as detrimental as lignin factors, but xylan-O-acetylation was inversely correlated with product yield and qualities. CONCLUSION: These results illustrate the role of generic features of lignocellulosic recalcitrance towards acidic pretreatments and fractionation, exemplified in the OrganoCat strategy. Discriminating between types of lignocellulosic biomass and highlighting important compositional variables, the improved understanding of how these parameters affect OrganoCat products will ameliorate bioeconomic concepts from agricultural production to chemical products. Herein, a methodological approach is proposed.

Immobilized lipase-CLEA aggregates encapsulated in lentikats® as robust biocatalysts for continuous processes in deep eutectic solvents.

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.

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.

OrganoCat Fractionation of Empty Fruit Bunches from Palm Trees into Lignin, Sugars, and Cellulose-Enriched Pulp.

The palm oil industry produces large amounts of empty fruit bunches (EFB) as waste. EFB are very recalcitrant toward further processing, although their valorization could create novel incentives and bio-economic opportunities for the industries involved. Herein, EFB have been successfully subjected to the OrganoCat pretreatment-using 2,5-furandicarboxylic acid as the biogenic catalyst-to fractionate and separate this lignocellulosic material into its main components in a single step. The pretreatment of EFB leads to the deacetylation and depolymerization of noncellulosic polysaccharides and to the partial delignification of the cellulosic fiber. The OrganoCat processing of EFB yielded 45 ± 0.5 wt % cellulose-enriched pulp, 20 ± 0.7 wt % extracted lignin, 3.8 ± 0.2 wt % furfural, and 11 ± 0.6 wt % hydrolyzed sugars. The obtained EFB-pulp showed high accessibility to cellulases, resulting in a glucan conversion of 73 ± 2% after 72 h (15 ± 2% after 1 h) with commercial cellulase cocktail (Accellerase 1500). Overall, the results suggest that the treatment of the EFB material using OrganoCat may create promising paths for the full valorization of EFBs.

Cyclopentyl Methyl Ether (CPME): A Versatile Eco-Friendly Solvent for Applications in Biotechnology and Biorefineries.

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.