Deep Eutectic Solvents As Smart Cosubstrate in Alcohol Dehydrogenase-Catalyzed Reductions

catalysts

Communication Deep Eutectic Solvents as Smart Cosubstrate in Alcohol Dehydrogenase-Catalyzed Reductions

1,2, 1, 2 Santiago Nahuel Chanquia † , Lei Huang †, Guadalupe García Liñares , Pablo Domínguez de María 3 and Selin Kara 1,*

1 Department of Engineering, Biocatalysis and Bioprocessing Group, Aarhus University, 8000 Aarhus, Denmark; [email protected] (S.N.C.); [email protected] (L.H.) 2 Organic Chemistry Department, UMYMFOR-CONICET, Laboratory of Biocatalysis, Faculty of Exact and Natural Sciences, University of Buenos Aires, Buenos Aires C1428EGA, Argentina; [email protected] 3 Sustainable Momentum, SL. Av. Ansite 3, 4-6, Las Palmas de Gran Canaria, 35011 Canary Islands, Spain; [email protected] * Correspondence: [email protected]; Tel.: +45-2237-8964 The authors contributed equally. † Received: 20 July 2020; Accepted: 29 August 2020; Published: 3 September 2020

Abstract: Alcohol dehydrogenase (ADH) catalyzed reductions in deep eutectic solvents (DESs) may become efficient and sustainable alternatives to afford alcohols. This paper successfully explores the ADH-catalyzed reduction of ketones and aldehydes in a DES composed of choline chloride and 1,4-butanediol, in combination with buffer (Tris-HCl, 20% v/v). 1,4-butanediol (a DES component), acts as a smart cosubstrate for the enzymatic cofactor regeneration, shifting the thermodynamic equilibrium to the product side. By means of the novel DES media, cyclohexanone reduction was 1 1 optimized to yield maximum productivity with low enzyme amounts (in the range of 10 g L− d− ). Notably, with the herein developed reaction media, cinnamaldehyde was reduced to cinnamyl alcohol, an important compound for the fragrance industry, with promising high productivities of 1 1 ~75 g L− d− .

Keywords: deep eutectic solvents; alcohol dehydrogenases; smart cosubstrate; reductions; cinnamyl alcohol

1. Introduction Oxidoreductases (EC1) are becoming increasingly important catalysts in organic synthesis given their high activity and selectivity when applied in redox processes. Reactions ranging from C = O and C = C reductions to hydroxylations, epoxidations, and Baeyer-Villiger reactions are catalyzed by these enzymes [1–5]. In particular, the application of alcohol dehydrogenases (ADHs, EC 1.1.1.1) represents a sustainable and eﬃcient method for the synthesis of (optically active) alcohols starting from carbonyl compounds as inexpensive substrates [6,7]. ADHs are cofactor-dependent (nicotinamide cofactors (NAD(P)H)) and mediate as electron donors/acceptors in oxidations or reductions. For economic reasons, cofactors must be used in catalytic amounts combined with an in situ regeneration system [8], which normally implies a second enzymatic reaction, usually mediated by glucose dehydrogenase (GDH) [9] or formate dehydrogenase (FDH) and an adequate ancillary cosubstrate. Alternatively, chemical, electrochemical, or photochemical regeneration methods have been proposed [10–12], as well as synthetic nicotinamide cofactors in stoichiometric amounts [13]. In this area, the ADH mediated oxidation of simple alcohols for cofactor regenerations is a common and eﬃcient approach, particularly useful when the same ADH is used in both reactions, conforming a biocatalytic version of the Meerwein–Ponndorf–Verley (MPV)

Catalysts 2020, 10, 1013; doi:10.3390/catal10091013 www.mdpi.com/journal/catalysts Catalysts 2020, 10, x FOR PEER REVIEW 2 of 8 of the Meerwein–Ponndorf–Verley (MPV) reduction [14–17]. However, these combined cycles often present drawbacks related to their reversibility and poor thermodynamic driving force, which forces the use of a surplus of the cosubstrate or the carrying out of a coproduct removal. Using an excess amount of cosubstrate (e.g., isopropanol, ethanol, etc.) might enhance the solubility of a main substrate/product;Catalysts 2020, 10, 1013 nevertheless, it is still not an ideal approach in terms of atom economy. 2 To of 8 overcome these issues, the use of smart cosubstrates has been put forth. The main concept behind this relatively new development consists of shifting the equilibrium of a certain reaction by transformingreduction [14 one–17]. of However, the substrates these combinedinto thermodynamically cycles often present and/or drawbacks kinetically related inert to coproducts their reversibility [18– 20].and poor thermodynamic driving force, which forces the use of a surplus of the cosubstrate or the carryingBiocatalysis out of in a coproductnon-conventional removal. reaction Using media, an excess such amountas solvent of-free cosubstrate systems, (e.g.,organic isopropanol, solvents, ionicethanol, liquids etc.) (ILs) might, or enhancesupercritical the solubility fluids, is of becoming a main substrate a well-established/product; nevertheless, strategy to cope it is stillwith not the an challengesideal approach that modern in terms synthesis of atom economy.entails [21]. To Herein, overcome deep these eutectic issues, solvents the use (DESs), of smart a new cosubstrates type of ILshas [22], been hold put promising forth. The features main concept as reaction behind solvents this relatively [23,24]. newA DES development is composed consists of a hydrogen of shifting bon thed donorequilibrium (HBD) ofand a certaina hydrogen reaction bond by acceptor transforming (HBA), one typically of the substrates a quaternary into ammonium thermodynamically salt. The and DES/or preparationkinetically inertis straightforward coproducts [18—–b20oth]. components must be gently mixed to form a liquid—which is advantageousBiocatalysis over in traditional non-conventional ILs both reactionin terms media, of simplicity such as and solvent-free environmental systems, impact. organic To solvents,reduce theionic inherent liquids high (ILs), viscosity or supercritical of DESs, the fluids, addition is becoming of water a as well-established cosolvent (up to strategy 20% v/v to) enables cope with a low the viscouschallenges media, that while modern keeping synthesis the non entails-conventional [21]. Herein, nature deep of eutecticit (i.e., a solvents substrate (DESs),-dissolving a new system) type of [25].ILs [These22], hold low promising viscous DES features-water as mixtures reaction solventsare particularly [23,24]. useful A DES when is composed continuous of a hydrogenprocesses bondare envisageddonor (HBD) [26,27 and]. a hydrogen bond acceptor (HBA), typically a quaternary ammonium salt. The DES preparationBased on is the straightforward—both recent work of us and components other groups must [20,28 be–30 gently], herein mixed the tocombination form a liquid—which of the smart is cosubstrateadvantageous concept over traditional with a DES, ILs to both set in up terms an of efficient simplicity and and enviro environmentalnmentally-friendly impact. synthetic To reduce alternative,the inherent is addressed high viscosity for the of first DESs, time. the ADH addition from ofhorse water liver as (HLADH), cosolvent (upwhich to can 20% acceptv/v) enables a broad a rangelow viscousof diols media,as substrates while [31 keeping–34], was the selected non-conventional as the prototypical nature of enzyme, it (i.e., as a substrate-dissolvingit also can catalyze reactionssystem) [in25 DES]. These-aqueou lows viscous systems DES-water [35]. As reaction mixtures media, are particularly a DES consisting useful whenof choline continuous chloride processes (ChCl) andare 1,4 envisaged-butanediol [26, 27(1,4].-BD) was chosen. Acting as smart cosubstrate, 1,4-BD shifts the equilibrium of the MPVBased reduction on the when recent being work oxidized of us and to γ other-butyrolactone groups [20 (GBL),,28–30 a], thermodyn herein theamically combination stable of and the kineticallysmart cosubstrate inert coproduct concept (Schemewith a DES, 1). toThus, set up the an reaction efficient solvent and environmentally-friendly becomes a part of the reaction, synthetic avoidingalternative, the addition is addressed of any for other the firstreagents, time. which ADH greatly from horse simplifies liver the (HLADH), process and which makes can it accept much a morebroad efficient range of and diols sustainable. as substrates Analo [31gous–34], was approaches selected have as the been prototypical reported enzyme, in the literature as it also for can glycerolcatalyze [25] reactions and for inglucose DES-aqueous [30]. systems [35]. As reaction media, a DES consisting of choline chloride (ChCl) and 1,4-butanediol (1,4-BD) was chosen. Acting as smart cosubstrate, 1,4-BD shifts the 2.equilibrium Results and of Discussion the MPV reduction when being oxidized to γ-butyrolactone (GBL), a thermodynamically stable and kinetically inert coproduct (Scheme1). Thus, the reaction solvent becomes a part of the To validate the concept of using smart cosubstrates as part of the DES-water media, the HLADH- reaction, avoiding the addition of any other reagents, which greatly simplifies the process and makes it catalyzed reduction of cyclohexanone (CHO) to cyclohexanol (CHL) was chosen as model reaction much more efficient and sustainable. Analogous approaches have been reported in the literature for (Scheme 1). A choline chloride—1,4-butanediol DES was formed and mixed with 20% buffer (v/v) to glycerol [25] and for glucose [30]. decrease viscosity and to provide the enzyme with the necessary water for its activity [35].

SchemeScheme 1. 1. AlcoAlcoholhol dehydrogenase dehydrogenase from from horse horse liver liver (HLADH (HLADH)-catalyzed)-catalyzed reduction reduction of of cyclohexanone cyclohexanone (CHO)(CHO) using using deepdeep eutectic eutectic solvent solvent (DES)-bu (DES)-ﬀbufferer mixture mixture while while using using 1,4-butanediol 1,4-butanediol as a DES as hydrogen a DES hydrogenbond donor bond component. donor component.

2. Results and Discussion

To validate the concept of using smart cosubstrates as part of the DES-water media, the HLADH-catalyzed reduction of cyclohexanone (CHO) to cyclohexanol (CHL) was chosen as model reaction (Scheme1). A choline chloride—1,4-butanediol DES was formed and mixed with Catalysts 2020, 10, 1013 3 of 8 Catalysts 2020, 10, x FOR PEER REVIEW 3 of 8

20%HLADH buffer ( vwas/v) toproduced decrease and viscosity purified and starting to provide from thethe enzymerespective with plasmid, the necessary and its activity water forwas its characterizedactivity [35]. for a range of CHO concentrations from 0 mM to 500 mM. A clear excess substrate inhibitionHLADH effect was was produced observed, and whereby purified high starting Ki/KM from value the (65.5) respective indicated plasmid, no severe and itsinhibition, activity wasKi beingcharacterized the inhibition for a range constant of CHO and K concentrationsM the Michaelis from-Menten 0 mM constant. to 500 mM. Kinetic A clear parameters excess substrate for this enzyme are shown in Figure S1. In a previous work, the attractive concept of using DES both as inhibition effect was observed, whereby high Ki/KM value (65.5) indicated no severe inhibition, Ki being solvent and for cofactor regeneration has been studied [30]. However, to the best of our knowledge, the inhibition constant and KM the Michaelis-Menten constant. Kinetic parameters for this enzyme are smartshown cosubstrates in Figure S1. in combination In a previous with work, DES the and attractive redox biocatalysis concept of usinghave never DES bothbeen asreported solvent before. and for cofactorFirstly, regeneration three different has been DESs studied of molar [30 ].ratio However, of ChCl to to the 1,4 best-BD of at our 1:3, knowledge, 1:4, and 1:5 smart were cosubstrates prepared andin combinationthe formation with of DESa liquid and phase redox was biocatalysis visually have evaluated. never beenTo perform reported the before. biocatalytic reactions, DES withFirstly, proportions three diff erentof 1:4 DESsand 1:5 of were molar used, ratio as of they ChCl formed to 1,4-BD a largely at 1:3, stable 1:4, and liquid 1:5 were phase prepared over time. and Conversely,the formation the of 1:3 a liquidcombination phase was formed visually two evaluated.phases after To being perform stored the at biocatalytic room temperature reactions, for DES a few with hoursproportions. of 1:4 and 1:5 were used, as they formed a largely stable liquid phase over time. Conversely, theOnce 1:3 combination DES proportions formed were two chosen, phases afterthe enzymatic being stored reaction at room was temperature conducted to for assess a few which hours. DES was a Oncemore appropriate DES proportions non-conventional were chosen, media the enzymatic(1:4 or 1:5), reactionin combination was conducted with an amount to assess of buffer which ofDES 20% was (v/v a) more to keep appropriate the viscosity non-conventional low [25]. Gratifyingly, media (1:4 both or 1:5), DES- inwater combination mixtures withwere an enzyme amount- compatible,of buffer of and 20% quantitative (v/(v) to keep product the viscosity (CHL) formation low [25]. Gratifyingly,was observed both after DES-water 24 h. The medium mixtures DES were (ChCl:1,4enzyme-compatible,-BD/1:4) was and chosen quantitative for further product studies (CHL) [36]. For formation the enzymatic was observed reaction, after full 24 conversion h. The medium was observedDES (ChCl:1,4-BD in both cases/1:4) wasafter chosen 24 h reaction for further time studies (Figure [36 1].). ForThe the inhibition enzymatic of reaction,HLADH fullby conversion1,4-BD at higherwas observed concentrations in both cases that after has 24 been h reaction previously time (Figure reported1). The[37] inhibition did not alleviate of HLADH to by reach 1,4-BD full at conversions.higher concentrations that has been previously reported [37] did not alleviate to reach full conversions.

(a) (b)

FigureFigure 1. 1.HLADHHLADH-catalyzed-catalyzed CHO CHO reduction reduction in two in twodifferent diﬀerent mixtures mixtures of DES of- DES-bubuffer (80:20,ﬀer (80:20, v/v): (vb/v) ): DES(b) DES(ChCl:1,4 (ChCl:1,4-BD-BD/1:4);/1:4); (a) DES (a) DES (ChCl:1,4 (ChCl:1,4-BD-BD/1:5)./1:5). Reaction Reaction conditions: conditions: 100 100mM mM CHO, CHO, 1 mM 1 mM NAD NAD+, 1+ ,

mg/mL1 mg/mL purified puriﬁed HLADH. HLADH. 25 25°C ,◦ andC, and 1200 1200 rpm. rpm.

EncouragedEncouraged by the the promising promising results results in the in new the DES-bu new DESffer- mediabuffer containing media containing a smart cosubstrate, a smart cosubstrate,the optimum the HLADH optimum loading HLADH for loading the system for the was system subsequently was subsequently evaluated. evaluated. To that end, To increasingthat end, increasingconcentrations concentrations of the purified of the enzyme purified were enzyme used were (0.05 used to 2 mg (0.05/mL) to to2 mg/mL) find out theto find best out reaction the best rate reactionwith the rate minimum with the amount minimum possible amount of enzyme, possible as of shown enzyme, in as Figure shown2. Allin Figure progress 2. curvesAll progress using curvesdifferent using enzyme different loadings enzyme are shown loadings in Figure are shown S2. Based in Figure on these S2. results Based we on have these chosen results the we following have chosenreaction the conditions following for reaction HLADH-catalyzed conditions for reduction HLAD ofH- CHOcatalyzed for the reduction further experiments:of CHO for ChCl:1,4-BDthe further + + experiments:1:4 as DES; HLADH, ChCl:1,4- 0.25BD 1:4 mg / asmL; DES; NAD HLADH,, 1 mM; 0.25 temperature: mg/mL; NAD 25 ◦C;, 1 stirring: mM; temperature: 1200 rpm;Tris-HCl 25 °C ; stirring:buffer volume 1200 rpm; percentage, Tris-HCl andbuffer 20% volumev/v. percentage, and 20% v/v.

Catalysts 2020, 10, x FOR PEER REVIEW 4 of 8 Catalysts 2020, 10, x FOR PEER REVIEW 4 of 8 Catalysts 2020, 10, 1013 4 of 8

Figure 2. Relation between reaction rate constants a andnd amount of HLADH used. Figure 2. Relation between reaction rate constants and amount of HLADH used. UsingUsing these chosen reaction conditions,conditions, a quantitative conversion of CHO to CHL was achieved 1 1 after 55 hUh leadingsing leading these to to ~10chosen ~10 g Lg − Lreaction−d1 −d−.1. PerformingPerforming conditions the the, a reactionquantitative reaction at at higher higherconversion concentrations concentrations of CHO of to CHO ofCHL CHO (e.g., was (e.g. 500achieved, mM500 −1 −1 ormMafter 1000 or 5 mM) 1000h leading led, mM) however, to led, ~10 however, g toL unsuccessful d . toPerforming unsuccessful results, the presumablyreaction results, at presumably higher due to concentrations substrate due to inhibitionsubstrate of CHO (Figure inhibition (e.g., S1) 500 and(FiguremM the or S1) cosubstrate 1000 and mM)the cosubstrate 1,4-BD led, however, inhibition 1,4- BD to [ 20inhibition unsu]. Theccessful fact [20]. that results, The adding fact presumably that water adding to the due water DES to tosubstrate system the DES leads inhibition system to a non-conventionalleads(Figure to a S1) non and-conventional the but cosubstrate low viscous but low1,4 media- BDviscous inhibition may media open[20]. themay optionThe open fact ofthe that setting option adding up of continuous watersetting to up the processescontinuous DES system in whichproleadscesses low to ina substrate nonwhich-conventional low loadings substrate can but loadings be low pumped viscous can in be media an pumped eﬃ cientmay in manneropen an efficient the [ 26option,27 manner]. of setting [26,27 ].up continuous proThecesses synthetic in which concept low substrate including loadings DES and can a besmart pumped cosubstrate in an efficient was extended manner to [26,27 other ]. substrates as well.The Whereas synthetic benzaldehyde concept including and and ethyl ethyl DES 4 4-chloro-3-oxobutanoate -andchloro a smart-3-oxobutanoate cosubstrate did didwas not notextended lead lead to to to any other conversion, substrates presumablyas well. Whereas due due to to lowbenzaldehyde low solubility solubility in and the in theDESethyl DES system, 4-chloro system, cinnamaldehyde-3- oxobutanoate cinnamaldehyde was did reduced wasnot lead reduced to to cinnamyl any to conversion, cinnamyl alcohol (~25%alcoholpresumably conversion, (~25% conversion, due 48 to h low reaction 48 solubility h reaction time, in Scheme time, the DES 2Scheme). system, 2). cinnamaldehyde was reduced to cinnamyl alcohol (~25% conversion, 48 h reaction time, Scheme 2).

Scheme 2. HLADH-catalyzed cinnamaldehyde reduction in DES-buﬀer (80:20, v/v). Scheme 2. HLADH-catalyzed cinnamaldehyde reduction in DES-buffer (80:20, v/v). DES: ChCl:1,4-BD/1:4. SchemeDES: ChCl:1,4-BD 2. HLADH/1:4.-catalyzed cinnamaldehyde reduction in DES-buffer (80:20, v/v). DES: ChCl:1,4-BD/1:4. Cinnamyl alcohol is a valuable industrial compound used as aroma chemical, e.g., derivatized as estersCinnamylCinnamyl [38]. Given alcohol alcohol the is is ainherent valuablea valuable instability industrial industrial of compound compound the substrate used used and as as aroma thearoma product chemical, chemica [39,40l, e.g., e.g.], derivatized biocatalytic, derivatized assynthesisas esters esters [of38 [38]. cinnamyl]. Given Given thealcohol the inherent inherent are put instability instability forth (using of of the natural the substrate substrate catalysts and and during the the product product the process). [39 [39,40,40], Remarkably,], biocatalytic biocatalytic synthesisverysynthesis promising of of cinnamyl cinnamyl productivities alcohol alcohol are ( are~75 put putg forthL forth−1 d− (using1) ( usingwere natural obtainednatural catalysts catalysts with the during during novel the thelow process). process). viscous Remarkably, Remarkably,DES-smart 1 −1 1−1 verycosubstratevery promising promising system, productivities productivities suggesting (~75 that (~75 ga Lgcontinuous− L d −d )) were were process obtained obtained might with with be the envisagedthe novel novel low lowfor viscous theviscous production DES-smart DES-smart of cosubstratethiscosubstrate type of system, compounds,system, suggesting suggesting for which thatthat a environmentallya continuouscontinuous processprocess-friendly might media be envisaged envisaged and reaction for for the the conditions production production are of ofneeded.this this type type For of ofwork compounds,-up procedures, for which which the environmentally-friendlyaddition environmentally of an excess-friendly of water media media to and andthe reaction media reaction breaks conditions conditions the DES are are needed.structure,needed. For andFor work-up workwould-up allow procedures, procedures, the extraction the the addition addition of the ofcinnamyl of an an excess excess alcohol of of water water with to solventsto the the media media (e.g. breaks, breaksethyl theacetate). the DES DES structure,Thestructure, evaporation and and would wouldof the allow waterallow the excessthe extraction extraction would of create of the the cinnamyl thecinnamyl DES mixture alcohol alcohol withagain with solvents [41].solvents (e.g., (e.g. ethyl, ethyl acetate). acetate). The evaporation of the water excess would create the DES mixture again [41]. The evaporation of the water excess would create the DES mixture again [41].

Catalysts 2020, 10, 1013 5 of 8

3. Materials and Methods

3.1. Chemicals and Materials Chemicals, cultivation media components, and reagents were purchased from Sigma–Aldrich (Søborg, Denmark), Carl Roth (Karlsruhe, Germany), and VWR (Søborg, Denmark) and used as received. Ni-NTA affinity resin was ordered from Expedeon (Cambridgeshire, UK) and BCA protein quantification kit (PierceTM) was obtained from Thermo Fischer Scientific (Schwerte, Germany).

3.2. Enzyme Production HLADH gene was present in a Novagen pET-28b plasmid. For transformation, 1 µL of plasmid solution was added to 50 µL of Agilent BL21-Gold (DE3) competent cells in a 1.5 mL plastic micro-centrifuge tube, mixed gently and then the mixture was incubated on ice for half an hour. Afterwards, the cells were incubated for 30 s at 42 ◦C and put on ice again for five minutes. Next, 800 µL of lysogeny broth (LB) liquid medium was added and left to incubate for 1 h at 37 ◦C and 180 rpm. Afterwards, 200 µL of the above-given preparation was added to Petri dishes with LB agar plates (+50 µg/mL kanamycin) and left to incubate overnight at 37 ◦C. Sterilized baffled flasks were filled with 20 mL of LB liquid medium (+50 µg/mL kanamycin). Next, a colony was picked from the agar plate and added to each flask containing the liquid medium. The liquid cultures were left to incubate overnight ® at 150 rpm and 37 ◦C in a New Brunswick™ Innova 44 (Eppendorf AG, Juelich, Germany) incubator. Later, 400 mL of Terrific Broth (TB) medium (+50 µg/mL kanamycin), and 4 mL of the previous pre-culture were added to 2-L baffled flasks and incubated at 37 ◦C and 150 rpm for 2.5 h. Cells were induced by adding 200 µL of a 1 M solution of isopropyl β-d-1-thiogalactopyranoside (IPTG) followed by incubation for 24 h at 25 ◦C and 150 rpm. The cultures were then divided in centrifuge plastic bottles and centrifuged at 9000 rpm and 4 ◦C for 10 min in a Sorvall LYNX 4000 Superspeed Centrifuge (Thermo Fischer Scientific, Schwerte, Germany). The cell pellets were next suspended in a lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) and sonicated using a Labsonic M (Sartorius, Goettingen, Germany) with MS 73 probe (60% amplitude: 4 4 min, 0.4 cycles on ice, 4 min break in between). Then, cell debris × and the soluble fraction were separated by centrifugation at 16,000 rpm for 45 min at 4 ◦C, obtaining a cell free extract (CFE), which was purified using a Ni-TNA column to obtain a solution of the pure HLADH, which was lyophilized and stored at 20 C. − ◦ 3.3. Kinetic Assay Enzyme kinetics were measured for lyophilized purified HLADH using a Cary 60 UV-Vis spectrophotometer (Agilent Technologies, Glostrup, Denmark) and thermostated at 25 ◦C with a PCB 1500 Water Peltier System (Agilent Technologies, Glostrup, Denmark) at 340 nm, using a NADH concentration of 0.1 mM and 0–500 mM model substrate CHO. Enzyme concentration ranged from 0.0005 to 0.005 mg/mL. The assays for the negative controls were performed without the substrate and without the enzyme.

3.4. DES Preparation DESs were prepared by adding the correspondent components, ChCl and 1,4-BD, to a ﬂask, which was magnetically stirred at 400 rpm IKA Magnetic stirrer (IKA, Staufen, Germany) and heated with a water bath at 80 ◦C for 1 h.

3.5. Reduction of Cyclohexanone To perform the reactions, stock solutions of NAD+ and HLADH were prepared by dissolving both the cofactor and the lyophilized enzyme in Tris-HCl (50 mM, pH 7.5), and incubated at 25 ◦C for 30 min. Then, 800 µL of the selected pure DES, 100 µL of Tris-HCl (50 mM, pH 7.5), 10 µL of Catalysts 2020, 10, 1013 6 of 8 cyclohexanone (CHO), and 100 µL of the stock solution of NAD+ and HLADH were added in glass vials and incubated at 25 ◦C and 1200 rpm in a heating thermo shaker (Hettich Benelux, ProﬁLab24 GmbH, Berlin, Germany). This methodology was also followed for other substrates (benzaldehyde, 4-chloro-3-oxobutanoate, and cinnamaldehyde) screened.

3.6. GC Analysis Conversion of the substrates and formation of the products were followed by gas chromatography (GC) (Schimadzu GC2030, Holm & Halby, Brøndby, Denmark) analysis. Samples of 50 µL were taken from the reaction vials and added to 1.5 mL plastic micro-centrifuge tubes, followed by the addition of 250 µL of ethyl acetate (containing 2 mM methyl benzoate as an internal standard), vortexed and centrifuged for 1 min at 13,000 rpm. Then, 200 µL was taken from the upper organic phase and transferred to another plastic micro-centrifuge tube. A small amount of MgCl2 was added for drying, the tubes were agitated manually, centrifuged for 1 min at 13,000 rpm, and 150 µL was taken and transferred to GC glass vials with a micro-insert for low volume. The GC method used can be found in the Supplementary Materials section.

4. Conclusions The successful use of a DES as solvent and as a smart cosubstrate for cofactor regeneration in redox systems has been successfully explored for the first time. Promising results for the reduction of a series of carbonyl compounds were achieved, leading to full conversions when enzyme loadings were properly optimized. The particular composition of the used DES enabled a highly efficient process system, as the formed by-product (GBL) is inert, pushing the equilibrium to the formation of the desired alcohol. Of particular relevance is the production of cinnamyl alcohol, for which 1 1 proof-of-concept productivities (~75 g L− d− ) are highly promising. Further optimizations for this DES media may include the assessment of higher substrate loadings (e.g., step-wise addition), enzyme immobilization, and set-up of continuous (non-viscous) DES-water media. Moreover, expanding the substrate range, as well as using other ADH enzymes (e.g., variants with higher robustness for the DES media), may become an important line of research. In a broader sense, the combination of DES and smart cosubstrate systems may create powerful synergies to be applied for efficient sustainable chemical processes.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4344/10/9/1013/s1, Figure S1: Enzyme kinetic assay of HLADH toward CHO, Table S1: Kinetic parameters for the obtained HLADH, Table S2: Details of GC method used in this study, Figure S2: HLADH-catalyzed CHO reduction progress in DES (ChCl:1,4-BD/1:4)-Buffer (80:20, v/v) media with different enzyme loading. Figure S3: Example of a GC chromatography spectrum. Author Contributions: Conceptualization: S.K.; experimental work: S.N.C. and L.H.; writing: S.N.C., L.H., G.G.L., P.D.d.M., and S.K. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by Deutsche Forschungsgemeinschaft (DFG) grant number: KA 4399/3-1 and Aarhus Universitets Forskningsfond (AUFF) Starting Grant. Acknowledgments: The authors thank Diederik Johannes Opperman (University of the Free State, South Africa) for the recombinant plasmid containing HLADH gene. Furthermore, we would also like to thank Kim Møller Johansen and Thomas Dyekjær from Department of Engineering at Aarhus University for their technical assistance. Conflicts of Interest: The authors declare no conflict of interest.

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