Crosslinked Aggregates of Fusion Enzymes in Microaqueous Organic Media.

Baeyer-Villiger monooxygenases (BVMOs) are attractive for selectively oxidizing various ketones using oxygen into valuable esters and lactones. However, the application of BVMOs is restrained by cofactor dependency and enzyme instability combined with water-related downsides such as low substrate loading, low oxygen capacity, and water-induced side reactions. Herein, we described a redox-neutral linear cascade with in-situ cofactor regeneration catalyzed by fused alcohol dehydrogenase and cyclohexanone monooxygenase in aqueous and microaqueous organic media. The cascade conditions have been optimized regarding substrate concentrations as well as the amounts of enzymes and cofactors with the Design of Experiments (DoE). The carrier-free immobilization technique, crosslinked enzyme aggregates (CLEAs), was applied to fusion enzymes. The resultant fusion CLEAs were proven to function in microaqueous organic systems, in which the enzyme ratios, water contents (0.5-5 vol. %), and stability have been systematically studied. The fusion CLEAs showed promising operational (up to 5 cycles) and storage stability.

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.

Exploring the Temperature Effect on Enantioselectivity of a Baeyer-Villiger Biooxidation by the 2,5-DKCMO Module: The SLM Approach.

Temperature is a crucial parameter for biological and chemical processes. Its effect on enzymatically catalysed reactions has been known for decades, and stereo- and enantiopreference are often temperature-dependent. For the first time, we present the temperature effect on the Baeyer-Villiger oxidation of rac-bicyclo[3.2.0]hept-2-en-6-one by the type II Bayer-Villiger monooxygenase, 2,5-DKCMO. In the absence of a reductase and driven by the hydride-donation of a synthetic nicotinamide analogue, the clear trend for a decreasing enantioselectivity at higher temperatures was observed. "Traditional" approaches such as the determination of the enantiomeric ratio (E) appeared unsuitable due to the complexity of the system. To quantify the trend, we chose to use the 'Shape Language Modelling' (SLM), a tool that allows the reaction to be described at all points in a shape prescriptive manner. Thus, without knowing the equation of the reaction, the substrate ee can be estimated that at any conversion.

Immobilization and Application of Fatty Acid Photodecarboxylase in Deep Eutectic Solvents.

Since its discovery in 2017, the fatty acid decarboxylase (FAP) photoenzyme has been the focus of extensive research, given its ability to convert fatty acids into alka(e)nes using merely visible blue light. Unfortunately, there are still some drawbacks that limit the applicability of this biocatalyst, such as poor solubility of the substrates in aqueous media, poor photostability, and the impossibility of reusing the catalyst for several cycles. In this work, we demonstrate the use of FAP in non-conventional media as a free enzyme and an immobilized preparation. Namely, its applicability in deep eutectic solvents (DESs) and a proof-of-concept immobilization using a commercial His-tag selective carrier, a thorough study of reaction and immobilization conditions in each case, as well as reusability studies are shown. We observed an almost complete selectivity of the enzyme towards C18 decarboxylation over C16 when used in a DES, with a product analytical yield up to 81 % when using whole cells. Furthermore, when applying the immobilized enzyme in DES, we obtained yields >10-fold higher than the ones obtained in aqueous media.

A Deep Eutectic Solvent Thermomorphic Multiphasic System for Biocatalytic Applications.

The applicability of a thermomorphic multiphasic system (TMS) composed of a hydrophobic deep eutectic solvent (DES) and an aqueous potassium phosphate buffer with a lower critical solution temperature (LCST) phase change for homogeneous biocatalysis was investigated. A lidocaine-based DES with the fatty acid oleic acid as a hydrogen-bond donor was studied. Phase diagrams were determined and presented within this study. We tested different additional components to the solvent system and observed a decrease in the cloud point of approximately 0.026 °C per concentration unit. Distribution studies revealed a clear distribution of the protein in the aqueous buffer phase (>95 %), whereas the hydrophobic substrate and educt accumulated (>95 %) in the DES-enriched layer. Finally, a reduction catalyzed by horse liver alcohol dehydrogenase was performed in a larger-scale experiment, and the biocatalyst could be recycled by simply removing the DES phase for three recycling runs.

Transmembrane Shuttling of Photosynthetically Produced Electrons to Propel Extracellular Biocatalytic Redox Reactions in a Modular Fashion.

Many biocatalytic redox reactions depend on the cofactor NAD(P)H, which may be provided by dedicated recycling systems. Exploiting light and water for NADPH-regeneration as it is performed, e.g. by cyanobacteria, is conceptually very appealing due to its high atom economy. However, the current use of cyanobacteria is limited, e.g. by challenging and time-consuming heterologous enzyme expression in cyanobacteria as well as limitations of substrate or product transport through the cell wall. Here we establish a transmembrane electron shuttling system propelled by the cyanobacterial photosynthesis to drive extracellular NAD(P)H-dependent redox reactions. The modular photo-electron shuttling (MPS) overcomes the need for cloning and problems associated with enzyme- or substrate-toxicity and substrate uptake. The MPS was demonstrated on four classes of enzymes with 19 enzymes and various types of substrates, reaching conversions of up to 99 % and giving products with >99 % optical purity.

Divorce in the two-component BVMO family: the single oxygenase for enantioselective chemo-enzymatic Baeyer-Villiger oxidations.

Two-component flavoprotein monooxygenases consist of a reductase and an oxygenase enzyme. The proof of functionality of the latter without its counterpart as well as the mechanism of flavin transfer remains unanswered beyond doubt. To tackle this question, we utilized a reductase-free reaction system applying purified 2,5-diketocamphane-monooxygenase I (2,5-DKCMO), a FMN-dependent type II Baeyer-Villiger monooxygenase, and synthetic nicotinamide analogues (NCBs) as dihydropyridine derivatives for FMN reduction. This system demonstrated the stand-alone quality of the oxygenase, as well as the mechanism of FMNH2 transport by free diffusion. The efficiency of this reductase-free system strongly relies on the balance of FMN reduction and enzymatic (re)oxidation, since reduced FMN in solution causes undesired side reactions, such as hydrogen peroxide formation. Design of experiments allowed us to (i) investigate the effect of various reaction parameters, underlining the importance to balance the FMN/FMNH2 cycle, (ii) optimize the reaction system for the enzymatic Baeyer-Villiger oxidation of rac-bicyclo[3.2.0]hept-2-en-6-one, rac-camphor, and rac-norcamphor. Finally, this study not only demonstrates the reductase-independence of 2,5-DKCMO, but also revisits the terminology of two-component flavoprotein monooxygenases for this specific case.

Chromoselective Photocatalysis Enables Stereocomplementary Biocatalytic Pathways*.

Controlling the selectivity of a chemical reaction with external stimuli is common in thermal processes, but rare in visible-light photocatalysis. Here we show that the redox potential of a carbon nitride photocatalyst (CN-OA-m) can be tuned by changing the irradiation wavelength to generate electron holes with different oxidation potentials. This tuning was the key to realizing photo-chemo-enzymatic cascades that give either the (S)- or the (R)-enantiomer of phenylethanol. In combination with an unspecific peroxygenase from Agrocybe aegerita, green light irradiation of CN-OA-m led to the enantioselective hydroxylation of ethylbenzene to (R)-1-phenylethanol (99 % ee). In contrast, blue light irradiation triggered the photocatalytic oxidation of ethylbenzene to acetophenone, which in turn was enantioselectively reduced with an alcohol dehydrogenase from Rhodococcus ruber to form (S)-1-phenylethanol (93 % ee).

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.

Convergent Cascade Catalyzed by Monooxygenase-Alcohol Dehydrogenase Fusion Applied in Organic Media.

With the aim of applying redox-neutral cascade reactions in organic media, fusions of a type II flavin-containing monooxygenase (FMO-E) and horse liver alcohol dehydrogenase (HLADH) were designed. The enzyme orientation and expression vector were found to influence the overall fusion enzyme activity. The resulting bifunctional enzyme retained the catalytic properties of both individual enzymes. The lyophilized cell-free extract containing the bifunctional enzyme was applied for the convergent cascade reaction consisting of cyclobutanone and butane-1,4-diol in different microaqueous media with only 5 % (v/v) aqueous buffer without any addition of external cofactor. Methyl tert-butyl ether and cyclopentyl methyl ether were found to be the best organic media for the synthesis of γ-butyrolactone, resulting in about 27 % analytical yield.

Kinetic insights into ϵ-caprolactone synthesis: Improvement of an enzymatic cascade reaction.

A computational approach for the simulation and prediction of a linear three-step enzymatic cascade for the synthesis of ϵ-caprolactone (ECL) coupling an alcohol dehydrogenase (ADH), a cyclohexanone monooxygenase (CHMO), and a lipase for the subsequent hydrolysis of ECL to 6-hydroxyhexanoic acid (6-HHA). A kinetic model was developed with an accuracy of prediction for a fed-batch mode of 37% for substrate cyclohexanol (CHL), 90% for ECL, and >99% for the final product 6-HHA. Due to a severe inhibition of the CHMO by CHL, a batch synthesis was shown to be less efficient than a fed-batch approach. In the fed-batch synthesis, full conversion of 100 mM CHL was 28% faster with an analytical yield of 98% compared to 49% in case of the batch synthesis. The lipase-catalyzed hydrolysis of ECL to 6-HHA circumvents the inhibition of the CHMO by ECL enabling a 24% higher product concentration of 6-HHA compared to ECL in case of the fed-batch synthesis without lipase. Biotechnol. Bioeng. 2017;114: 1215-1221. © 2017 Wiley Periodicals, Inc.

Evaluation of the Substrate Scope of Benzoic Acid (De)carboxylases According to Chemical and Biochemical Parameters.

The enzymatic carboxylation of phenolic compounds has been attracting increasing interest in recent years, owing to its regioselectivity and technical potential as a biocatalytic equivalent for the Kolbe-Schmitt reaction. Mechanistically the reaction was demonstrated to occur through electrophilic aromatic substitution/water elimination with bicarbonate as a cosubstrate. The effects of the substituents on the phenolic ring have not yet been elucidated in detail, but this would give detailed insight into the substrate-activity relationship and would provide predictability for the acceptance of future substrates. In this report we show how the kinetic and (apparent) thermodynamic behavior can be explained through the evaluation of linear free energy relationships based on electronic, steric, and geometric parameters and through the consideration of enzyme-ligand interactions. Moreover, the similarity between the benzoic acid decarboxylases and the amidohydrolases superfamily is investigated, and promiscuous hydrolytic activity of the decarboxylase in the context of the hydrolysis of an activated ester bond has been established.

Recent trends and novel concepts in cofactor-dependent biotransformations.

Cofactor-dependent enzymes catalyze a broad range of synthetically useful transformations. However, the cofactor requirement also poses economic and practical challenges for the application of these biocatalysts. For three decades, considerable research effort has been devoted to the development of reliable in situ regeneration methods for the most commonly employed cofactors, particularly NADH and NADPH. Today, researchers can choose from a plethora of options, and oxidoreductases are routinely employed even on industrial scale. Nevertheless, more efficient cofactor regeneration methods are still being developed, with the aim of achieving better atom economy, simpler reaction setups, and higher productivities. Besides, cofactor dependence has been recognized as an opportunity to confer novel reactivity upon enzymes by engineering their cofactors, and to couple (redox) biotransformations in multi-enzyme cascade systems. These novel concepts will help to further establish cofactor-dependent biotransformations as an attractive option for the synthesis of biologically active compounds, chiral building blocks, and bio-based platform molecules.

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.

Antimicrobial properties and biocompatibility of semi-synthetic carbohydrate-based ionic hydrogels.

Hydrogels have gained significant interest in the last decades, especially in the medical sector, due to their versatile properties. While hydrogels from naturally occurring polysaccharides (e.g. cellulose) are well-known, those produced from polymerizable carbohydrate-based monomers remain underexplored. However, these semi-synthetic hydrogels offer the great advantage of having adjustable properties for customization depending on their application. The objective of this study was to characterize semi-synthetic carbohydrate-based ionic hydrogels produced from GVIM-I (glucosyl vinyl imidazolium iodide). The antimicrobial activity was evaluated using the disk diffusion method, which demonstrated that all samples exhibit inhibitory effects on the growth of Candida auris. In vitro biocompatibility was determined by cell viability studies with L929 mouse fibroblasts, and a correlation was observed between eluate concentration and cell viability. In particular, the type of initiator system employed for polymerization was found to affect cell viability. The direct contact assessments showed that specific pre-treatments of the hydrogels resulted in higher cell viability than non-treated hydrogels. The results also revealed the impact of crosslinker concentration and type and identified poly(ethylene glycol)diacrylate (PEGDA) 575 as a promising crosslinker for future medical applications. LC-MS analysis of the wash medium identified unreacted GVIM-I as the leached material, which is presumed to be the cause of the observed cytotoxicity. Overall, the study provides valuable insights into the characteristics of GVIM-I based hydrogels and sheds light on the factors that influence their cytotoxicity and potential for medical application.

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.

Development of a Human Recombinant Collagen for Vat Polymerization-Based Bioprinting.

In light-based 3D-bioprinting, gelatin methacrylate (GelMA) is one of the most widely used materials, as it supports cell attachment, and shows good biocompatibility and degradability in vivo. However, as an animal-derived material, it also causes safety concerns when used in medical applications. Gelatin is a partial hydrolysate of collagen, containing high amounts of hydroxyproline. This causes the material to form a thermally induced gel at ambient temperatures, a behavior also observed in GelMA. This temperature-dependent gelation requires precise temperature control during the bioprinting process to prevent the gelation of the material. To avoid safety concerns associated with animal-derived materials and reduce potential issues caused by thermal gelation, a recombinant human alpha-1 collagen I fragment was expressed in Komagataella phaffii without hydroxylation. The resulting protein was successfully modified with methacryloyl groups and underwent rapid photopolymerization upon ultraviolet light exposure. The developed material exhibited slightly slower polymerization and lower storage modulus compared to GelMA, while it showed higher stretchability. However, unlike the latter, the material did not undergo physical gelation at ambient temperatures, but only when cooled down to below 10°C, a characteristic that has not been described for comparable materials so far. This gelation was not caused by the formation of triple-helical structures, as shown by the absence of the characteristic peak at 220 nm in CD spectra. Moreover, the developed recombinant material facilitated cell adherence with high cell viability after crosslinking via light to a 3D structure. Furthermore, desired geometries could be easily printed on a stereolithographic bioprinter.

3D printed and stimulus responsive drug delivery systems based on synthetic polyelectrolyte hydrogels manufactured via digital light processing.

Hydrogels are three-dimensional hydrophilic polymeric networks absorbing up to and even more than 90 wt% of water. These superabsorbent polymers retain their shape during the swelling process while enlarging their volume and mass. In addition to their swelling behavior, hydrogels can possess other interesting properties, such as biocompatibility, good rheological behavior, or even antimicrobial activity. This versatility qualifies hydrogels for many medical applications, especially drug delivery systems. As recently shown, polyelectrolyte-based hydrogels offer beneficial properties for long-term and stimulus-responsive applications. However, the fabrication of complex structures and shapes can be difficult to achieve with common polymerization methods. This obstacle can be overcome by the use of additive manufacturing. 3D printing technology is gaining more and more attention as a method of producing materials for biomedical applications and medical devices. Photopolymerizing 3D printing methods offer superior resolution and high control of the photopolymerization process, allowing the fabrication of complex and customizable designs while being less wasteful. In this work, novel synthetic hydrogels, consisting of [2-(acryloyloxy) ethyl]trimethylammonium chloride (AETMA) as an electrolyte monomer and poly(ethylene glycol)-diacrylate (PEGDA) as a crosslinker, 3D printed via Digital Light Processing (DLP) using a layer height of 100 µm, are reported. The hydrogels obtained showed a high swelling degree q∞m,t ∼ 12 (24 h in PBS; pH 7; 37 °C) and adjustable mechanical properties with high stretchability (εmax ∼ 300%). Additionally, we embedded the model drug acetylsalicylic acid (ASA) and investigated its stimulus-responsive drug release behaviour in different release media. The stimulus responsiveness of the hydrogels is mirrored in their release behavior and could be exploited in triggered as well as sequential release studies, demonstrating a clear ion exchange behavior. The received 3D-printed drug depots could also be printed in complex hollow geometry, exemplarily demonstrated via an individualized frontal neo-ostium implant prototype. Consequently, a drug-releasing, flexible, and swellable material was obtained, combining the best of both worlds: the properties of hydrogels and the ability to print complex shapes.

Correction: 3D printed and stimulus responsive drug delivery systems based on synthetic polyelectrolyte hydrogels manufactured via digital light processing.

Correction for '3D printed and stimulus responsive drug delivery systems based on synthetic polyelectrolyte hydrogels manufactured via digital light processing' by Sonja Vaupel et al., J. Mater. Chem. B, 2023, DOI: https://doi.org/10.1039/d3tb00285c.

Robust Light Driven Enzymatic Oxyfunctionalization via Immobilization of Unspecific Peroxygenase.

Unspecific peroxygenases have attracted interest in synthetic chemistry, especially for the oxidative activation of C-H bonds, as they only require hydrogen peroxide (H2 O2 ) instead of a cofactor. Due to their instability in even small amounts of H2 O2 , different strategies like enzyme immobilization or in situ H2 O2 production have been developed to improve the stability of these enzymes. While most strategies have been studied separately, a combination of photocatalysis with immobilized enzymes was only recently reported. To show the advantages and limiting factors of immobilized enzyme in a photobiocatalytic reaction, a comparison is made between free and immobilized enzymes. Adjustment of critical parameters such as (i) enzyme and substrate concentration, (ii) illumination wavelength and (iii) light intensity results in significantly increased enzyme stabilities of the immobilized variant. Moreover, under optimized conditions a turnover number of 334,500 was reached.