catalysts

Article Kinetic Resolution of Racemic Amines to Enantiopure (S)-amines by a Biocatalytic Cascade Employing and Dehydrogenase

Mahesh D. Patil , Sanghan Yoon, Hyunwoo Jeon, Taresh P. Khobragade, Sharad Sarak, Amol D. Pagar, Yumi Won and Hyungdon Yun *

Department of Systems Biotechnology, Konkuk University, Seoul 05029, Korea * Correspondence: [email protected]; Tel.: +82-2-450-0496; Fax: +82-2-450-0686

 Received: 4 June 2019; Accepted: 10 July 2019; Published: 12 July 2019 

Abstract: Amine dehydrogenases (AmDHs) efficiently catalyze the NAD(P)H-dependent asymmetric reductive amination of prochiral carbonyl substrates with high enantioselectivity. AmDH-catalyzed can also be used for the kinetic resolution of racemic amines to obtain enantiopure amines. In the present study, kinetic resolution was carried out using a coupled- cascade consisting of AmDH and (AlaDH). AlaDH efficiently catalyzed the conversion of pyruvate to alanine, thus recycling the nicotinamide cofactors and driving the reaction forward. The ee values obtained for the kinetic resolution of 25 and 50 mM rac-α-methylbenzylamine using the purified enzymatic systems were only 54 and 43%, respectively. The use of whole-cells apparently reduced the / inhibition, and the use of only 30 and 40 mgDCW/mL of whole-cells co-expressing AmDH and AlaDH efficiently resolved 100 mM of rac-2-aminoheptane and rac-α-methylbenzylamine into the corresponding enantiopure (S)-amines. Furthermore, the applicability of the reaction protocol demonstrated herein was also successfully tested for the efficient kinetic resolution of wide range of racemic amines.

Keywords: amine dehydrogenase; alanine dehydrogenase; chiral amines; kinetic resolution; whole-cell biotransformations; oxidative deamination

1. Introduction Enantiopure chiral amines are important precursors of numerous small molecule pharmaceuticals, agrochemicals and fine chemicals. It has been estimated that more than one third of the 200 most prescribed small molecule drugs contain chiral amine precursors [1–3]. The asymmetric synthesis of amines from prochiral carbonyls and has also been acknowledged as one of the most highly desired transformations industrially by the ACS Green Chemistry Institute Pharmaceutical Roundtable [4]. The increasing demand for enantiomerically pure compounds and the simultaneous imposition of environmental restrictions by many countries necessitates the effective integration of traditional chemical syntheses with biocatalytic ‘greener’ methods [5–9]. Traditional organo-catalytic methods for the synthesis of chiral amines use toxic intermediates and require environmentally harsh conditions. Moreover, the purification of toxic metals is warranted, which complicates the synthetic methods and its cost [10–12]. A surge of interest has occurred in recent years in the use of superior biocatalytic alternatives to the chemical syntheses of chiral amines [13,14]. such as transaminases [15], imine reductases [16], amine oxidases [17], P450 monooxygenases [18], lipases [19], and berberine bridge enzyme [20] have been successfully employed for chiral amines synthesis. More recently, amine dehydrogenases (AmDHs)—catalyzing the reductive amination of ketones using ammonia and generating only

Catalysts 2019, 9, 600; doi:10.3390/catal9070600 www.mdpi.com/journal/catalysts Catalysts 2019, 9, 600 2 of 14 as byproduct—have been discovered [21]. Owing to the high enantioselectivity and atom efficiency, AmDH-mediated synthesis is very attractive and a potential biocatalytic approach for the synthesis of chiral amines. However, AmDHs are not ubiquitously present in nature. Nevertheless, Itoh et al. first reported the presence of AmDH in Streptomyces virginiae IFO 12827 [22]. However, this enzyme was unexplored for another decade owing to its poor enantioselectivity and the unavailability of the genetic information. Subsequently, thermostable AmDH from Petrotoga mobiliz DSM 10674 was discovered [23]. Furthermore, the group of Bommarius at Georgia Institute of Technology, U.S.A. generated the first engineered AmDH using dehydrogenase (LeuDH) from Bacillus stearothermophilus [21]. Bommarius’ group developed second AmDH based on phenylalanine dehydrogenase (PheDH) from Bacillus badius as a scaffold [24]. Furthermore, the Bommarius group generated a chimeric AmDH by domain shuffling of LeuDH and PheDH parental scaffolds. This chimeric AmDH substantially improved the substrate scope of the previously generated AmDHs and converted sterically hindered substrates to their corresponding enantiopure (R)-amine products [25]. Using a similar engineering strategy to that used previously, more AmDHs were generated from L-amino acid dehydrogenase scaffolds from Rhodococcus sp. M4, Exiguobacterium sibiricum, Caldalkalibacillus thermarum and Lysinibacillus fusiformis [26–29]. These engineered AmDHs have been successfully employed in tandem with other enzymes to obtain various enantiopure compounds. For instance, AmDHs in combination with alcohol dehydrogenases have been used in a -neutral two-enzyme cascade for the asymmetric transformation of racemic secondary alcohols into chiral amines [27,30]. AmDH-catalyzed reductive amination of ketones has been widely used for the synthesis of chiral amines. Until very recently, when Mayol et al. [31] characterized six naturally occurring AmDHs which exhibited (S)-selectivity, all the previously generated AmDHs showed exclusive (R)-selectivity owing to their generation from (L)-amino acid dehydrogenase parents. The scarce availability of (S)-selective AmDHs for the generation of (S)-amines can be overcome by (R)-AmDH-mediated kinetic resolution of racemic amines using oxidative deamination. Nevertheless, AmDH-catalyzed oxidative deamination is also an interesting approach that can be used for the kinetic resolution of racemic amines to obtain enantiopure (S)-amines. We have previously reported the specific deamination of the R-amines from racemic mixtures leaving behind the unreacted enantiopure S-amines using a whole-cell biocatalyst expressing AmDH and NADH-oxidase [32]. However, one of the major problems related with this system was the requirement of very high quantities of a whole-cell biocatalyst. For example, the minimum amount of whole-cells required to resolve only 20 mM racemic methylbenzylamine (rac-MBA) into enantiopure (S)-amine was 100 mgDCW/mL. Also, the different optimum pH of AmDH and NADH-oxidase poses a hurdle for the efficient coupled-enzyme reaction. We envisaged the development of an improved method for the oxidative deamination of racemic amines for the synthesis of their enantiopure (S)-amine counterparts. We herein report a two-enzyme cascade employing AmDH and alanine dehydrogenase (AlaDH) for the kinetic resolution of racemic amines (Figure1). The specificity of enantioselective AmDHs was exploited for resolving a racemic mixture to obtain only the enantiopure (S)-amines. The major advantage with the use of AlaDH is that it efficiently converts pyruvate to alanine at the expense of NADH, thereby regenerating nicotinamide cofactors, and ultimately, shifting the equilibrium of the primary reaction toward product formation. Catalysts 2018, 8, x FOR PEER REVIEW 2 of 15 generating only water as byproduct—have been discovered [21]. Owing to the high enantioselectivity and atom efficiency, AmDH-mediated synthesis is very attractive and a potential biocatalytic approach for the synthesis of chiral amines. However, AmDHs are not ubiquitously present in nature. Nevertheless, Itoh et al. first reported the presence of AmDH in Streptomyces virginiae IFO 12827 [22]. However, this enzyme was unexplored for another decade owing to its poor enantioselectivity and the unavailability of the genetic information. Subsequently, thermostable AmDH from Petrotoga mobiliz DSM 10674 was discovered [23]. Furthermore, the group of Bommarius at Georgia Institute of Technology, U.S.A. generated the first engineered AmDH using (LeuDH) from Bacillus stearothermophilus [21]. Bommarius’ group developed second AmDH based on phenylalanine dehydrogenase (PheDH) from Bacillus badius as a scaffold [24]. Furthermore, the Bommarius group generated a chimeric AmDH by domain shuffling of LeuDH and PheDH parental scaffolds. This chimeric AmDH substantially improved the substrate scope of the previously generated AmDHs and converted sterically hindered substrates to their corresponding enantiopure (R)-amine products [25]. Using a similar engineering strategy to that used previously, more AmDHs were generated from L-amino acid dehydrogenase scaffolds from Rhodococcus sp. M4, Exiguobacterium sibiricum, Caldalkalibacillus thermarum and Lysinibacillus fusiformis [26–29]. These engineered AmDHs have been successfully employed in tandem with other enzymes to obtain various enantiopure compounds. For instance, AmDHs in combination with alcohol dehydrogenases have been used in a redox-neutral two-enzyme cascade for the asymmetric transformation of racemic secondary alcohols into chiral amines [27,30]. AmDH-catalyzed reductive amination of ketones has been widely used for the synthesis of chiral amines. Until very recently, when Mayol et al. [31] characterized six naturally occurring AmDHs which exhibited (S)-selectivity, all the previously generated AmDHs showed exclusive (R)-selectivity owing to their generation from (L)-amino acid dehydrogenase parents. The scarce availability of (S)-selective AmDHs for the generation of (S)-amines can be overcome by (R)-AmDH-mediated kinetic resolution of racemic amines using oxidative deamination. Nevertheless, AmDH-catalyzed oxidative deamination is also an interesting approach that can be used for the kinetic resolution of racemic amines to obtain enantiopure (S)-amines. We have previously reported the specific deamination of the R-amines from racemic mixtures leaving behind the unreacted enantiopure S-amines using a whole-cell biocatalyst expressing AmDH and NADH-oxidase [32]. However, one of the major problems related with this system was the requirement of very high quantities of a whole-cell biocatalyst. For example, the minimum amount of whole-cells required to resolve only 20 mM racemic methylbenzylamine (rac-MBA) into enantiopure (S)-amine was 100 mgDCW/mL. Also, the different optimum pH of AmDH and NADH-oxidase poses a hurdle for the efficient coupled-enzyme reaction. We envisaged the development of an improved method for the oxidative deamination of racemic amines for the synthesis of their enantiopure (S)-amine counterparts. We herein report a two-enzyme cascade employing AmDH and alanine dehydrogenase (AlaDH) for the kinetic resolution of racemic amines (Figure 1). The specificity of enantioselective AmDHs was exploited for resolving a racemic mixture to obtain only the enantiopure (S)-amines. The major advantage with the use of AlaDH is that it efficiently converts pyruvate to alanine at the expense of NADH, thereby Catalysts 2019 9 regenerating, , nicotinamide 600 cofactors, and ultimately, shifting the equilibrium of the primary reaction3 of 14 toward product formation.

Figure 1. The oxidative deamination of racemic amines to enantiopure (S)-amines using a two-enzyme cascade employing amine dehydrogenase and alanine dehydrogenase. (R)-AmDH catalyze the oxidative

deamination of (R)-amine to corresponding ketone, leaving behind enantiopure (S)-amine. Nicotinamide cofactors were recycled using alanine dehydrogenase.

2. Results and Discussion

2.1. Reactivity of Amine Dehydrogenases toward Racemic Amines To catalyze the oxidative deamination of racemic amines, two AmDHs, (1) A chimeric AmDH (Chi-AmDH) generated by the domain shuffling of the LeuDH and PheDH [25] and (2) Rs-AmDH (developed from L-phenylalanine dehydrogenase of Rhodococcus sp.) [26] were used in this study. The genes encoding these AmDHs were cloned in pET-24ma vector and transformed in E. coli BL21 (DE3). Following the induction of transformants by IPTG, the His-tagged proteins were purified as previously reported [33]. In order to explore the applicability of AmDHs for the kinetic resolution, the oxidative deamination potential of AmDHs was evaluated toward various racemic amines (Figure2A). Chi-AmDH and Rs-AmDH displayed varying activity toward racemic amine substrates. While the highest activity by Rs-AmDH was displayed toward 4-phenyl-butan-2-amine (a5), Chi-AmDH showed the highest activity heptan-2-amine (a3). Both enzymes also displayed good activity toward 1-(4-fluorophenyl)propan-2-amine (a6) and 1-m-tolylethanamine (a10) (Figure2B). The successful oxidative deamination catalyzed by AmDHs suggested their potential for the kinetic resolution of chiral amines to obtain enantiopure (S)-amines. Catalysts 2019, 9, 600 4 of 14 Catalysts 2018, 8, x FOR PEER REVIEW 4 of 15

FigureFigure 2.2. ((AA)) RacemicRacemic amineamine substratessubstrates testedtested forfor thethe kinetickinetic resolutionresolution byby AmDH-AlaDHAmDH-AlaDH system;system; andand ((BB)) TheThe oxidativeoxidative deaminationdeamination potentialpotential ofof AmDHsAmDHs towardtoward variousvarious racemicracemic amines;amines; ReactionReaction + conditionsconditions forfor oxidativeoxidative deamination:deamination: 10 mMmM racemicracemic amineamine substrate,substrate, 11 mMmM NADNAD+,, 0.20.2 mgmg/mL/mL AmDHs,AmDHs, 100100 mMmM GlycineGlycine bubufferffer (pH(pH 10.0).10.0). 2727 mUmU/ /mgmgactivity activitywas was consideredconsidered as as 100%. 100%. 2.2. Kinetic Resolution of Racemic Amines Using a Purified AmDH/AlaDH Coupling System 2.2. Kinetic Resolution of Racemic Amines Using a Purified AmDH/AlaDH Coupling System In a two-enzyme cascade reported herein for the kinetic resolution, the oxidative deamination of the RIn-amines a two-enzyme from racemic cascade mixtures reported to their herein corresponding for the kinetic ketones, resolution, leaving the behind oxidative the deamination enantiopure (ofS)-amines, the R-amine was catalyzeds from racemic by (R)-AmDHs. mixtures Theto overalltheir corresponding efficiency of the ketones, reaction leaving largely dependsbehind the on theenantiopure availability (S)-amines, of nicotinamide was catalyzed cofactors by and(R)-AmDHs. proficient The recycling overall thereof.efficiency Moreover, of the reaction the e ffilargelycient recyclingdepends on of cofactorsthe availability is necessary of nicotinamide to overcome cofactors the product and proficient inhibition recycling of the primary thereof. biocatalystMoreover, the by theefficient recycling and to shiftof cofactors the reaction is necessary equilibrium to towardovercome product the formation.product inhibition Herein, weof usedthe primary alanine biocatalyst by the cofactor and to shift the reaction equilibrium toward product formation. Herein, we used alanine dehydrogenase for the regeneration of nicotinamide cofactors. To study the kinetic

Catalysts 2019, 9, 600 5 of 14

Catalystsdehydrogenase 2018, 8, x FOR PEER for REVIEW the regeneration of nicotinamide cofactors. To study the kinetic resolution5 of 15 of racemic amines, rac-α-MBA (rac-a7) and rac-2-aminoheptane (rac-a3) were selected as representative resolutionaromatic of racemic and aliphatic amines, amines, rac-α-MBA respectively. (rac-a7) and rac-2-aminoheptane (rac-a3) were selected as representativeSince thearomatic stability and and aliphatic activity amines, of the biocatalysts respectively. are dependent on the pH, they can critically affect theSince overall the stability outcome and of theactivity biocatalytic of the biocatalysts reaction. Especially are dependent for reactions on the pH, catalyzed they can by critically the enzymatic affect the overall outcome of the biocatalytic reaction. Especially for reactions catalyzed by the cascades, wherein each constituent enzyme exhibits different optimum pH, the pH of the reaction enzymatic cascades, wherein each constituent enzyme exhibits different optimum pH, the pH of the should be carefully determined so that activity of the enzymes is not severely compromised [10,32,33]. reaction should be carefully determined so that activity of the enzymes is not severely compromised Thus, the effect of varying pH on the activities of AmDHs and AlaDH was evaluated (Figure3). [10,32,33]. Thus, the effect of varying pH on the activities of AmDHs and AlaDH was evaluated In the case of AlaDH, marginal improvement in activity was observed with the increase in pH of (Figure 3). In the case of AlaDH, marginal improvement in activity was observed with the increase in Tris buffer (100 mM) from pH 7.0 to 9.0. The optimum activity of AlaDH was observed at pH 10.0 pH of Tris buffer (100 mM) from pH 7.0 to 9.0. The optimum activity of AlaDH was observed at pH (Glycine buffer, 100 mM). For AmDHs, steep increase in activity was observed with the increase in 10.0 (Glycine buffer, 100 mM). For AmDHs, steep increase in activity was observed with the increase pH beyond 9.0. The maximum activity for Chi-AmDH and Rs-AmDH was observed at pH 10.5 and 9.5, in pH beyond 9.0. The maximum activity for Chi-AmDH and Rs-AmDH was observed at pH 10.5 respectively. Thus, the kinetic resolution using Chi-AmDH-AlaDH and Rs-AmDH-AlaDH was carried and 9.5, respectively. Thus, the kinetic resolution using Chi-AmDH-AlaDH and Rs-AmDH-AlaDH at pH 10.0 and 9.5, respectively. was carried at pH 10.0 and 9.5, respectively.

FigureFigure 3. Effect 3. E offfect pH of on pH the on activity the activity of (A of) AlaDH; (A) AlaDH; (B) Chi-AmDH; (B) Chi-AmDH; and Rs-AmDH. and Rs-AmDH. Reactions Reactions + conditions:conditions: For AmDH For AmDH assay: assay: 10 mM 10 rac- mMa7 rac- (rac-a7a3(rac--fora3 Rs-AmDH-for Rs-AmDH),), 1 mM 1NAD mM+ NAD, 0.2 mg/mL, 0.2 mg AmDH,/mL AmDH, 100 mM100 mMTris/HCl Tris/ HClbuffer bu (pHffer 7.0–9.0), (pH 7.0–9.0), 100 mM 100 Glycin mM Glycinee buffer bu (pHffer 9.0–11.0); (pH 9.0–11.0); For AlaDH For AlaDH assay: assay:10 mM 10 mM pyruvate,pyruvate, 1 mM 1 mMNADH, NADH, 0.2 mg/mL 0.2 mg AlaDH,/mL AlaDH, 100 mM 100 Tris/HCl mM Tris (pH/HCl 7.0–9.0), (pH 7.0–9.0), 100 mM 100 Glycine mM Glycine buffer buffer (pH (pH9.0–11.0). 9.0–11.0). Since AlaDH-catalyzed conversion of pyruvate to alanine helps to drive forward the primary Since AlaDH-catalyzed conversion of pyruvate to alanine helps to drive forward the primary reaction of oxidative deamination, it was necessary to determine the optimal concentration of pyruvate reaction of oxidative deamination, it was necessary to determine the optimal concentration of pyruvateneeded needed to catalyze to catalyze the effi thecient efficient kinetic kinetic resolution resolution of racemic of racemic amines. amines. To evaluate To evaluate the eff ectthe ofeffect pyruvate, α of pyruvate,the kinetic the resolution kinetic resolution of 10 mM of rac-2-aminoheptane 10 mM rac-2-aminoheptane (rac-a3) and (rac- rac-a3) -MBAand rac- (rac-α-MBAa7) was (rac- carrieda7) out + was incarried 100 mM out Glycine in 100 bumMffer Glycine (pH 9.5 buffer/10.0) containing(pH 9.5/10.0) 1 mM containing NAD with1 mM AmDHs NAD+ (1with mg /AmDHsmL) and (1 AlaDH mg/mL)(1 mg and/mL) AlaDH with varying (1 mg/mL) concentration with varying of pyruvate. concentration It was observedof pyruvate. that It increasing was observed concentration that of increasingpyruvate concentration improved the of enantiomericpyruvate improved excess the (ee) enantiomeric of remained (excessS)-a3 and (ee) (ofS)- remaineda7. The ee (valuesS)-a3 and obtained (S)-a7.for The (S)- a3ee valuesand (S )-obtaineda7 with for the (S use)-a3 of and 5 mM (S)-a7 pyruvate with the (2:1 use of of racemic 5 mM pyruvate substrate: (2:1 pyruvate) of racemic were 58 substrate:and 41%, pyruvate) respectively. were 58 The and increase 41%, respectively. in concentration The ofincrease pyruvate in concentration beyond 20 mM of (1:2pyruvate of racemic beyondsubstrate: 20 mM pyruvate) (1:2 of racemic sufficed substrate: to obtain py theruvate) enantiopure sufficed (S )-toa3 obtainand ( Sthe)-a7 enantiopurewith excellent (S)-eea3values, and i.e., (S)-a7>99% with (Figure excellent4). ee values, i.e., >99% (Figure 4).

CatalystsCatalysts 20182019,, 89,, x 600 FOR PEER REVIEW 6 6of of 15 14 Catalysts 2018, 8, x FOR PEER REVIEW 6 of 15

Figure 4. Effect of pyruvate concentration on the kinetic resolution of (A) rac-a3 by Chi-AmDH-AlaDH FigureFigure 4. Effect4. Effect of pyruvateof pyruvate concentration concentration on theon kineticthe kinetic resolution resolution of (A )of rac-(Aa3) rac-by a3 by and Rs-AmDH-AlaDH; and (B) rac-a7 by Chi-AmDH–AlaDH. Reaction conditions: 10 mM rac-a7/rac-a3, Chi-AmDH-AlaDHChi-AmDH-AlaDH and andRs-AmDH-AlaDH; Rs-AmDH-AlaDH; and (andB) rac-(Ba7) rac-bya7 Chi-AmDH--AlaDH. by Chi-AmDH--AlaDH. Reaction Reaction 1 mM NAD+, 1 mg/mL AmDH, 1 mg/mL AlaDH, 5–30 mM Pyruvate, 100 mM Glycine buffer conditions: 10 mM rac-a7/rac-a3, 1 mM NAD+, 1 mg/mL+ AmDH, 1 mg/mL AlaDH, 5-30 mM conditions:(pH 10.0/9.5). 10 mM rac-a7/rac-a3, 1 mM NAD , 1 mg/mL AmDH, 1 mg/mL AlaDH, 5-30 mM Pyruvate,Pyruvate, 100 mM 100 GlycinemM Glycine buffer buffer (pH 10.0/9.5). (pH 10.0/9.5). The successful resolution of 10 mM of rac-a3 and rac-a7 encouraged us to test the applicability of The successful resolution of 10 mM of rac-a3 and rac-a7 encouraged us to test the applicability the presentThe successful coupled resolution enzymatic of system 10 mM for of therac- resolutiona3 and rac- ofa7 higher encouraged concentration us to test of the racemic applicability amines. of the present coupled enzymatic system for the resolution of higher concentration of racemic ofThus, the thepresent kinetic coupled resolution enzymatic of 25 and system 50 mM rac-fora3 theand resolution rac-a7 was of carried higher out concentration in 100 mM Glycine of racemic buffer amines. Thus, the kinetic resolution of 25 and 50 mM rac-a3 and rac-a7 was carried out in 100 mM amines.(pH 9.5 /Thus,10.0) containingthe kinetic 1resolution mM NAD of+ with25 and AmDHs 50 mM (1 rac- mga3/mL) and and rac- AlaDHa7 was (1carried mg/mL) out (Figurein 100 mM5A). Glycine buffer (pH 9.5/10.0) containing 1 mM NAD+ with AmDHs (1 mg/mL) and AlaDH (1 mg/mL) GlycineThe results buffer of (pH this 9.5/10.0) study showed containing that 1 25mM and NAD 50+ mMwith rac-AmDHsa3 could (1 mg/mL) be successfully and AlaDH resolved (1 mg/mL) into (Figure 5A). The results of this study showed that 25 and 50 mM rac-a3 could be successfully (Figureenantiopure 5A). (SThe)-a3 resultsby both of the this combinations, study showed i.e., Chi-AmDH-AlaDHthat 25 and 50 mM and rac- Rs-AmDH-AlaDH.a3 could be successfully As rac-a7 resolved into enantiopure (S)-a3 by both the combinations, i.e. Chi-AmDH-AlaDH and resolvedis not recognized into enantiopure as a substrate ( byS)-a3 Rs-AmDH, by both the the resolution combinations, of 25 and 50i.e. mM Chi-AmDH-AlaDH rac-a7 was studied usingand Rs-AmDH-AlaDH. As rac-a7 is not recognized as a substrate by Rs-AmDH, the resolution of 25 and Rs-AmDH-AlaDH. As rac-a7 is not recognized as a substrate by Rs-AmDH, thea7 resolution of 25 andee 50 mMChi-AmDH-AlaDH rac-a7 was studied system. using The Chi-AmDH-AlaDH reaction profile of system. the kinetic The resolution reaction profile of rac- of showedthe kinetic that the 50 mM rac-a7 was studied using Chi-AmDH-AlaDH system. The reaction profile of the kinetic resolutionvalues of obtained rac-a7 showed for 25 andthat 50the mM ee values rac-a7 obtainedwere 44 for and 25 30%, and respectively50 mM rac-a7 at were 12 h 44 (Figure and 30%,5B). The ee resolution of rac-a7 showed that the ee values obtained for 25 and 50 mM rac-a7 were 44 and 30%, respectivelyvalues marginally at 12 h (Figure improved 5B). withThe ee the values reaction marginally time and improved reached up-to with 54the and reaction 43% after time 24 and h with 25 respectively at 12 h (Figure 5B). The ee values marginally improved with the reaction time and reachedand up-to 50 mM 54rac- anda7 43%, respectively. after 24 h with 25 and 50 mM rac-a7, respectively. reached up-to 54 and 43% after 24 h with 25 and 50 mM rac-a7, respectively.

Figure 5. (A) Kinetic resolution of increased concentration of rac-a3 and (B) Time profile of the kinetic Figure 5. (A) Kinetic resolution of increased concentration of rac-a3 and (B) Time profile of the resolution of rac-a7 (25 and 50 mM) using AmDH-AlaDH purified enzyme system. Reaction conditions: kineticFigure resolution 5. (A )of Kinetic rac-a7 (25resolution and 50 mM)of increased using AmDH-AlaDH concentration purified of rac- enzymea3 and (systemB) Time. Reaction profile of the 25/50 mM rac-a7 (or rac-a3), 1 mM NAD+, 1 mg/mL AmDH, 1 mg/mL AlaDH, 50/100 mM Pyruvate, conditions:kinetic 25/50resolution mM rac of-a7 rac- (ora7 rac (25-a3 and), 1 50mM mM) NAD using+, 1 mg/mL AmDH-AlaDH AmDH, 1 purifiedmg/mL AlaDH, enzyme 50/100 system mM. Reaction 100 mM Glycine buffer (pH 9.5/10.0), 37 ◦C, 200 rpm for 24 h. Pyruvate,conditions: 100 mM 25/50 Glycine mM bufferrac-a7 (pH(or rac 9.5/10.0),-a3), 1 mM37 °C, NAD 200 +rpm, 1 mg/mL for 24 h.AmDH, 1 mg/mL AlaDH, 50/100 mM Pyruvate,The unsuccessful 100 mM Glycine resolution buffer of (pH higher 9.5/10.0), concentration 37 °C, 200 of rpm rac- fora7 24implied h. that the enzymatic reactions The unsuccessful resolution of higher concentration of rac-a7 implied that the enzymatic demonstrated herein could suffer from substrate/product inhibition. It has also been reported that reactionsThe demonstrated unsuccessful herein resolution could sufferof higher from concentration substrate/product of rac- inhibitia7 impliedon. It hasthat also the beenenzymatic reactions demonstrated herein could suffer from substrate/product inhibition. It has also been

CatalystsCatalysts 20192018,, 98,, 600x FOR PEER REVIEW 77 of 1415

reported that product inhibition can limit the applicability of the enzymes even with the use of productfavorable inhibition substrates can [28]. limit For the applicabilityinstance, native of the L-amino enzymes acid even products with the have use of been favorable reported substrates to impose [28]. Forsevere instance, product native inhibitionL-amino upon acid va productsrious amino have acid been dehydrogenases reported to impose [34,35]. severe Nevertheless, product inhibitionstrategies uponsuch as various biphasic amino reaction acid dehydrogenasessystem has been [ 34success,35]. Nevertheless,fully used to overcome strategies suchthe product as biphasic inhibition reaction in systemAmDH-catalyzed has been successfully asymmetric used synthesis to overcome of amines the product [36]. inhibition in AmDH-catalyzed asymmetric synthesisOur ofprevious amines studies [36]. for the kinetic resolution of racemic amines using Chi-AmDH-NADH oxidaseOur system previous also studies reported for thethe kineticstrong resolutioninhibition of racemicthe enzymes amines by usingracemic Chi-AmDH-NADH amine substrates oxidaseand/or corresponding system also reported ketones the [32]. strong We inhibition herein studied of the enzymesthe product by racemic inhibition amine of Rs-AmDH substrates andin the/or correspondingpresence of varying ketones concentration [32]. We herein of 2-heptanone studied the productranging inhibitionfrom 2–30 of mM Rs-AmDH (Figure 6A). in the As presence alanine ofis varyingalso produced concentration in the ofAmDH-AlaDH 2-heptanone ranging catalyzed from reac 2–30tions, mM (Figurethe effect6A). of As varying alanine isconcentration also produced of inalanine the AmDH-AlaDH (10–100 mM) on catalyzed the activity reactions, of Rs-AmDH the effect was of varying evaluated concentration (Figure 6B). of Rs-AmDH alanine (10–100 retained mM) 61 onand the 65% activity of its of Rs-AmDHoriginal activity was evaluated in the presen (Figurece6B). of Rs-AmDH15 mM 2-heptanone retained 61 andand 65% 50 ofmM its originalalanine, activityrespectively. in the presence of 15 mM 2-heptanone and 50 mM alanine, respectively. Also,Also, thethe inhibitioninhibition ofof anotheranother constituentconstituent enzymeenzyme ofof thethe coupledcoupled enzymeenzymereaction, reaction,i.e., i.e. alaninealanine dehydrogenasedehydrogenase by substrate and product was examinedexamined (Figure6 6C).C). TheThe eeffectffect ofof 1010 mMmM racemicracemic amineamine substratessubstrates (rac-(rac-a3 andand rac-rac-a7a7),), thethe correspondingcorresponding ketonesketones (2-heptanone(2-heptanone andand acetophenone)acetophenone) waswas studiedstudied onon thethe activityactivity ofof AlaDH.AlaDH. TheThe lossloss ofof AlaDHAlaDH activityactivity inin presence ofof ketonesketones 2-heptanone2-heptanone andand acetophenoneacetophenone was was higher higher than than that th inat the in presencethe presence of amines of amines rac-a3 rac-anda3 rac- anda7. rac- Thea7 relative. The relative activity ofactivity AlaDH of was AlaDH 63 and was 84% 63 inand the 84% presence in the ofpresence 10 mM rac-of 10a3 mMand rac- rac-a3a7 ,and respectively. rac-a7, respectively. On the other On hand, the theother relative hand, activities the relative of AlaDHactivities in of the AlaDH presence in th ofe 10 presence mM 2-heptanone of 10 mM and2-heptanone acetophenone and acetophenone were only 44 andwere 46%, only respectively. 44 and 46%, respectively.

Figure 6. Inhibition of Rs-AmDH by (A) 2-heptanone; Reaction conditions- 10 mM rac-a3, 0–30 mM 2-heptanone,Figure 6. Inhibition 1 mM NAD of Rs-AmDH+, 0.2 mg/mL by Rs-AmDHs,(A) 2-heptanone; Buffer- Reaction Glycine conditions- buffer (100 mM, 10 mM pH 9.5);rac-a3 (B, )0-30 Alanine; mM Reaction2-heptanone, conditions- 1 mM 10NAD mM+, rac-0.2 a3mg/mL, 0–100 Rs-AmDHs, mM alanine, Buffer- 1 mM NAD Glycine+, 0.2 buffer mg/mL (100 Rs-AmDHs, mM, pH 1009.5); mM (B) GlycineAlanine; buReactionffer (pH conditions- 9.5) and (10C) mM Inhibition rac-a3, 0-100 of AlaDH mM alanine, by amines 1 mM rac- NADa3 and+, 0.2rac- mg/mLa7; and Rs-AmDHs, ketones 2-heptanone100 mM Glycine and acetophenonebuffer (pH 9.5) (10 and mM (C each),) Inhibition Control-activity of AlaDH of by AlaDH amines in rac- thea3 absence and rac- ofa7 amines; and andketones ketones. 2-heptanone and acetophenone (10 mM each), Control- activity of AlaDH in the absence of amines and ketones.

Catalysts 2019, 9, 600 8 of 14

2.3. Kinetic Resolution of Racemic Amines Using E. coli Whole Cells Co-Expressing AmDH and AlaDH As enzymes are intracellularly produced, keeping them in their natural environment creates apparent advantages for biocatalytic reactions [37]. The mechanical disruption of microbial cells and subsequent downstream processing for the purification of desired protein in a homogeneous form is very laborious and costly [38–42]. It has been reported that use of purified enzyme system for biocatalytic purposes costs approximately ten times more than that by whole-cell biotransformations [43,44]. Although the use of purified enzymes is limited for their application on large scales, another form of biocatalysts, i.e., cell-free extracts, is feasible if expression levels of the target protein are sufficient [45,46]. However, cell free extracts are unstable. Thus, whole cells expressing the enzyme of interest for the biosynthetic applications are advantageous [47]. Besides the cost effective and simpler handling of whole-cell biotransformations, an added advantage includes the utility of the enzyme production machinery of the fully functional living organism [48,49]. Also, the implementation of biocatalytic syntheses using whole-cell biotransformations is advantageous regarding the regeneration of the redox cofactors [50]. Moreover, whole-cell biocatalysts show less susceptibility to substrate and/or product inhibition due to the diffusional barrier of the cell membrane [51]. The industrial applicability of the biocatalytic processes can be greatly improved with the use of immobilized enzyme systems owing to the improved operational stability and recyclability of the enzymes [52,53]. Moreover, recent years have seen the development of systems comprising of coimmobilized enzymes and cofactors to increase the operational life-span, thereby increasing the cost effectiveness of the processes [54,55]. For example, the group of Lopez-Gallego developed a novel system for the co-immobilization of enzymes and the requisite phosphorylated cofactors on various porous matrices to obtain a self-sufficient heterogenous enzymatic system which is independent of the exogenous addition of the cofactors [55]. The unsuccessful resolution of rac-a7 using the purified enzyme system implied that the coupled-enzyme reactions could suffer from the substrate/product inhibition. Therefore, to catalyze the efficient kinetic resolution of racemic amines, recombinant E. coli cells expressing AmDHs and AlaDH were used as whole-cell biocatalysts. Whole cell biotransformation systems constituting two or more cells, each expressing different protein, may impose the mass transfer limitations [56,57]. In order to develop a single-cell system expressing AmDH and AlaDH, the genes encoding Chi-AmDH-AlaDH and AlaDH were cloned into pET-24ma and pET-Duet vectors, respectively [Since the substrate scope of Chi-AmDH-AlaDH is much better than Rs-AmDH, we only used Chi-AmDH-AlaDH whole cell system]. The plasmids were co-transformed into E. coli cells to generate Chi-AmDH-AlaDH system. Following the induction of transformants by IPTG, cells were grown overnight at 20 ◦C. The cells were centrifuged (4000 g, 20 min) and washed twice with 50 mM Tris/HCl buffer (pH 7.0). Chi-AmDH-AlaDH cells × were resuspended in 100 mM Glycine buffer pH 10.0 and used for whole-cell biotransformations. To commence the whole-cell biotransformations, kinetic resolutions of 10 mM rac-a3 and rac-a7 were carried out using 10 mgDCW/mL cells. Since cofactor regeneration is efficient in whole cells, these biotransformations were carried out without any external addition of NAD+. Both the racemic substrates were efficiently resolved into corresponding (S)-amines with excellent ee of >99% [Data not shown]. Since our previous studies reported that the kinetic resolution of rac-a7 is more challenging [32], we attempted the kinetic resolution of higher concentrations (20, 30 and 50 mM) of rac-a7. The results of these whole-cell biotransformations revealed that the rac-a7, even at the higher concentrations, can be resolved into enantiopure (S)-a7. While the ee values for the resolution of 25 and 50 mM rac-a7 using the purified enzymatic system were 54 and 43%, respectively at 24 h (Figure5B), ee values obtained using 30 mgDCW/mL whole cells of Chi-AmDH-AlaDH were >99% after 12 h for the resolution of 20, 30 and 50 mM rac-a7, implying that whole-cell biotransformations were highly efficient than purified enzymatic systems (Figure7). Also, the whole cell biotransformation could efficiently resolve 50 mM rac-a3 with excellent ee values (>99%; data not shown). These results were in a good agreement with previous studies by Pushpanath et al. [28], who reported that the lyophilized Catalysts 2019, 9, 600 9 of 14

Catalysts 2018, 8, x FOR PEER REVIEW 9 of 15 whole-cells yielded better conversion values than those by lyophilized lysate for the asymmetric lyophilized whole-cells yielded better conversion values than those by lyophilized lysate for the synthesis of amines from prochiral ketones. asymmetric synthesis of amines from prochiral ketones. 120

100

80 20 mM 30 mM 50 mM

(%) 60 S ee

40

20

0 0 5 10 15 20 25 Time (h) Figure 7. Reaction profiles for the kinetic resolution of 20, 30 and 50 mM rac-a7 using Chi-AmDH-AlaDH Figure 7. Reaction profiles for the kinetic resolution of 20, 30 and 50 mM rac-a7 using whole cell system; Reaction conditions: (20/30 or 50 mM rac-a7, 40/60 or 100 mM pyruvate), 100 mM Chi-AmDH-AlaDH whole cell system; Reaction conditions: (20/30 or 50 mM rac-a7, 40/60 or 100 glycine buffer pH 10.0, 30 mgDCW/mL Chi-AmDH-AlaDH cells, 37 ◦C, 200 rpm for 24 h). mM pyruvate), 100 mM glycine buffer pH 10.0, 30 mgDCW/mL Chi-AmDH-AlaDH cells, 37 °C, 200 rpmThe for successful 24 h). resolution of 50 mM rac-a3 and rac-a7 encouraged us to further evaluate the efficiency of this reaction cascade for the resolution of higher concentration of racemic amines. Thus,The to determinesuccessful resolution the optimal of amount50 mM ofrac- wholea3 and cell rac- biocatalysta7 encouraged required us to to further obtain evaluate enantiopure the efficiency(S)-amines of from this reaction 100 mM cascade rac-a3 forand the rac- resolutia7 substrates,on of higher reactions concentration were carried of racemic out usingamines. varying Thus, to determine the optimal amount of whole cell biocatalyst required to obtain enantiopure (S)-amines concentration of whole cell catalyst ranging from 10–60 mgDCW/mL (Figure8). The ee of (S)-amines from 100 mM rac-a3 and rac-a7 substrates, reactions were carried out using varying concentration of increased with the increasing amount of Chi-AmDH-AlaDH whole-cell catalyst. While 30 mgDCW/mL wholecells su cellfficed catalyst to completely ranging from resolve 10-60 100 mg mMDCW rac-/mLa3 (Figureinto enantiopure 8). The ee of (S)- (Sa3)-amines, the minimum increased number with the of increasing amount of Chi-AmDH-AlaDH whole-cell catalyst. While 30 mgDCW/mL cells sufficed to cells required to resolve 100 mM rac-a7 were 40 mgDCW/mL. These results corroborated the results of completelythe previous resolve studies 100 which mM exhibited rac-a3 into the enantiopure higher inhibition (S)-a3 of, the Chi-AmDH minimum by number acetophenone of cells thanrequired that byto resolve2-heptanone 100 mM [32]. rac- Thea7 higher were e 40fficiency mgDCW of/mL. the coupledThese results Chi-AmDH-AlaDH corroborated systemthe results demonstrated of the previous herein studiesis worth which emphasizing, exhibited given the thathigher the minimuminhibition numberof Chi-AmDH of previously by acetophenone reported Chi-AmDH-NOX than that by 2-heptanone [32]. The higher efficiency of the coupled Chi-AmDH-AlaDH system demonstrated cells required to completely resolve only 50 mM rac-a3 and rac-a7 were 60 and 100 mg DCW/mL [32]. herein is worth emphasizing, given that the minimum number of previously reported On the other hand, only 30 and 40 mg DCW/mL Chi-AmDH-AlaDH cells sufficed to completely resolve Chi-AmDH-NOX100 mM of rac-a3 andcells rac- requireda7, respectively to completely (Figure resolve8). only 50 mM rac-a3 and rac-a7 were 60 and 100 mg DCWIn/mL order [32]. to investigate On the other the hand, applicability only 30 of and the coupled-enzyme40 mg DCW/mL Chi-AmDH-AlaDH system designed herein, cells sufficed the kinetic to completelyresolution ofresolve various 100 racemic mM of aminerac-a3 and substrates rac-a7, torespectively their corresponding (Figure 8). enantiopure (S)-amines was performed [Table1]. All the racemic substrates, except a1 and a2, were successfully resolved into their corresponding (S)-amine form with excellent ee of >99% (Figures S3–S11). The kinetic resolution of a1 and a2 resulted in ee values of 19 and 59%, respectively. Although substrate specificity suggested the varying activity of AmDH toward various racemic amine substrates, a clear correlation between conversion and the reactivity of substrates could not be demonstrated. Nevertheless, it can be stated that AlaDH-catalyzed conversion of pyruvate to alanine efficiently shifted the equilibrium of the oxidative deamination in such a fashion that the kinetic resolution of racemic substrates was catalyzed independent of their activity toward AmDH.

CatalystsCatalysts2019 2018, 9,, 600 8, x FOR PEER REVIEW 1010 of 14of 15

120

a3 100 a7

80 (%)

S 60 ee

40

20

0 10 20 30 40 60 Whole-cell catalyst (mgDCW/mL) Figure 8. Effect of concentration of whole-cell catalyst on the kinetic resolution of 100 mM rac-a3 Figure 8. Effect of concentration of whole-cell catalyst on the kinetic resolution of 100 mM rac-a3 and and rac-a7. Reaction conditions: (100 mM rac-a3 or rac-a7, 200 mM Pyruvate), 100 mM glycine buffer rac-a7. Reaction conditions: (100 mM rac-a3 or rac-a7, 200 mM Pyruvate), 100 mM glycine buffer pH pH 10.0, 10–60 mgDCW/mL Chi-AmDH-AlaDH cells, 37 ◦C, 200 rpm for 24 h). 10.0, 10-60 mgDCW/mL Chi-AmDH-AlaDH cells, 37°C, 200 rpm for 24 h). Table 1. Kinetic resolution of various racemic amines. In order to investigate the applicability of the coupled-enzyme system designed herein, the Substrate Conv. (%) [a] eeS (%) kinetic resolution of various racemic amine substrates to their corresponding enantiopure (S)-amines was performed [Tablea1 1].[b] All the racemic17 substrates, 18.5 except a1 and a2, were successfully [b] resolved into their correspondinga2 (S)-amine form37 with excellent 59 ee of >99% (Figure S3–Figure S11). a4 [b] 51 >99 The kinetic resolution of a1 and a2 resulted in ee values of 19 and 59%, respectively. Although a5 [b] 51 >99 substrate specificity suggested a6the[b] varying activity51 of AmDH>99 toward various racemic amine substrates, a clear correlation betweena8 [c] conversion51 and the> 99reactivity of substrates could not be demonstrated. Nevertheless, it a9can[c] be stated 51that AlaDH-catalyzed>99 conversion of pyruvate to [c] alanine efficiently shifted the equia10librium of the52 oxidative >deamination99 in such a fashion that the [c] kinetic resolution of racemic substratesa11 was cata51lyzed independent>99 of their activity toward AmDH. Reaction conditions: 20 mM rac-a1–a11, 40 mM pyruvate, 100 mM glycine buffer pH 10.0, 30 mgDCW/mL Chi-AmDH-AlaDH cells, 37 ◦C, 200 rpm for 24 h; a1–a6 were analyzed by C18 symmetry column after derivatization with GITC; a8–a11 were analyzedTable by 1. Crownpack Kinetic resolution CR (+) column. of various [a] Conversion racemic amines was defined. as percentage ratio of the difference between initial racemic amine substrate and final enantiopure amine to initial racemic amine [a] S [Initial racemic amine substrateSubstratef inal enantiopure Conv. amine product (%)] ee (%) substrate; Conv. = − 100. [b] Conversions and ee were determined [Initial racemica1 amine [b] substrate ] 17 × 18.5 by C18 symmetry column after derivatization with GITC. [c] Conversions and ee were determined by Crownpack [b] CR (+) column. a2 37 59 a4 [b] 51 >99 3. Materials and Methods a5 [b] 51 >99 a6 [b] 51 >99 3.1. Chemicals and Media a8 [c] 51 >99 All the racemic amine substratesa9 [c] (rac- a1–a1151), derivatizing>99 agent 2,3,4,6-Tetra-O-acetyl-β -d-glucopyranosyl isothiocynate (GITC),a10 nicotinamide [c] 52 cofactors (NADH>99 and NAD+) were purchased from Sigma-Aldrich (Yongin, Korea). Alla11 the [c] other chemical51 and reagents>99 used were of analytical grade. Reaction conditions: 20 mM rac-a1–a11, 40 mM pyruvate, 100 mM glycine buffer pH 10.0, 30 3.2. EnzymemgDCW Expression/mL Chi-AmDH-AlaDH and Purification cells, 37 °C, 200 rpm for 24 h; a1–a6 were analyzed by C18 symmetry Thecolumn gene encodingafter derivatization Chi-AmDH with [25 GITC;] and Rs-AmDHa8–a11 were [26 analyzed] was synthesized by Crownpack by Bionics CR (+) (Seoul, column. Korea). [a] Conversion was defined as percentage ratio of the difference between initial racemic amine substrate The genes were cloned into IPTG-inducible pET-24ma vector, expressed and purified as previously and final enantiopure amine to initial racemic amine substrate; 𝐶𝑜𝑛𝑣. = reported[ [33, 51]. ] 𝑥 100 . [b] Conversions and ee were [ ]

Catalysts 2019, 9, 600 11 of 14

3.3. Co-Expression of Chi-AmDH and AlaDH The gene encoding AmDH was cloned into pET24ma vector and AlaDH in pET-duet vector. Both the plasmids were transformed by heat shock method to E. coli BL21 cells. The cells were grown in the presence of Ampicillin (100 µg/mL) and kanamycin (50 µg/mL). Once OD600 of the cells reached 0.6–0.8, IPTG was added (0.1 mM final concentration) and cells were cultivated overnight. The cells were centrifuged and washed with tris buffer (20 mM, pH 7.0). Cells were centrifuged again, resuspended in Glycine buffer (100 mM, pH 10.0) and used for whole-cell transformations.

3.4. Representative Procedure for Whole-Cell Biotransformations for the Kinetic Resolution of Racemic Amines E. coli cells co-transformed with plasmids pET-24ma and pET-duet harboring Chi-AmDH and AlaDH, respectively were cultivated overnight following their induction by IPTG. The induced cells were centrifuged (4000 g, 20 min, 4 C). The cell pellet was washed twice with tris buffer (20 mM, × ◦ pH 7.0) and recollected by centrifugation. The washed were resuspended in Glycine buffer (100 mM, pH 10.0) and used for whole-cell biotransformations. To assess the kinetic resolution of racemic amine substrates, reaction was carried out containing 10 mM substrate (rac-a1–a11; stock in DMSO), 20 mM pyruvate, 30 mgDCW/mL E. coli cells co-expressing Chi-AmDH and AlaDH and Glycine buffer (100 mM, pH 10.0) in a final volume of 1 mL in 1.5 mL microcentrifuge tubes. This reaction mixture was incubated at 37 ◦C and 200 rpm for 24 h. Biotransformation were initiated. After 24 h, the reaction mixture was centrifuged (10,000 g, 20 min) to remove the cellmass and the supernatant was quantified by HPLC × for the remaining (S)-amines.

3.5. Analysis of Amines The analysis of kinetic resolution of racemic amines to their enantiopure (S)-amines was performed as previously reported [32,33].

4. Conclusions Since their generation by protein engineering strategies from L-amino acid dehydrogenase parent scaffolds, amine dehydrogenases have been used for the reductive amination of prochiral ketone to chiral amines. Nevertheless, (R)-AmDH-catalyzed oxidative deamination can also be efficiently used for the kinetic resolution of racemic amines to enantiopure (S)-amines. Kinetic resolution was performed using a coupled-enzyme reaction cascade consisting of AmDH and AlaDH. The use of AlaDH was advantageous not only for cofactor regeneration, but also for the effective conversion of pyruvate to alanine, thereby shifting the reaction equilibrium of the primary reaction toward product formation. Another advantage of this reaction protocol is that the optimum pHs for both the enzymes are close to each other, implying that reactions using coupled-enzyme systems can be efficiently performed without compromising the activities of the constituent enzymes. Although a purified enzyme system could not resolve higher concentrations of challenging racemic substrate such as a7, the whole-cell biotransformations efficiently resolved racemic amines substrates. By using the whole-cell system expressing AmDH and AlaDH, 100 mM each of rac-2-aminoheptane and rac-α-MBA were successfully resolved into (S)-form with >99% ee, respectively. Furthermore, this reaction protocol enabled the kinetic resolution of wide range of chiral amines to their corresponding (S)-amines with excellent enantioselectivity (ee > 99%). One of the major disadvantages with the use of pyruvate is its cost, which ultimately affects the overall cost of the biocatalytic process. Nevertheless, the recent discovery of enantiocomplementary (S)-AmDHs [31] suggests that the applicability of the protocols such as that demonstrated herein could also be efficiently extended to generate enantiopure (R)-amines.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4344/9/7/600/s1, Figure S1: SDS-PAGE analysis of purified AmDHs and AlaDH, Figure S2: SDS-PAGE analysis of whole-cells expressing AmDHs and AlaDH, Figures S3–S13: HPLC analyses of kinetic resolution of rac-a1–rac-a11 (red line) to (S)-a1–(S)-a11 (black line), Table S1: Amine retention times. Catalysts 2019, 9, 600 12 of 14

Author Contributions: H.Y. and M.D.P. designed the experiments of the project; M.D.P., S.Y., T.P.K. and S.S. performed the experiments, H.J., A.D.P. and Y.W. contributed reagents/materials/analysis tools. M.D.P. drafted this manuscript; H.Y. proofread the manuscript and supervised the studies. Funding: This work was supported by the Ministry of Trade, Industry and Energy of South Korea (MOTIE, Korea) under the Industrial Technology Innovation Program (No. 10062550 and No. 10076343). Conflicts of Interest: The authors declare no financial or commercial conflict of interest.

References

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