Cofactor Regeneration Research Articles

Cover Picture: Bioelectrocatalytic Cofactor Regeneration Coupled to CO2 Fixation in a Redox‐Active Hydrogel for Stereoselective C−C Bond Formation (Angew. Chem. Int. Ed. 38/2021)

Redox-active polymers for wiring NADPH recycling and CO2-fixating enzymes create a bioelectrocatalytic volume on electrodes for the regio- and stereoselective incorporation of CO2 into complex molecules via C−C bond formation at high current densities. Details of the study are presented in the Research Article by Michael Richter, Nicolas Plumeré et al. on page 21056.

Cell-Free Total Biosynthesis of Plant Terpene Natural Products using an Orthogonal Cofactor Regeneration System.

Here we report the one-pot, cell-free enzymatic synthesis of the plant monoterpene nepetalactol starting from the readily available geraniol. A pair of orthogonal cofactor regeneration systems permitted NAD+-dependent geraniol oxidation followed by NADPH-dependent reductive cyclization without isolation of intermediates. The orthogonal cofactor regeneration system maintained a high ratio of NAD+ to NADH and a low ratio of NADP+ to NADPH. The overall reaction contains four biosynthetic enzymes, including a soluble P450; and five accessory and cofactor regeneration enzymes. Furthermore, addition of a NAD+-dependent dehydrogenase to the one-pot mixture led to ~1 g/L of nepetalactone, the active cat- attractant in catnip.

Bioelektrokatalytische Cofaktor‐Regeneration und CO2‐Fixierung in einem redoxaktiven Hydrogel durch stereoselektive C‐C‐Bindungsknüpfung

AbstractDie nachhaltige Abscheidung und Umwandlung von Kohlendioxid (CO2) ist ein Schlüssel für das Erreichen einer kreislauforientierten Kohlenstoffwirtschaft. Die Bioelektrokatalyse verspricht, dieses Ziel zu erreichen. Die funktionale Integration von CO2‐fixierenden Enzymen in Materialien zur Elektrodenfunktionalisierung für die Elektrosynthese stereochemisch komplexer Moleküle muss jedoch erst noch demonstriert werden. Hier wird die stromgetriebene regio‐ und stereoselektive Inkorporation von CO2 in Crotonyl‐CoA durch eine NADPH‐abhängige enzymatische reduktive Carboxylierung gezeigt. Die Coimmobilisierung einer Ferredoxin‐NADP+‐Reduktase und einer Crotonyl‐CoA‐Carboxylase/‐Reduktase in einem mit 2,2′‐Viologen modifizierten Hydrogel ermöglichte ein gekoppeltes NADPH‐Recycling samt stereoselektiver Bildung von (2S)‐Ethylmalonyl‐CoA, einem Zwischenprodukt zu Multi‐Kohlenstoff‐Produkten aus CO2, mit 92±6 % faradayscher Effizienz und mit einer Rate von 1,6±0,4 μmol cm−2 h−1.

Bioelectrocatalytic Cofactor Regeneration Coupled to CO2 Fixation in a Redox-Active Hydrogel for Stereoselective C-C Bond Formation.

The sustainable capture and conversion of carbon dioxide (CO2) is key to achieving a circular carbon economy. Bioelectrocatalysis, which aims at using renewable energies to power the highly specific, direct transformation of CO2 into value added products, holds promise to achieve this goal. However, the functional integration of CO2‐fixing enzymes onto electrode materials for the electrosynthesis of stereochemically complex molecules remains to be demonstrated. Here, we show the electricity‐driven regio‐ and stereoselective incorporation of CO2 into crotonyl‐CoA by an NADPH‐dependent enzymatic reductive carboxylation. Co‐immobilization of a ferredoxin NADP+ reductase and crotonyl‐CoA carboxylase/reductase within a 2,2′‐viologen‐modified hydrogel enabled iterative NADPH recycling and stereoselective formation of (2S)‐ethylmalonyl‐CoA, a prospective intermediate towards multi‐carbon products from CO2, with 92±6 % faradaic efficiency and at a rate of 1.6±0.4 μmol cm−2 h−1. This approach paves the way for realizing even more complex bioelectrocatalyic cascades in the future.

One-Pot Highly Efficient Synthesis of N-Enrich Graphene Quantum Dots as a Photocatalytic Platform for NAD+/NADP+ Reduction.

An efficient photocatalytic regeneration of nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) has been carried out by two-electron reduction and protonation of NAD+ /NADP+ , induced by photons in the visible light region. This functional artificial photosynthetic counterpart of the complete energy-trapping occurring in natural photosystem I (PS I) is achieved with nitrogen-enrich graphene quantum dot (N-EGQD) as the light-harvesting photocatalyst. In buffer aqueous solution, this compound photo catalytically recycles a rhodium hydride complex of the type [Cp*Rh(bpy)H]+ (Cp* = pentamethylcyclopentadienyl, bpy = 2,2'-bipyridine) which can mediate hydride transfer processes leading to nucleotide co-factor reduction. Very promising yields of 73.31%/78.45% of NADH/NADPH with the excellent thermal stability of N-EGQD photocatalyst is observed. Thus, in this work, an efficient light-harvesting photocatalyst is synthesized for the regeneration of nicotinamide cofactor that has pharmaceuticals application.

Modular Assembly of Phosphite Dehydrogenase and Phenylacetone Monooxygenase for Tuning Cofactor Regeneration.

The use of multienzyme complexes can facilitate biocatalytic cascade reactions by employing fusion enzymes or protein tags. In this study, we explored the use of recently developed peptide tags that promote complex formation of the targeted proteins: the dimerization-docking and anchoring domain (RIDD–RIAD) system. These peptides allow self-assembly based on specific protein–protein interactions between both peptides and allow tuning of the ratio of the targeted enzymes as the RIAD peptide binds to two RIDD peptides. Each of these tags were added to the C-terminus of a NADPH-dependent Baeyer–Villiger monooxygenase (phenylacetone monooxygenase, PAMO) and a NADPH-regenerating enzyme (phosphite dehydrogenase, PTDH). Several RIDD/RIAD-tagged PAMO and PTDH variants were successfully overproduced in E. coli and subsequently purified. Complementary tagged enzymes were mixed and analyzed for their oligomeric state, stability, and activity. Complexes were formed in the case of some specific combinations (PAMORIAD–PTDHRIDD and PAMORIAD/RIAD–PTDHRIDD). These enzyme complexes displayed similar catalytic activity when compared with the PTDH–PAMO fusion enzyme. The thermostability of PAMO in these complexes was retained while PTDH displayed somewhat lower thermostability. Evaluation of the biocatalytic performance by conducting conversions revealed that with a self-assembled PAMO–PTDH complex less PTDH was required for the same performance when compared with the PTDH–PAMO fusion enzyme.

Engineering cellular metabolite transport for biosynthesis of computationally predicted tropane alkaloid derivatives in yeast

Microbial biosynthesis of plant natural products (PNPs) can facilitate access to valuable medicinal compounds and derivatives. Such efforts are challenged by metabolite transport limitations, which arise when complex plant pathways distributed across organelles and tissues are reconstructed in unicellular hosts without concomitant transport machinery. We recently reported an engineered yeast platform for production of the tropane alkaloid (TA) drugs hyoscyamine and scopolamine, in which product accumulation is limited by vacuolar transport. Here, we demonstrate that alleviation of transport limitations at multiple steps in an engineered pathway enables increased production of TAs and screening of useful derivatives. We first show that supervised classifier models trained on a tissue-delineated transcriptome from the TA-producing plant Atropa belladonna can predict TA transporters with greater efficacy than conventional regression- and clustering-based approaches. We demonstrate that two of the identified transporters, AbPUP1 and AbLP1, increase TA production in engineered yeast by facilitating vacuolar export and cellular reuptake of littorine and hyoscyamine. We incorporate four different plant transporters, cofactor regeneration mechanisms, and optimized growth conditions into our yeast platform to achieve improvements in de novo hyoscyamine and scopolamine production of over 100-fold (480 μg/L) and 7-fold (172 μg/L). Finally, we leverage computational tools for biosynthetic pathway prediction to produce two different classes of TA derivatives, nortropane alkaloids and tropane N-oxides, from simple precursors. Our work highlights the importance of cellular transport optimization in recapitulating complex PNP biosyntheses in microbial hosts and illustrates the utility of computational methods for gene discovery and expansion of heterologous biosynthetic diversity.

Production of Glucose 6-Phosphate From a Cellulosic Feedstock in a One Pot Multi-Enzyme Synthesis.

Glucose 6-phosphate is the phosphorylated form of glucose and is used as a reagent in enzymatic assays. Current production occurs via a multi-step chemical synthesis. In this study we established a fully enzymatic route for the synthesis of glucose 6-phosphate from cellulose. As the enzymatic phosphorylation requires ATP as phosphoryl donor, the use of a cofactor regeneration system is required. We evaluated Escherichia coli glucokinase and Saccharomyces cerevisiae hexokinase (HK) for the phosphorylation reaction and Pseudomonas aeruginosa polyphosphate kinase 2 (PPK2) for ATP regeneration. All three enzymes were characterized in terms of temperature and pH optimum and the effects of substrates and products concentrations on enzymatic activities. After optimization of the conditions, we achieved a 85% conversion of glucose into glucose 6-phosphate using the HK/PPK2 activities within a 24 h reaction resulting in 12.56 g/l of glucose 6-phosphate. Finally, we demonstrated the glucose 6-phosphate formation from microcrystalline cellulose in a one-pot reaction comprising Aspergillus niger cellulase for glucose release and HK/PPK2 activities. We achieved a 77% conversion of released glucose into glucose 6-phosphate, however at the expense of a lower glucose 6-phosphate yield of 1.17 g/l. Overall, our study shows an alternative approach for synthesis of glucose 6-phosphate that can be used to valorize biomass derived cellulose.

Sequential co-immobilization of multienzyme nanodevices based on SpyCatcher and SpyTag for robust biocatalysis

Sequential immobilization of multiple enzymes can not merely improve the enzymatic activity and enzyme stability but also facilitate the recovery for reuse, well imitating the natural enzyme complexes. Here, we developed a novel approach of preparing sequential multienzyme nanomachineries based on chemical crosslinking and protein assembly, and then characterized the nanodevice. In the preparation process, KgBDH-SC (SpyCatcher-fused (2R,3R)-2,3-butanediol hydrogenase) was firstly immobilized onto the functionalized silicon dioxide nanoparticles with glutaraldehyde, and then FDH-ST (SpyTag-fused formate hydrogenase) was immobilized by the spontaneous protein reaction between SpyTag and SpyCatcher to achieve the sequential co-immobilized enzymes FDH-ST-SC-Kg[email protected]2, which can asymmetrically reduce 2-hydroxyacetophenone to (R)-1-phenyl-1,2-ethanediol by cofactor regeneration. In comparison to the mixture of free enzymes, the sequential co-immobilized enzymes created under the design conditions performed around 50% activity recovery. Furthermore, FDH-ST-SC-Kg[email protected]2 presented wider pH tolerance and greater organic solvents tolerance than the mixture of free enzymes as well as retained 51.7% of overall activity after being recycled eight times. Finally, the sequential co-immobilized enzyme enabled the biosynthesis of (R)-1-phenyl-1,2-ethanediol from 80 mM 2-hydroxyacetophenone in the phosphate buffer reaction system, with 87.4% yield and over 99% optical purity.

Efficient Production of Phenyllactic Acid by Whole-cell Biocatalysis with Cofactor Regeneration System

Efficient Production of Phenyllactic Acid by Whole-cell Biocatalysis with Cofactor Regeneration System

A Redox-Neutral, Two-Enzyme Cascade for the Production of Malate and Gluconate from Pyruvate and Glucose

A triple mutant of NADP(H)-dependent malate dehydrogenase from thermotolerant Thermococcus kodakarensis has an altered cofactor preference for NAD+, as well as improved malate production compared to wildtype malate dehydrogenase. By combining mutant malate dehydrogenase with glucose dehydrogenase from Sulfolobus solfataricus and NAD+/NADH in a closed reaction environment, gluconate and malate could be produced from pyruvate and glucose. After 3 h, the yield of malate was 15.96 mM. These data demonstrate the feasibility of a closed system capable of cofactor regeneration in the production of platform chemicals.

Enhancing recombinant Chaetomium thermophilium Formate Dehydrogenase Expression with CRISPR Technology.

Genetic manipulation of Escherichia coli influences the regulation of bacterial metabolism, which could be useful for the production of different targeted products. The RpoZ gene encodes for the ω subunit of the RNA polymerase (RNAP) and is involved in the regulation of the relA gene pathway. RelA is responsible for the production of guanosine pentaphosphate (ppGpp), which is a major alarmone in the stringent response. Expression of relA is reduced in the early hours of growth of RpoZ mutant E. coli. In the absence of the ω subunit, ppGpp affinity to RNAP is decreased; thus, rpoZ gene deleted E. coli strains show a modified stringent response. We used the E. coli K-12 MG1655 strain that lacks rpoZ (JEN202) to investigate the effect of the modified stringent response on recombinant protein production. However, the absence of the ω subunit results in diminished stability of the RNA polymerase at the promoter site. To avoid this, we used a deactivated CRISPR system that targets the ω subunit to upstream of the promoter site in the expression plasmid. The expression plasmid encodes for Chaetomium thermophilum formate dehydrogenase (CtFDH), a valuable enzyme for cofactor regeneration and CO2 reduction. A higher amount of CtFDH from the soluble fraction was purified from the JEN202 strain compared to the traditional BL21(DE3) method, thus offering a new strategy for batch-based recombinant enzyme production.

In situ cofactor regeneration enables selective CO2 reduction in a stable and efficient enzymatic photoelectrochemical cell

Mimicking natural photosynthesis by direct photoelectrochemical (PEC) reduction of CO2 to chemicals and fuels requires complex cell assemblies with limitations in selectivity, efficiency, cost, and stability. Here, we present a breakthrough cathode utilizing an oxygen tolerant formate dehydrogenase enzyme derived from clostridium carboxidivorans and coupled to a novel and efficient in situ nicotinamide adenine dinucleotide (NAD+/NADH) regeneration mechanism through interfacial electrochemistry on g-C3N4 films. We demonstrate stable (20 h) aerobic PEC CO2-to-formate reduction at close to 100 % faradaic efficiency and unit selectivity in a bio-hybrid PEC cell of minimal engineering with optimized Ta3N5 nanotube photoanode powered by simulated sunlight with a solar to fuel efficiency of 0.063 %, approaching that of natural photosynthesis.

Genome-wide gene expression changes of Pseudomonas veronii 1YdBTEX2 during bioaugmentation in polluted soils

BackgroundBioaugmentation aims to use the capacities of specific bacterial strains inoculated into sites to enhance pollutant biodegradation. Bioaugmentation results have been mixed, which has been attributed to poor inoculant growth and survival in the field, and, consequently, moderate catalytic performance. However, our understanding of biodegradation activity mostly comes from experiments conducted under laboratory conditions, and the processes occurring during adaptation and invasion of inoculants into complex environmental microbiomes remain poorly known. The main aim of this work was thus to study the specific and different cellular reactions of an inoculant for bioaugmentation during adaptation, growth and survival in natural clean and contaminated non-sterile soils, in order to better understand factors limiting bioaugmentation.ResultsAs inoculant we focused on the monoaromatic compound-degrading bacterium Pseudomonas veronii 1YdBTEX2. The strain proliferated in all but one soil types in presence and in absence of exogenously added toluene. RNAseq and differential genome-wide gene expression analysis illustrated both a range of common soil responses such as increased nutrient scavenging and recycling, expression of defense mechanisms, as well as environment-specific reactions, notably osmoprotection and metal homeostasis. The core metabolism of P. veronii remained remarkably constant during exponential growth irrespective of the environment, with slight changes in cofactor regeneration pathways, possibly needed for balancing defense reactions.ConclusionsP. veronii displayed a versatile global program, enabling it to adapt to a variety of soil environments in the presence and even in absence of its target pollutant toluene. Our results thus challenge the widely perceived dogma of poor survival and growth of exogenous inoculants in complex microbial ecosystems such as soil and provide a further basis to developing successful bioaugmentation strategies.

Scalable production and application of Pichia pastoris whole cell catalysts expressing human cytochrome P450 2C9

BackgroundCurrently, the numerous and versatile applications in pharmaceutical and chemical industry make the recombinant production of cytochrome P450 enzymes (CYPs) of great biotechnological interest. Accelerating the drug development process by simple, quick and scalable access of human drug metabolites is key for efficient and targeted drug development in response to new and sometimes unexpected medical challenges and needs. However, due its biochemical complexity, scalable human CYP (hCYP) production and their application in preparative biotransformations was still in its infancy.ResultsA scalable bioprocess for fine-tuned co-expression of hCYP2C9 and its essential complementary human cytochrome P450 reductase (hCPR) in the yeast Pichia pastoris (Komagataella phaffii) is presented. High-throughput screening (HTS) of a transformant library employing a set of diverse bidirectional expression systems with different regulation patterns and a fluorimetric assay was used in order to fine-tune hCYP2C9 and hCPR co-expression, and to identify best expressing clonal variants. The bioprocess development for scalable and reliable whole cell biocatalyst production in bioreactors was carried out based on rational optimization criteria. Among the different alternatives studied, a glycerol carbon-limiting strategy at high µ showed highest production rates, while methanol co-addition together with a decrease of µ provided the best results in terms of product to biomass yield and whole cell activity. By implementing the mentioned strategies, up to threefold increases in terms of production rates and/or yield could be achieved in comparison with initial tests. Finally, the performance of the whole cell catalysts was demonstrated successfully in biotransformation using ibuprofen as substrate, demonstrating the expected high selectivity of the human enzyme catalyst for 3′hydroxyibuprofen.ConclusionsFor the first time a scalable bioprocess for the production of hCYP2C9 whole cell catalysts was successfully designed and implemented in bioreactor cultures, and as well, further tested in a preparative-scale biotransformation of interest. The catalyst engineering procedure demonstrated the efficiency of the employment of a set of differently regulated bidirectional promoters to identify transformants with most effective membrane-bound hCYP/hCPR co-expression ratios and implies to become a model case for the generation of other P. pastoris based catalysts relying on co-expressed enzymes such as other P450 catalysts or enzymes relying on co-expressed enzymes for co-factor regeneration.

Chemical Tagging Assisted Mass Spectrometry Analysis Enables Sensitive Determination of Phosphorylated Compounds in a Single Cell.

Polar phosphorylated metabolites are involved in a variety of biological processes and play vital roles in energetic metabolism, cofactor regeneration, and nucleic acid synthesis. However, it is often challenging to interrogate polar phosphorylated metabolites and compounds from biological samples. Liquid chromatography-mass spectrometry (LC/MS) now plays a central role in metabolomic studies. However, LC/MS-based approaches have been hampered by the issues of the low ionization efficiencies, low in vivo concentrations, and less chemical stability of polar phosphorylated metabolites. In this work, we synthesized paired reagents of light and heavy isotopomers, 2-(diazomethyl)phenyl)(9-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)methanone (DMPI) and d3-(2-(diazomethyl)phenyl)(9-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)methanone (d3-DMPI). The paired reagents of DMPI and d3-DMPI carry diazo groups that can efficiently and selectively react with the phosphate group on polar phosphorylated metabolites under mild conditions. As a proof of concept, we found that the transfer of the indole heterocycle group from DMPI/d3-DMPI to ribonucleotides led to the significant increase of ionization efficiencies of ribonucleotides during LC/MS analysis. The detection sensitivities of these ribonucleotides increased by 25-1137-fold upon DMPI tagging with the limits of detection (LODs) being between 7 and 150 amol. With the developed method, we achieved the determination of all the 12 ribonucleotides from a single mammalian cell and from a single stamen of Arabidopsis thaliana. The method provides a valuable tool to investigate the dynamic changes of polar phosphorylated metabolites in a single cell under particular conditions.

DFT Mechanism Studies: Biomimetic 1,4-NADH Chemoselective, Co-factor Regeneration with [Cp*Rh(bpy)H]+, in Tandem with the Biocatalysis Pathways of a Core Model of the (HLADH)-Zn(II) Mediated Enzyme, in the Enantioselective Reduction of Achiral Ketones to Chiral S-Alcohols

Quantum Chemical (QC) calculations, utilizing Density Functional Theory (DFT), were performed to investigate the mechanistic aspects of the chemoselective catalyzed reaction of [Cp*Rh(bpy)H]+ with the biomimetic NAD+ analogues, N-benzylnicotinamide triflate, 1, and β-nicotinamide ribose-5′-methyl phosphate, 2, in the conversion to their 1,4-NADH analogues, 1,4-dihydro-N-benzylnicotinamide, 4, and β-1,4-dihydronicotinamide-5′-ribose methyl phosphate, 5. This reaction was in tandem with the 1,4-NADH dependent HLADH-Zn(II)-catalyzed reduction of achiral ketones to chiral S-alcohols. The [Cp*Rh(bpy)H]+ complex, and not its equilibrium tautomer, [η4-Cp*HRh(bpy)]+, was found to control the hydride transfer during the biomimetic NAD+/1,4-NADH conversion, through the non-covalent interactions of the biomimetic co-factors with [Cp*Rh(bpy)H]+. The thermodynamics and kinetics for the chiral reduction of the Zn(II) bound ketones, 2-pentanone and 4-phenyl-2-butanone, with co-factor, 4, catalyzed by Zn(SCH3)2(Imidazole), a core model of the Zn(II)-based catalytic center of HLADH, was also investigated by the evaluation of two possible reaction pathways: (1) formation of a ZnH from the C4-H hydride transfer of co-factor, 4, followed by reaction of the postulated ZnH with the bound 2-pentanone or 4-phenyl-2-butanone substrate, and (2), the direct C4-H transfer to the bound achiral ketone substrates, to provide the dominant chiral alcohols, S-2-pentanol or S-4-phenyl-2-butanol. The latter pathway was found most viable, and DFT calculations also revealed an essential η2-coordination of the 5,6 double bond of co-factor, 4, to the HLADH-Zn(II) metal ion center, upon imidazole decomplexation, providing an asymmetric differentiation of S-η2-5,6-1,4-NADH-Zn(II) binding. A proposed new paradigm for the Zn(II)'s non-innocent role in the HLADH-Zn(II) biocatalysis reduction mechanism, for enantioselective hydride transfer to a Zn(II) bound ketone, providing S-alcohols.

Design of artificial metalloenzymes for the reduction of nicotinamide cofactors

Artificial metalloenzymes result from the insertion of a catalytically active metal complex into a biological scaffold, generally a protein devoid of other catalytic functionalities. As such, their design requires efforts to engineer substrate binding, in addition to accommodating the artificial catalyst. Here we constructed and characterised artificial metalloenzymes using alcohol dehydrogenase as starting point, an enzyme which has both a cofactor and a substrate binding pocket. A docking approach was used to determine suitable positions for catalyst anchoring to single cysteine mutants, leading to an artificial metalloenzyme capable to reduce both natural cofactors and the hydrophobic 1-benzylnicotinamide mimic. Kinetic studies revealed that the new construct displayed a Michaelis-Menten behaviour with the native nicotinamide cofactors, which were suggested by docking to bind at a surface exposed site, different compared to their native binding position. On the other hand, the kinetic and docking data suggested that a typical enzyme behaviour was not observed with the hydrophobic 1-benzylnicotinamide mimic, with which binding events were plausible both inside and outside the protein. This work demonstrates an extended substrate scope of the artificial metalloenzymes and provides information about the binding sites of the nicotinamide substrates, which can be exploited to further engineer artificial metalloenzymes for cofactor regeneration. Synopsis about graphical abstractThe manuscript provides information on the design of artificial metalloenzymes based on the bioconjugation of rhodium complexes to alcohol dehydrogenase, to improve their ability to reduce hydrophobic substrates. The graphical abstract presents different binding modes and results observed with native cofactors as substrates, compared to the hydrophobic benzylnicotinamide.

Enzymatic direct carboxylation under supercritical CO2

Thermodynamically-constrained carboxylation reaction requires high temperature and pressure in industrial processes. Research endeavors are aimed towards developing safer and greener alternatives. Enzymatic systems are considered as potential substitutes. In this study, we conducted direct carboxylation via a bi-enzymatic reaction with Thermoplasma acidophilum isocitrate dehydrogenase (TaIDH) and T. acidophilum glucose dehydrogenase (TaGDH) to produce isocitric acid from of 2-ketoglutaric acid under supercritical CO2. Coupling with the TaGDH reaction (for cofactor regeneration) enabled the equilibrium of the reactions to shift towards carboxylation, resulting in a 16-fold increase in yield. Furthermore, the yield was increased by optimizing the reaction parameters such as pH, substrate concentration, temperature, and time, and by testing various metal ions. A preparative scale reaction was conducted successfully.

Towards a Novel Computer-Aided Optimization of Microreactors: Techno-Economic Evaluation of an Immobilized Enzyme System

Immobilized multi-enzyme cascades are increasingly used in microfluidic devices. In particular, their application in continuous flow reactors shows great potential, utilizing the benefits of reusability and control of the reaction conditions. However, capitalizing on this potential is challenging and requires detailed knowledge of the investigated system. Here, we show the application of computational methods for optimization with multi-level reactor design (MLRD) methodology based on the underlying physical and chemical processes. We optimize a stereoselective reduction of a diketone catalyzed by ketoreductase (Gre2) and Nicotinamidadenindinukleotidphosphat (NADPH) cofactor regeneration with glucose dehydrogenase (GDH). Both enzymes are separately immobilized on magnetic beads forming a packed bed within the microreactor. We derive optimal reactor feed concentrations and enzyme ratios for enhanced performance and a basic economic model in order to maximize the techno-economic performance (TEP) for the first reduction of 5-nitrononane-2,8-dione.