Redox Biocatalysis Research Articles

Redox Biocatalysis in Lidocaine-based Hydrophobic Deep Eutectic Solvents: Non-conventional Media Outperform Aqueous Conditions.

Redox biocatalysis is an important pillar of the chemical industry. Yet, the enzymes' nature restricts most reactions to aqueous conditions, where the limited substrate solubility leads to unsustainable diluted biotranformations. Non-aqueous media represent a strategic solution to conduct intensified biocatalytic routes. Deep eutectic solvents (DESs) are designable solvents that can be customized to meet specific application needs. Within the large design space of combining DES components (and ratios), hydrophobic DESs hold the potential to be both enzyme-compatible - keeping the enzymes' hydration -, and solubilizers for hydrophobic reactants. We explored two hydrophobic DESs, lidocaine/oleic acid, and lidocaine/decanoic acid, as reaction media for carbonyl reduction catalyzed by horse liver alcohol dehydrogenase, focusing on the effect of water contents and on maximizing substrate loadings. Enzymes remained highly active and stable in the DESs with 20 wt.% buffer, whereas the reaction performance in DESs outperformed the pure buffer system with hydrophobic substrates (e.g., cinnamaldehyde to form the industrially relevant cinnamyl alcohol), with a 2-fold higher specific activity. Notably, the cinnamaldehyde reduction was for the first time performed at 800 mM (~100 g[[EQUATION]]L-1) with full conversion, which opens up new avenues to industrial applications of hydrophobic DESs for enzyme catalysis.

Immobilised Alcohol Dehydrogenase and Ene‐Reductase Work in Concert to Reduce Cyclohex‐2‐enone in Bulk Organic Solvent

AbstractEnone reduction by ene‐reductase (ERED) has not been reported yet in bulk organic media probably due to expected challenges with the cofactor recycling using a second enzyme and the exchange of the NAD(P)‐cofactor between the two enzymes. Herein, the combination of an ERED from Zymomonas mobilis (NCR‐ERED) and Thermoanaerobacter ethanolicus alcohol dehydrogenase (TeSADH) has been found as an efficient biocatalytic redox system for the reduction of cyclohex‐2‐enone into cyclohexanol in bulk organic solvents. Both enzymes were successfully immobilised on three EziG supports, finding Coral as the most promising carrier for both of them. The highest enzymatic activities were found in hydrophobic solvents such as toluene and isooctane. It was observed that the control of water content and post‐immobilisation treatments enabled retention of enzyme activity. Both enzymes were co‐immobilised on the same bead together with NADP+, leading to the production of a self‐sufficient catalytic system, although the external supplementation of the nicotinamide cofactor was necessary for an efficient enzyme reuse. In this case, no ERED activity loss was detected after two additional cycles, while the ADH experienced less than 20 % activity loss upon reuse.

Enantiodivergent kinetic resolution of 4-substituted 1,2,3,4-tetrahydroquinolines employing amine oxidase

Optically pure 1,2,3,4-tetrahydroquinolines (THQs) represent a class of important motifs in many natural products and pharmaceutical agents. While recent advances on redox biocatalysis have demonstrated the great potential of amine oxidases, all the transformations focused on 2-substituted THQs. The corresponding biocatalytic method for the preparation of chiral 4-substituted THQs is still challenging due to the poor activity and stereoselectivity of the available enzyme. Herein, we developed a biocatalytic kinetic resolution approach for enantiodivergent synthesis of 4-phenyl- or alkyl-substituted THQs. Through structure-guided protein engineering of cyclohexylamine oxidase derived from Brevibacterium oxidans IH-35 A (CHAO), the variant of CHAO (Y215H/Y214S) displayed improved specific activity toward model substrate 4-phenyl substituted THQ (0.14 U/mg, 13-fold higher than wild-type CHAO) with superior (R)-stereoselectivity (E > 200). Molecular dynamics simulations show that CHAO Y215H/Y214S allows a suitable substrate positioning in the expanded binding pocket to be facilely accessed, enabling enhanced activity and stereoselectivity. Furthermore, a series of 4-alkyl-substituted THQs can be transformed by CHAO Y215H/Y214S, affording R-isomers with good yields (up to 50 %) and excellent enantioselectivity (up to ee > 99 %). Interestingly, the monoamine oxidase from Pseudomonas fluorescens Pf0–1 (PfMAO1) with opposite enantioselectivity was also mined. Together, this system enriches the kinetic resolution methods for the synthesis of chiral THQs.

Design of a cofactor self-sufficient whole-cell biocatalyst for enzymatic asymmetric reduction via engineered metabolic pathways and multi-enzyme cascade.

NAD(P)H-dependent oxidoreductases are crucial biocatalysts for synthesizing chiral compounds. Yet, the industrial implementation of enzymatic redox reactions is often hampered by an insufficient supply of expensive nicotinamide cofactors. Here, a cofactor self-sufficient whole-cell biocatalyst was developed for the enzymatic asymmetric reduction of 2-oxo-4-[(hydroxy)(-methyl)phosphinyl] butyric acid (PPO) to L-phosphinothricin (L-PPT). The endogenous NADP+ pool was significantly enhanced by regulating Preiss-Handler pathway toward NAD(H) synthesis and, in the meantime, introducing NAD kinase to phosphorylate NAD(H) toward NADP+. The intracellular NADP(H) concentration displayed a 2.97-fold increase with the strategy compared with the wild-type strain. Furthermore, a recombinant multi-enzyme cascade biocatalytic system was constructed based on the Escherichia coli chassis. In order to balance multi-enzyme co-expression levels, the strategy of modulating rate-limiting enzyme PmGluDH by RBS strengths regulation successfully increased the catalytic efficiency of PPO conversion. Finally, the cofactor self-sufficient whole-cell biocatalyst effectively converted 300mM PPO to L-PPT in 2h without the need to add exogenous cofactors, resulting in a 2.3-fold increase in PPO conversion (%) from 43% to 100%, with a high space-time yield of 706.2gL-1d-1 and 99.9% ee. Overall, this work demonstrates a technological example for constructing a cofactor self-sufficient system for NADPH-dependent redox biocatalysis.

Light-driven redox biocatalysis on gram-scale in Synechocystis sp. PCC 6803 via an in vivo cascade.

The photosynthetic light reaction in cyanobacteria constitutes a highly attractive tool for productive biocatalysis, as it can provide redox reactions with high-energy reduction equivalents using sunlight and water as sources of energy and electrons, respectively. Here, we describe the first artificial light-driven redox cascade in Synechocystis sp. PCC 6803 to convert cyclohexanone to the polymer building block 6-hydroxyhexanoic acid (6-HA). Co-expression of a Baeyer-Villiger monooxygenase (BVMO) and a lactonase, both from Acidovorax sp. CHX100, enabled this two-step conversion with an activity of up to 63.1 ± 1.0 U/gCDW without accumulating inhibitory ε-caprolactone. Thereby, one of the key limitations of biocatalytic reactions, that is, reactant inhibition or toxicity, was overcome. In 2 L stirred-tank-photobioreactors, the process could be stabilized for 48 h, forming 23.50 ± 0.84 mm (3.11 ± 0.12 g/L) 6-HA. The high specificity enabling a product yield (YP/S ) of 0.96 ± 0.01 mol/mol and the remarkable biocatalyst-related yield of 3.71 ± 0.21 g6-HA /gCDW illustrate the potential of producing this non-toxic product in a synthetic cascade. The fine-tuning of the energy burden on the catalyst was found to be crucial, which indicates a limitation by the metabolic capacity of the cells possibly being compromised by biocatalysis-related reductant withdrawal. Intriguingly, energy balancing revealed that the biotransformation could tap surplus electrons derived from the photosynthetic light reaction and thereby relieve photosynthetic sink limitation. This study shows the feasibility of light-driven biocatalytic cascade operation in cyanobacteria and highlights respective metabolic limitations and engineering targets to unleash the full potential of photosynthesis.

LUMO-Mediated Se and HOMO-Mediated Te Nanozymes for Selective Redox Biocatalysis.

Selenium (Se) and tellurium (Te) nanomaterials with novel chain-like structures have attracted widespread interest owing to their intriguing properties. Unfortunately, the still-unclear catalytic mechanisms have severely limited the development of biocatalytic performance. In this work, we developed chitosan-coated Se nanozymes with a 23-fold higher antioxidative activity than Trolox and bovine serum albumin coated Te nanozymes with stronger prooxidative biocatalytic effects. Based on density functional theory calculations, we first propose that the Se nanozyme with Se/Se2- active centers favored reactive oxygen species (ROS) clearance via a LUMO-mediated mechanism, while the Te nanozyme with Te/Te4+ active centers promoted ROS production through a HOMO-mediated mechanism. Furthermore, biological experiments confirmed that the survival rate of γ-irritated mice treated with the Se nanozyme was maintained at 100% for 30 days by inhibiting oxidation. However, the Te nanozyme had the opposite biological effect via promoting radiation oxidation. The present work provides a new strategy for improving the catalytic activities of Se and Te nanozymes.

Redox cascade reaction for kinetic resolution of racemic α-methylbenzylamine and biosynthesis of α-phenylethanol.

Chiral α-methylbenzylamine and α-phenylethanol are important building blocks for the industrial production of optically active drugs, bioactive compounds. Methods for the simultaneous synthesis of chiral α-methylbenzylamine and α-phenylethanol remain rare. Herein, a biocatalytic redox cascade reaction composed of ω-transaminase, aldo-keto reductase, and glutamate dehydrogenase for chiral α-methylbenzylamine and α-phenylethanol synthesis from racemic α-methylbenzylamine was constructed. A novel ω-transaminase and two different chiral aldo-keto reductases were demonstrated in the cascade reaction. The cosubstrate and redox equivalents were regenerated simultaneously by glutamate dehydrogenase. Using the approach, (R)-α-phenylethanol, (S)-α-phenylethanol, and (R)-α-methylbenzylamine were prepared with excellent stereoselectivity (ee > 99.7%). Furthermore, semi-preparative-scale biotransformation of racemic α-methylbenzylamine was conducted. The production of (R)-α-phenylethanol reached 26.05mM at 24h, and the production of (S)-α-phenylethanol reached 25.44mM at 32h. Taken together, a novel idea was proposed for the efficient and green synthesis of chiral α-methylbenzylamine and α-phenylethanol, which had great potential for industrial application. KEY POINTS: • Excellent stereoselectivity chiral α-methylbenzylamine and α-phenylethanol were synthesized. • A novel ω-transaminase demonstrated the catalysis toward (S)-α-methylbenzylamine. • Two novel aldo-keto reductases demonstrated the conversion toward acetophenone.

Redox Biocatalysis: Quantitative Comparisons of Nicotinamide Cofactor Regeneration Methods.

Enzymatic processes, particularly those capable of performing redox reactions, have recently been of growing research interest. Substrate specificity, optimal activity at mild temperatures, high selectivity, and yield are among the desirable characteristics of these oxidoreductase catalyzed reactions. Nicotinamide adenine dinucleotide (phosphate) or NAD(P)H-dependent oxidoreductases have been extensively studied for their potential applications like biosynthesis of chiral organic compounds, construction of biosensors, and pollutant degradation. One of the main challenges associated with making these processes commercially viable is the regeneration of the expensive cofactors required by the enzymes. Numerous efforts have pursued enzymatic regeneration of NAD(P)H by coupling a substrate reduction with a complementary enzyme catalyzed oxidation of a co-substrate. While offering excellent selectivity and high total turnover numbers, such processes involve complicated downstream product separation of a primary product from the coproducts and impurities. Alternative methods comprising chemical, electrochemical, and photochemical regeneration have been developed with the goal of enhanced efficiency and operational simplicity compared to enzymatic regeneration. Despite the goal, however, the literature rarely offers a meaningful comparison of the total turnover numbers for various regeneration methodologies. This comprehensive Review systematically discusses various methods of NAD(P)H cofactor regeneration and quantitatively compares performance across the numerous methods. Further, fundamental barriers to enhanced cofactor regeneration in the various methods are identified, and future opportunities are highlighted for improving the efficiency and sustainability of commercially viable oxidoreductase processes for practical implementation.

Photoelectrocatalytic biosynthesis fuelled by microplastics

Biocatalytic artificial photosynthesis integrates photocatalysis and redox biocatalysis to synthesize value-added chemicals using solar energy. However, this nature-inspired approach suffers from sluggish rates of reaction because of challenging water oxidation kinetics. Here we report photoelectrochemical biosynthetic reactions that use non-recyclable real-world poly(ethylene terephthalate) (PET) microplastics as an electron feedstock. A Zr-doped haematite photoanode extracts electrons from hydrolysed PET solutions obtained from post-consumer PET waste, such as drinks bottles, and transfers the electrons to the bioelectrocatalytic site. Carbon-based cathodes receive the electrons to activate redox enzymes (for example, unspecific peroxygenase, L-glutamate dehydrogenase and ene-reductase from the old yellow enzyme family) that drive various organic synthetic reactions. These reactions include oxyfunctionalization of C–H bonds, amination of C=O bonds and asymmetric hydrogenation of C=C bonds. These photoelectrocatalytic–biocatalytic hybrid reactions achieve total turnover numbers of 362,000 (unspecific peroxygenase), 144,000 (L-glutamate dehydrogenase) and 1,300 (old yellow enzyme). This work presents a photoelectrocatalytic approach for integrating environmental remediation and biocatalytic photosynthesis towards sustainable solar-to-chemical synthesis.

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.

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.

Glycolic acid as an alternative carbon and energy source for redox biocatalysis

Glycolic acid as an alternative carbon and energy source for redox biocatalysis

Glycolic acid as an alternative carbon and energy source for redox biocatalysis

Glycolic acid as an alternative carbon and energy source for redox biocatalysis

Supramolecular Enzymatic Labeling for Aptamer Switch-Based Electrochemical Biosensor.

Here we report a novel labeling strategy for electrochemical aptasensors based on enzymatic marking via supramolecular host–guest interactions. This approach relies on the use of an adamantane-modified target-responsive hairpin DNA aptamer as an affinity bioreceptor, and a neoglycoconjugate of β-cyclodextin (CD) covalently attached to a redox enzyme as a labeling element. As a proof of concept, an amperometric aptasensor for a carcinoembryonic antigen was assembled on screen-printed carbon electrodes modified with electrodeposited fern-like gold nanoparticles/graphene oxide and, by using a horseradish peroxidase-CD neoglycoenzyme as a biocatalytic redox label. This aptasensor was able to detect the biomarker in the concentration range from 10 pg/mL to 1 ng/mL with a high selectivity and a low detection limit of 3.1 pg/mL in human serum samples.

Maximizing Photosynthesis-Driven Baeyer–Villiger Oxidation Efficiency in Recombinant Synechocystis sp. PCC6803

Photosynthesis-driven whole-cell biocatalysis has great potential to contribute to a sustainable bio-economy since phototrophic cells use light as the only energy source. It has yet to be shown that phototrophic microorganisms, such as cyanobacteria, can combine the supply of high heterologous enzyme levels with allocation of sufficient reduction equivalents to enable efficient light-driven redox biocatalysis. Here, we demonstrated that the heterologous expression of an NADPH-dependent Baeyer–Villiger monooxygenase (BVMO) gene from Acidovorax sp. CHX100 turns Synechocystis sp. PCC6803 into an efficient oxyfunctionalization biocatalyst, deriving electrons and O2 from photosynthetic water oxidation. Several expression systems were systematically tested, and a PnrsB-(Ni2+)–controlled expression based on a replicative plasmid yielded the highest intracellular enzyme concentration and activities of up to 60.9 ± 1.0 U gCDW−1. Detailed analysis of reaction parameters, side reactions, and biocatalyst durability revealed—on the one hand—a high in vivo BVMO activity in the range of 6 ± 2 U mgBVMO−1 and—on the other hand—an impairment of biocatalyst performance by product toxicity and by-product inhibition. Scale-up of the reaction to 2-L fed-batch photo-bioreactors resulted in the stabilization of the bioconversion over several hours with a maximal specific activity of 30.0 ± 0.3 U g CDW−1, a maximal volumetric productivity of 0.21 ± 0.1 gL−1 h−1, and the formation of 1.3 ± 0.1 gL−1 of ε-caprolactone. Process simulations based on determined kinetic data revealed that photosynthesis-driven cyclohexanone oxidation on a 2-L scale under high-light conditions was kinetically controlled and not subject to a limitation by photosynthesis.

Boosting the Productivity of H2-Driven Biocatalysis in a Commercial Hydrogenation Flow Reactor Using H2 From Water Electrolysis

Translation of redox biocatalysis into a commercial hydrogenation flow reactor, with in-built electrolytic H2 generation, was achieved using immobilized enzyme systems. Carbon-supported biocatalysts were first tested in batch mode, and were then transferred into continuous flow columns for H2-driven, NADH-dependent asymmetric ketone reductions. The biocatalysts were thus handled comparably to heterogeneous metal catalysts, but operated at room temperature and 1–50 bar H2, highlighting that biocatalytic strategies enable implementation of hydrogenation reactions under mild–moderate conditions. Continuous flow reactions were demonstrated as a strategy for process intensification; high conversions were achieved in short residence times, with a high biocatalyst turnover frequency and productivity. These results show the prospect of using enzymes in reactor infrastructure designed for conventional heterogeneous hydrogenations.

Solar-Assisted eBiorefinery: Photoelectrochemical Pairing of Oxyfunctionalization and Hydrogenation Reactions.

Inspired by natural photosynthesis, biocatalytic photoelectrochemical (PEC) platforms are gaining prominence for the conversion of solar energy into useful chemicals by combining redox biocatalysis and photoelectrocatalysis. Herein, we report a dual biocatalytic PEC platform consisting of a molybdenum (Mo)-doped BiVO4 (Mo:BiVO4 ) photoanode and an inverse opal ITO (IO-ITO) cathode that gives rise to the coupling of peroxygenase and ene-reductase-mediated catalysis, respectively. In the PEC cell, the photoexcited electrons generated from the Mo:BiVO4 are transferred to the IO-ITO and regenerate reduced flavin mononucleotides to drive ene-reductase-catalyzed trans-hydrogenation of ketoisophrone to (R)-levodione. Meanwhile, the photoactivated Mo:BiVO4 evolves H2 O2 in situ via a two-electron water-oxidation process with the aid of an applied bias, which simultaneously supplies peroxygenases to drive selective hydroxylation of ethylbenzene into enantiopure (R)-1-phenyl-1-hydroxyethane. Thus, the deliberate integration of PEC systems with redox biocatalytic reactions can simultaneously produce valuable chemicals on both electrodes using solar-powered electrons and water.

Solar‐Assisted eBiorefinery: Photoelectrochemical Pairing of Oxyfunctionalization and Hydrogenation Reactions

AbstractInspired by natural photosynthesis, biocatalytic photoelectrochemical (PEC) platforms are gaining prominence for the conversion of solar energy into useful chemicals by combining redox biocatalysis and photoelectrocatalysis. Herein, we report a dual biocatalytic PEC platform consisting of a molybdenum (Mo)‐doped BiVO4 (Mo:BiVO4) photoanode and an inverse opal ITO (IO‐ITO) cathode that gives rise to the coupling of peroxygenase and ene‐reductase‐mediated catalysis, respectively. In the PEC cell, the photoexcited electrons generated from the Mo:BiVO4 are transferred to the IO‐ITO and regenerate reduced flavin mononucleotides to drive ene‐reductase‐catalyzed trans‐hydrogenation of ketoisophrone to (R)‐levodione. Meanwhile, the photoactivated Mo:BiVO4 evolves H2O2 in situ via a two‐electron water‐oxidation process with the aid of an applied bias, which simultaneously supplies peroxygenases to drive selective hydroxylation of ethylbenzene into enantiopure (R)‐1‐phenyl‐1‐hydroxyethane. Thus, the deliberate integration of PEC systems with redox biocatalytic reactions can simultaneously produce valuable chemicals on both electrodes using solar‐powered electrons and water.

Carbon as a Simple Support for Redox Biocatalysis in Continuous Flow.

A continuous packed bed reactor for NADH-dependent biocatalysis using enzymes co-immobilized on a simple carbon support was optimized to 100% conversion in a residence time of 30 min. Conversion of pyruvate to lactate was achieved by co-immobilized lactate dehydrogenase and formate dehydrogenase, providing in situ cofactor recycling. Other metrics were also considered as optimization targets, such as low E factors between 2.5–11 and space-time yields of up to 22.9 g L–1 h–1. The long-term stability of the biocatalytic reactor was also demonstrated, with full conversion maintained over more than 30 h of continuous operation.

Biocatalytic hydrogenations on carbon supports.

We describe the use of carbon as a versatile support for H2-driven redox biocatalysis for NADH-dependent CX bond reductions in batch and flow reactions. In each case, carbon is providing an electronic link between enzymes for H2 oxidation and reduction of the biological cofactor NAD+, as well as a support for a multi-enzyme biocatalysis system. Carbon nanopowders offer high surface areas for enzyme immobilization and good dispersion in aqueous solution for heterogeneous batch reactions. Difficulties in handling multi-wall carbon nanotubes in aqueous solution are overcome by growing them on quartz tubes to form carbon nanotube column reactors, and we show that these facilitate simple translation of H2-driven biocatalysis into flow processes. Using this flow reactor design, high conversions (90%) and total enzyme turnover numbers up to 54,000 could be achieved. Use of an entirely heterogeneous biocatalysis system simplifies recovery and re-use of the enzymes; combined with highly atom-efficient cofactor recycling, this means that high product purity can be achieved. We demonstrate these methods as platform approaches for overcoming challenges with NADH-dependent biocatalysis.