The Kinetic Resolution of Racemic Amines Using a Whole-Cell Biocatalyst Co-Expressing Amine Dehydrogenase and NADH Oxidase

catalysts

Article The Kinetic Resolution of Racemic Amines Using a Whole-Cell Biocatalyst Co-Expressing Amine Dehydrogenase and NADH Oxidase

Hyunwoo Jeon 1,†, Sanghan Yoon 1,†, Md Murshidul Ahsan 2, Sihyong Sung 1, Geon-Hee Kim 1, Uthayasuriya Sundaramoorthy 1, Seung-Keun Rhee 2 and Hyungdon Yun 1,*

1 Department of Systems Biotechnology, Konkuk University, Seoul 05029, Korea; [email protected] (H.J.); [email protected] (S.Y.); [email protected] (S.S.); [email protected] (G.-H.K.); [email protected] (U.S.) 2 School of Biotechnology, Yeungnam University, Kyongsan 38541, Korea; [email protected] (M.M.A.); [email protected] (S.-K.R.) * Correspondence: [email protected]; Tel.: +82-2-450-0496; Fax: +82-2-450-0686 † Hyunwoo Jeon and Sanghan Yoon have contributed equally to this work.

Received: 9 August 2017; Accepted: 23 August 2017; Published: 25 August 2017

Abstract: Amine dehydrogenase (AmDH) possesses tremendous potential for the synthesis of chiral amines because AmDH catalyzes the asymmetric reductive amination of ketone with high enatioselectivity. Although a reductive application of AmDH is favored in practice, the oxidative route is interesting as well for the preparation of chiral amines. Here, the kinetic resolution of racemic amines using AmDH was ﬁrst extensively studied, and the AmDH reaction was combined with an NADH oxidase (Nox) to regenerate NAD+ and to drive the reaction forward. When the kinetic resolution was carried out with 10 mM rac-2-aminoheptane and 5 mM rac-α-methylbenzylamine (α-MBA) using puriﬁed enzymes, the enantiomeric excess (ee) values were less than 26% due to the product inhibition of AmDH by ketone and the inhibition of Nox by the substrate amine. The use of a whole-cell biocatalyst co-expressing AmDH and Nox apparently reduces the substrate and product inhibition, and/or it increases the stability of the enzymes. Fifty millimoles (50 mM) rac-2-aminoheptane and 20 mM rac-α-MBA were successfully resolved into the (S)-form with >99% ee using whole cells. The present study demonstrates the potential of a whole-cell biocatalyst co-expressing AmDH and Nox for the kinetic resolution of racemic amines.

Keywords: amine dehydrogenase; biocatalysts; chiral amine; kinetic resolution; oxidative deamination

1. Introduction Recently, considerable efforts have been made to economically obtain enantiomerically pure intermediates for drugs and agrochemicals. Among them, the chiral amines are of great interest to the pharmaceutical and fine-chemical industries, since enantiopure amines are important building blocks for pharmaceutical manufacturing [1]. Given the significance of chiral amines, their efficient synthesis in enantiopure form has become an attractive challenge to organic chemists and biologists in recent years [2–4]. Recently, many biocatalytic methods for the production of chiral amines have been developed using biocatalysts, such as lipase [5], amine oxidase [6], imine reductase [7], transaminase [8], ammonia lyases [9], Pictet-Spenglerase [10], barberine bridge enzyme [11], engineered P450 monooxygenase [12], and amine dehydrogenase (AmDH) [3]. Each enzyme has its own advantages and disadvantages for the synthesis of chiral amines ([13] and references therein for more details).

Catalysts 2017, 7, 251; doi:10.3390/catal7090251 www.mdpi.com/journal/catalysts Catalysts 2017, 7, 251 2 of 13

Among them, as AmDH catalyzes the asymmetric reductive amination of ketones, and uses cheap ammonia as the reagent and generates only water as by product, with high atom efficiency and enantioselectivity [14], AmDH is highly attractive and possesses tremendous potential for the synthesis of chiral amines [13]. Until now, only one NAD+-dependent AmDH found in nature has been reported, in Streptomyces virginiae, but it showed poor enantioselectivity and its DNA information is not available [15]. Recently, AmDHs have been created through protein engineering using the existing L-amino acid dehydrogenase. First, AmDH was engineered by the Bommarius group using leucine dehydrogenase (LeuDH) from Bacillus stearothomophilus to accept ketone substrates through the introduction of 2–4 point mutations [16]. Subsequently, a second amine dehydrogenase was developed based on the scaffold of phenylalanine dehydrogenase (PheDH) from Bacillus badius by the same group using a similar approach [17]. Further, a chimeric enzyme was generated by combining the N-terminal section (residues 1–149) of the second AmDH (PheDH variant) with residues 140–366 of the first AmDH (LeuDH variant), and the chimeric enzyme displayed good activity towards various amines, such as acetophenone and adamantyl methyl ketone, and converted them to (R)-amine products with excellent enantioselectivity [18]. Since then, different AmDHs have been developed using L-amino acid dehydrogenases from Rhodococcus sp. M4, Exiguobacterium sibiricum, and Caldalkalibacillus thermarum [14,19,20]. A systematic investigation of substrate acceptance, optimal reaction conditions, and the chemo- and stereoselectivity of AmDHs has been carried out [13,20]. In addition, AmDHs have been used in elegant hydrogen borrowing dual-enzyme cascade reactions for the synthesis of chiral amines from alcohols with “closed-loop” recycling of the cofactor [19,21]. Due to the importance of the reductive amination of ketones by AmDH, an extensive and detailed review on relevant studies has recently been published [3]. Although a reductive application of dehydrogenases is favored in practice, the oxidative route is interesting as well for the preparation of asymmetric compounds from prochiral substrates, such as D-amino acids with L-amino acid dehydrogenases, (S)-alcohols with (R)-specific alcohol dehydrogenases, or the conversion of hydroxy acids, amino acids, and alcohols into the corresponding keto compounds [22]. Until now, all reported AmDHs exhibit (R)-enantioselectivity because they were generated from an L-amino acid dehydrogenase scaffold. Therefore, (R)-amines are produced through reductive amination by AmDHs from ketones (for certain compounds, (S)-forms were generated due to the Cahn–Ingold–Prelog priority rules for naming chiral centers). Since (S)-specific AmDH is not available, (S)-amines cannot be synthesized via reductive amination. Although (S)-amines can be obtained through the kinetic resolution of racemic amines using oxidative deamination by (R)-specific AmDH, the kinetic resolution of amines using AmDH has not been extensively studied until recently. We describe herein the production of (S)-amines through the oxidative deamination of racemic amines by AmDH with help of an NADH oxidase (Nox) (Figure1). One of the main advantages of using Nox for NAD+ coenzyme regeneration is that it catalyzes an irreversible reaction, which then shifts the equilibrium of the primary reaction to the side of the product formation. Nox is a flavoprotein which oxidizes NADH to NAD+ via a coupled reaction of molecular oxygen to either water in a four-electron reduction or to hydrogen peroxide in a two-electron reduction [22]. Due to the toxicity of hydrogen peroxide [23], water-forming Nox from Lactobacillus brevis was used in this study [24]. Among the reported AmDHs, the chimeric enzyme [18] generated by the domain shuffling of the LeuDH and PheDH variants was used in this study. Catalysts 2017,, 7,, 251 3 of 13 Catalysts 2017, 7, 251 3 of 13

NH2 NH2 O NH2 AmDH NH2 O AmDH + + + NH4+ R1 R2 R1 R2 R1 R2 NH4 R1 R2 R1 R2 R1 R2

+ NAD+ NADH

1 H2O 1/2 O2 H2O NOX /2 O2 NOX Figure 1. R Figure 1. The kinetic resolution of amine by (R))-speciﬁc‐specific AmDH in combination with NADH oxidase (Nox). (R)-amine is oxidized into the corresponding ketone by AmDH utilizing NAD+. Additionally, (Nox). (R)‐amine is oxidized into the corresponding ketone by AmDH utilizing NAD+. Additionally, NAD+ is regenerated by Nox, and enantiomerically pure (S)-amine is obtained. NAD+ is regenerated by Nox, and enantiomerically pure (S)‐amine is obtained.

2. Results and Discussion

2.1. Substrate Specificity and Kinetic Parameters of AmDH 2.1. Substrate Specificity and Kinetic Parameters of AmDH The AmDH gene was cloned into a pET24ma vector with a C-terminal His6-tag, effectively The AmDH gene was cloned into a pET24ma vector with a C‐terminal His6‐tag, effectively expressed in the Escherichia coli BL21 cell (DE3), and subsequently purified on a Ni-NTA affinity column. expressed in the Escherichia coli BL21 cell (DE3), and subsequently purified on a Ni‐NTA affinity As mentioned above, the main interest of this study is the oxidative deamination of amines by AmDH. column. As mentioned above, the main interest of this study is the oxidative deamination of amines The oxidative deamination activity of AmDH towards various amines was examined in order to explore by AmDH. The oxidative deamination activity of AmDH towards various amines was examined in the applicability of the enzyme to the kinetic resolution of various amines (Figure2). The enzyme order to explore the applicability of the enzyme to the kinetic resolution of various amines (Figure 2). showed considerable activity towards the tested amines. The enzyme showed good activity towards The enzyme showed considerable activity towards the tested amines. The enzyme showed good (R)-α-methylbenzylamine (α-MBA, a2) and (S)-phenylalaninol (a9), but gave negligible activity for activity towards (R)‐α‐methylbenzylamine (α‐MBA, a2) and (S)‐phenylalaninol (a9), but gave (S)-α-MBA (a3) and (R)-phenylalaninol (a10). It suggested that the enantioselectivity of the enzyme is negligible activity for (S)‐α‐MBA (a3) and (R)‐phenylalaninol (a10). It suggested that the high, and the result is consistent with that reported previously [18]. The enzyme’s reactivity towards a enantioselectivity of the enzyme is high, and the result is consistent with that reported previously broad range of substrates and high enantioselectivity indicate the applicability of the enzyme in the [18]. The enzyme’s reactivity towards a broad range of substrates and high enantioselectivity indicate rac α a1 rac a12 productionthe applicability of chiral of the amines. enzyme After in the that, production- -MBA of ( chiral) and amines.-2-aminoheptane After that, rac (‐α‐MBA) were (a1 selected) and rac as‐ representative2‐aminoheptane aromatic (a12) were and aliphaticselected aminesas representative for further aromatic study. To and determine aliphatic the amines kinetic parametersfor further a1 ofstudy. the To enzyme, determine the initialthe kinetic rate parameters of the enzyme of the was enzyme, analyzed the initial with rate various of the concentrations enzyme was analyzed of or a12 (0–25 mM) in the presence of 1 mM NAD+ (Figure3 and Figure S2). The kinetic+ parameters with various concentrations of a1 or a12 (0–25 mM) in the presence of 1 mM NAD+ (Figures 3 and S2). wereThe kinetic determined parameters based were on the determined Lineweaver–Burk based on plot. the Lineweaver–Burk The Km and kcat values plot. The of AmDH Km and for kcata1 valueswere The kinetic parameters were determined based on the Lineweaver–Burk plot. The Km and kcat values −1 3.99of AmDH (±0.39) for mM a1 were and 3.99 0.96 (±0.39) (±0.043) mM min and 0.96, respectively. (±0.043) min The−1, respectively. Km and kcat Thevalues Km foranda12 kcat valueswere 8.17 for of AmDH for a1 were 3.99 (±0.39) mM and 0.96 (±0.043) min−1, respectively. The Km and kcat values for (±0.25) mM and 11.88 (±0.53) min−1, respectively.−1 a12 were 8.17 (±0.25) mM and 11.88 (±0.53) min−1, respectively.

NH2 NH2 NH2 NH2 NH2 NH2 NH2 NH2

a1 a2 a3 F a4 a1 a2 a3 F a4

NH NH2 NH2 NH2 NH2 NH2 2 NH2 NH2 F F

F a5 a6 a7 a8 F a5 a6 a7 a8 F F F F NH2 NH2 NH2 NH2 NH2 NH2 NH2 NH2 OH OH OH OH a11 a12 a9 a10 a11 a12 a9 a10 (a)

Figure 2. Cont.

CatalystsCatalysts2017 2017, 7,, 251 7, 251 4 of4 13of 13 Catalysts 2017, 7, 251 4 of 13

300300

275275

250250

120120 100100 80

Relative activity (%) Relative activity 80 Relative activity (%) Relative activity 6060 4040 2020 0 0 a1a1 a2 a2 a3 a3 a4 a4 a5 a5 a6 a6 a7 a7 a8 a8 a9 a9 a10 a10 a11 a11 a12 a12 Substrate Substrate (b()b) Figure 2. (a) Substrate spectrum; (b) Substrate specificity of AmDH. Oxidative deamination was FigureFigure 2. 2.( a()a Substrate) Substrate spectrum; spectrum; (b) ( Substrateb) Substrate speciﬁcity specificity of AmDH. of AmDH. Oxidative Oxidative deamination deamination was carried was carriedcarried out out in in 100 100 mM mM glycine glycine buffer buffer (pH (pH 10.0) 10.0) containing containing AmDH AmDH (0.15 (0.15 mg/mL), mg/mL), 5 mM5 mM substrate, substrate, and and out in 100 mM+ glycine buffer (pH 10.0) containing AmDH (0.15 mg/mL), 5 mM substrate, and 1 mM 1 mM NAD+ at 25 °C. One hundred percent (100%) corresponds to 44 mU/mg. NAD1 mM+ NADat 25 ◦ C.at 25 One °C. hundred One hundred percent percent (100%) (100%) corresponds corresponds to 44 mU/mg. to 44 mU/mg.

0.060.06 M/min) M/min)   0.040.04 2-aminoheptane2-aminoheptane (a12 (a12) ) -MBA-MBA (a1 (a1) )

0.020.02 Reaction rate ( Reaction rate (

0.000.00 00 5 5 10 10 15 15 20 20 25 25 Concentration (mM) Concentration (mM) Figure 3. Michaelis–Menten plot of the reaction rate depending on the substrate concentration. FigureFigure 3. 3.Michaelis–Menten Michaelis–Menten plot plot of of the the reaction reaction rate rate depending depending on on the the substrate substrate concentration. concentration. The The Thereactions reactions were were carried carried out out in in 100 100 mM mM Tris/HCl Tris/HCl (pH (pH 8.5) 8.5) containing containing substrate substrate (0–30 (0–30 mM), mM), AmDH AmDH (0.3 reactions were carried out in 100 mM Tris/HCl (pH 8.5) containing◦ substrate (0–30 mM), AmDH (0.3 (0.3 mg/mL for a12, 0.45 mg/mL for a1), and 1 mM NAD+ + at 25 C. mg/mLmg/mL for for a12 a12, 0.45, 0.45 mg/mL mg/mL for for a1 a1), and), and 1 mM1 mM NAD NAD+ at at25 25 °C. °C.

2.2.2.2.2.2. KineticKinetic Kinetic ResolutionResolution Resolution ofof of AminesAmines Amines UsingUsing Using aa Puriﬁed Purifieda Purified AmDH/Nox AmDH/Nox AmDH/Nox Coupling Coupling Coupling System System System + + InInIn thethe the kinetic kinetic kinetic resolution resolution resolution of amines of of amines amines by AmDH, by by AmDH, theAmDH, regeneration the the regeneration regeneration of cofactor of NADof cofactor cofactoris of NAD importance, NAD+ is isof of + + asimportance,importance, the reaction as as requiresthe the reaction reaction a stoichiometric requires requires a astoichiometric amountstoichiometric of NAD amount amount(Figure of of NAD1 ).NAD In+ addition(Figure (Figure 1). to1). In reducing In addition addition the to to + + costreducingreducing of the the enzymaticthe cost cost of of the the reaction, enzymatic enzymatic NAD reaction, reaction,cofactor NAD NAD regeneration+ cofactor cofactor regeneration regeneration simpliﬁes productsimplifies simplifies isolation, product product preventsisolation, isolation, + problemspreventsprevents ofproblems productproblems inhibitionof of product product by inhibition the inhibition cofactor, by andby the the can cofactor, favorablycofactor, and and drive can can the favorably reactionfavorably forward.drive drive the the For reaction NADreaction + forward.forward. For For NAD NAD+ regeneration, regeneration, Nox Nox from from Lactobacillus Lactobacillus brevis brevis was was used used [24]. [24]. The The reduced reduced byproduct byproduct of Nox is H2O, and this reaction is considered irreversible, which means that it is the driving force to of Nox is H2O, and this reaction is considered irreversible, which means that it is the driving force to

Catalysts 2017, 7, 251 5 of 13 Catalysts 2017, 7, 251 5 of 13 regeneration,move the reaction Nox from forwardLactobacillus in a coupled brevis AmDH/Noxwas used [24 ].reaction The reduced [25]. In byproduct the coupled of Noxenzyme is H 2reaction,O, and thisespecially reaction when is considered the optimum irreversible, pH of the which two meansenzymes that is it different, is the driving the pH force of the to move reaction the should reaction be forwardcarefully in determined a coupled AmDH/Nox to balance reactionthe two [enzyme25]. In the activities. coupled Therefore, enzyme reaction, the pH especially dependence when of the the optimumactivities pH of ofAmDH the two and enzymes Nox was is different,examined the (Figure pH of 4). the The reaction AmDH should and beNox carefully showed determined the highest toactivity balance at the pH two 10.0 enzyme and 7.5, activities. respectively. Therefore, The results the pHare dependencein good agreement of the activitieswith previously of AmDH reported and Noxresults was [18,26]. examined The (Figure specific4 ).activity The AmDH of AmDH and and Nox Nox showed was the48 mU/mg highest and activity 95 U/mg at pH at 10.0 the andoptimum 7.5, respectively.pH, respectively. The results are in good agreement with previously reported results [18,26]. The speciﬁc activity of AmDH and Nox was 48 mU/mg and 95 U/mg at the optimum pH, respectively.

100

AmDH, Tris/HCl buffer 40 AmDH, Glycine buffer Nox, Tris/HCl buffer

Relative activity (%) Relative activity Nox, Glycine buffer 20

0 7891011

pH FigureFigure 4. 4.Effect Effect of of pH pH on on the the activity activity of of AmDH AmDH and and Nox. Nox. For For the the AmDH AmDH activity activity assay, assay, the the reactions reactions werewere performed performed in in 100 100 mM mM Tris/HCl Tris/HCl buffer buffer (pH (pH 7.0–9.0) 7.0–9.0) or or 100 100 mM mM glycine glycine buffer buffer (pH (pH 9.0–11.0) 9.0–11.0) containingcontaining 20 20 mM mMa1 a1, 1, 1 mM mM NAD NAD+,+ and, and AmDH AmDH (0.15 (0.15 mg/mL). mg/mL). For For the the Nox Nox activity activity assay, assay, 0.2 0.2 mM mM NADHNADH and and 0.01 0.01 mg/mL mg/mL of of Nox Nox were were used. used.

Next,Next, the the kinetic kinetic resolution resolution of of 10 10 mM mMrac rac-2-aminoheptane‐2‐aminoheptane ( a12(a12)) was was carried carried out out in in 100 100 mM mM + Tris/HClTris/HCl buffer buffer (pH (pH 8.0–9.0) 8.0–9.0) containing containing 1 1 mM mM NAD NAD+ with with AmDH AmDH (1 (1 mg/mL) mg/mL) and and Nox Nox (1 (1 mg/mL). mg/mL). TheThe enantiomeric enantiomeric excess excess ( ee(ee)) value value of of a12 a12 was was 22%, 22%, 26%, 26%, and and 19% 19% at at pH pH 8.0, 8.0, 8.5, 8.5, and and 9.0, 9.0, respectively respectively (Figure(Figure5). 5). Also, Also, when when the kineticthe kinetic resolution resolution was carried was carried out with out 5 mM withrac 5- αmM-MBA rac (‐α‐a1)MBA at the ( conditiona1) at the describedcondition above described at pH above 8.5 forat pH 24 8.5 h, thefor 24ee valueh, the ee of valuea1 was of onlya1 was 20%. only The 20%. results The results suggest suggest that the that activitiesthe activities of AmDH of AmDH and and Nox Nox were were affected affected by theby the substrate substrate and/or and/or product. product. Several Several amino amino acid acid dehydrogenasesdehydrogenases have have been been observed observed toto suffersuffer from strong product inhibition, inhibition, often often by by their their native native L‐ L-aminoamino acid acid product product [20]. [20]. In In addition, addition, in the asymmetric synthesissynthesis ofof aminesamines from from ketones ketones by by AmDH, AmDH, productproduct inhibition inhibition has has been been previously previously suggested suggested by the by use the of ause biphasic of a reactionbiphasic system reaction to ostensiblysystem to overcomeostensibly inhibition overcome in inhibition AmDH-catalyzed in AmDH reactions‐catalyzed [27 reactions]. [27].

CatalystsCatalysts2017 2017, 7, 251, 7, 251 6 6of of 13 13 Catalysts 2017, 7, 251 6 of 13

30 30

25 25

20 20

(%) 15

(%) 15 ee

ee pH 8.0 10 pH 8.08.5 10 pH 8.59.0 pH 9.0 5 5

0 0 0 5 10 15 20 25 0 5 10 15 20 25 Time (h) Time (h) Figure 5. The kinetic resolution of 10 mM a12. Reaction conditions: 1 mM NAD+, 100 mM Tris/HCl Figure 5. The kinetic resolution of 10 mM a12. Reaction conditions: 1 mM NAD++, 100 mM Tris/HCl Figurebuffer 5. The (pH kinetic 8.0–9.0), resolution 1 mg/mL of AmDH, 10 mM a12and. 1 Reaction mg/mL of conditions: Nox. 1 mM NAD , 100 mM Tris/HCl bufferbuffer (pH (pH 8.0–9.0), 8.0–9.0), 1 mg/mL 1 mg/mL of of AmDH, AmDH, and and 1 1 mg/mL mg/mL of Nox. Therefore, the product inhibition of AmDH by ketones (2‐aminoheptanone or acetophenone) Therefore, the product inhibition of AmDH by ketones (2‐aminoheptanone or acetophenone) wasTherefore, examined the in product the presence inhibition of of10 AmDH mM a1 by and ketones a12 (Figure (2-aminoheptanone 6a). In the presence or acetophenone) of 2 mM was2‐ was examined in the presence of 10 mM a1 and a12 (Figure 6a). In the presence of 2 mM 2‐ examinedaminoheptanone in the presence and acetophenone, of 10 mM a1 and thea12 enzyme(Figure showed6a). In only the presence 60% and of49% 2 mM of its 2-aminoheptanone original activity. aminoheptanone and acetophenone, the enzyme showed only 60% and 49% of its original activity. These results indicate that the continuous removal of ketone in the reaction mixture could improve andThese acetophenone, results indicate the enzyme that the showed continuous only 60%removal and of 49% ketone of its in original the reaction activity. mixture These could results improve indicate the degree of conversion. Also, the activity inhibition of Nox by the substrate and product was thatthe the degree continuous of conversion. removal of Also, ketone the in activity the reaction inhibition mixture of Nox could by improve the substrate the degree and product of conversion. was examined (Figure 6b). The ketones did not significantly affect the activity of Nox, but the activity was Also,examined the activity (Figure inhibition 6b). The of ketones Nox by did the substratenot significantly and product affect the was activity examined of Nox, (Figure but 6theb). activity The ketones was dramatically reduced in the presence of amine (5 mM). The relative activities of Nox in the presence diddramatically not signiﬁcantly reduced affect in the the activity presence of of Nox, amine but (5 the mM). activity The was relative dramatically activities reducedof Nox in in the the presence presence of a1 and a12 were only 38% and 74% of its original activity, respectively. The unexpected poor of amineof a1 (5and mM). a12 Thewere relative only 38% activities and 74% of Nox of its in theoriginal presence activity, of a1 respectively.and a12 were The only unexpected 38% and 74%poor of conversion of the coupled AmDH/Nox reaction is partly due to product inhibition of AmDH by its originalconversion activity, of the respectively. coupled AmDH/Nox The unexpected reaction poor is conversionpartly due ofto theproduct coupled inhibition AmDH/Nox of AmDH reaction by ketone and activity inhibition of Nox by the amine. is partlyketone due and to activity product inhibition inhibition of ofNox AmDH by the by amine. ketone and activity inhibition of Nox by the amine.

100 100

2-heptanone 80 2-heptanoneacetophenone 80 acetophenone

60 60

40 40 Relative activity (%) activity Relative Relative activity (%) activity Relative 20 20

0 0 0246810 0246810 Concentration (mM) Concentration (mM) (a) (a) Figure 6. Cont.

Catalysts 2017, 7, 251 7 of 13 Catalysts 2017, 7, 251 7 of 13

120

100

40 Relative activity (%)

0 CON a12 2-heptanone a1 acetophenone

(b)

FigureFigure 6. Substrate 6. Substrate and and product product inhibition inhibition of of AmDH AmDH and and Nox; Nox; ( (aa)) EffectEffect ofof ketone concentration on on AmDH activity. Reaction conditions: 100 mM Tris/HCl buffer (pH 8.5), 1 mM NAD+, 10 mM a12 (or AmDH activity. Reaction conditions: 100 mM Tris/HCl buffer (pH 8.5), 1 mM NAD+, 10 mM a12 a1), 2‐heptanone (or acetophenone) (0–25 mM), 0.3 mg/mL of AmDH; (b) Effect of amine and ketone (or a1), 2-heptanone (or acetophenone) (0–25 mM), 0.3 mg/mL of AmDH; (b) Effect of amine and on Nox activity. Reaction conditions: 0.2 mM NADH, 5 mM amine or ketone, 100 mM Tris/HCl buffer ketone on Nox activity. Reaction conditions: 0.2 mM NADH, 5 mM amine or ketone, 100 mM Tris/HCl (pH 8.5), and 0.05 mg/mL of Nox. (CON: control, the Nox activity in the absence of amine and ketone.) buffer (pH 8.5), and 0.05 mg/mL of Nox. (CON: control, the Nox activity in the absence of amine and ketone.) 2.3. Kinetic Resolution of Amines Using E. coli Co‐Expressing AmDH and Nox

2.3. KineticThe Resolution immobilization of Amines of enzymes Using E. on coli a solid Co-Expressing support system AmDH improves and Nox catalyst stability, recycling, and longevity under industrially relevant process conditions [28]. For example, polyethyleneimine‐ activatedThe immobilization agarose beads were of enzymes used to non on‐covalently a solid supportattach phosphorylated system improves versions catalyst of the cofactors stability, recycling,NAD+, FAD and+, longevityand PLP, creating under an industrially association/dissociation relevant process equilibrium conditions of phosphorylated [28]. For and example, non‐ polyethyleneimine-activatedphosphorylated cofactors on agarose support beads [29]. were An increase used to non-covalentlyin turnover number attach was phosphorylated observed in versionscomparison of the cofactorsto non‐immobilized NAD+, FAD enzymes+, and PLP, or non creating‐immobilized an association/dissociation cofactors. However, equilibriumin this study, of phosphorylatedsubstrate and and product non-phosphorylated inhibitions of enzymes cofactors were on the support major [hurdles29]. An for increase carrying in turnover out the efficient number waskinetic observed resolution in comparison of racemic to non-immobilizedamines using AmDH enzymes and Nox. or non-immobilized Therefore, recombinant cofactors. Escherichia However, coli in thiscells study, co‐ substrateexpressing and AmDH product and inhibitions Nox were ofused enzymes as biocatalysts were the ( majorvide infra hurdles). The foruse carrying of whole out‐cells the efficientinstead kinetic of isolated resolution enzymes of racemic in biological amines usingprocesses AmDH has anda simple Nox. and Therefore, economical recombinant advantage,Escherichia as the coli costcells for co-expressing cell lysis and AmDH purification and Noxis reduced were used[30]. Another as biocatalysts advantage (vide of infra a whole). The‐cell use reaction of whole-cells system insteadis that of isolatedcofactor‐ enzymesdependent in enzymatic biological processesreactions can has be a simple effectively and economicalperformed by advantage, using endogenous as the cost for cellcofactors lysis and without purification adding costly is reduced cofactors [30]. [30]. Another Sometimes, advantage a whole of a cell whole-cell reaction reaction is less susceptible system is that to cofactor-dependentenzyme inhibition enzymatic by substrate reactions and product can be due effectively to the diffusion performed barrier by of using the cell endogenous membrane cofactors [31]. In order to develop a recombinant E. coli system expressing both enzymes in a single cell, the without adding costly cofactors [30]. Sometimes, a whole cell reaction is less susceptible to enzyme AmDH and Nox genes were cloned into pET24ma and pETDuet vectors, respectively. After inhibition by substrate and product due to the diffusion barrier of the cell membrane [31]. overnight induction at 20 °C, the cells were harvested, washed twice with 100 mM Tris/HCl buffer In order to develop a recombinant E. coli system expressing both enzymes in a single cell, the (pH 8.5), and used for the whole‐cell reaction. When kinetic resolutions of 10 mM a12 and 5 mM a1 AmDH and Nox genes were cloned into pET24ma and pETDuet vectors, respectively. After overnight were carried ◦out in the 100 mM Tris/HCl buffer (pH 8.5) with whole‐cell (20 mg dry cell weight induction(DCW)/mL) at 20 withoutC, the cellsadding were cofactor harvested, NAD+ washedfor 24 h, twiceboth of with the 100amines mM were Tris/HCl successfully buffer resolved (pH 8.5), andinto used the for (S) the‐form whole-cell with a high reaction. ee (>99%). When These kinetic results resolutions clearly showed of 10 that mM thea12 useand of whole 5 mM‐cella1 waswere carriedmuch out more in the efficient 100 mM than Tris/HCl the reaction buffer using (pH purified 8.5) with enzymes whole-cell that (20 gave mg 26% dry ee cell (Figure weight 6b). (DCW)/mL) The Shell + withoutgroup adding reported cofactor that in NADthe asymmetricfor 24 h, synthesis both of of the chiral amines amine were by AmDH, successfully the lyophilized resolved intowhole the‐ (S)-formcell gave with better a high conversionsee (>99%). both These in the results absence clearly and presence showed of that co‐solvent the use heptane of whole-cell than lyophilized was much morelysate efficient [20]. thanThe use the of reaction whole‐ usingcell apparently purified enzymesreduces the that substrate/product gave 26% ee (Figure inhibition,6b). Theand/or Shell it groupincreases reported the that stability in the of asymmetric the enzymes. synthesis When the of reactions chiral amine with 20, by 30, AmDH, 40, and the 50 lyophilized mM a12 were whole-cell carried gaveout better at pH conversions 8.5 using whole both‐cell in the(25 mg absence DCW/mL) and presence for 24 h, the of co-solvent ee values of heptane (S)‐a12 were than >99%, lyophilized 87%, lysate [20]. The use of whole-cell apparently reduces the substrate/product inhibition, and/or it Catalysts 2017, 7, 251 8 of 13

Catalysts 2017, 7, 251 8 of 13 increases the stability of the enzymes. When the reactions with 20, 30, 40, and 50 mM a12 were carried out48%, at pH and 8.5 30%, using respectively whole-cell (Figure (25 mg 7). DCW/mL) At initial forsubstrate 24 h, the concentrationsee values of ( Sof)- 20,a12 40,were and >99%, 50 mM, 87%, the 48%,produced and 30%, 2‐heptanone, respectively determined (Figure7). by At the initial consumed substrate a12 concentrations, was 14.0, 12.9, of and 20, 11.6 40, and mM, 50 respectively. mM, the producedWhen 2‐ 2-heptanone,heptanone is accumulated determined by to thesome consumed extent (11–14a12, wasmM), 14.0, the 12.9,reaction and does 11.6 not mM, seem respectively. to proceed Whenany 2-heptanonefurther. The ismain accumulated reason for to the some lower extent ee (11–14at higher mM), substrate the reaction concentrations does not seem seems to proceed to be the anyinhibition further. of The AmDH main by reason 2‐heptanone. for the lower ee at higher substrate concentrations seems to be the inhibition of AmDH by 2-heptanone.

100

80 20 mM 30 mM 60 40 mM 50 mM (%) ee 40

0 0 5 10 15 20 25

Time (h)

FigureFigure 7. Reaction7. Reaction profiles profiles of the of kinetic the kinetic resolution resolution at different at concentrationsdifferent concentrations of a12. Reaction of a12 conditions:. Reaction a12conditions:(0–50 mM), a12 100 (0–50 mM Tris/HCl mM), 100 buffer mM (pH Tris/HCl 8.5), whole-cell buffer (pH (25 8.5), mg dry whole cell‐ weightcell (25 (DCW)/mL). mg dry cell weight (DCW)/mL). The next step was to increase the final product’s concentration, which could be accomplished by enhancingThe next the step biocatalyst was to increase concentrations the final product’s in the reaction concentration, medium. which Compared could tobea12 accomplished, the kinetic by resolutionenhancing of thea1 is biocatalyst much more concentrations difficult due toin thethe poorreaction reactivity medium. of AmDH Compared for a1 to, aa12 more, the severe kinetic productresolution inhibition of a1 is by much the corresponding more difficult ketone, due to acetophenone, the poor reactivity and aof stronger AmDH inhibition for a1, a more of Nox severe by a1product(Figure 6inhibition). After the by concentrationsthe corresponding of a1ketone,and a12 acetophenone,were set at and 20 mM a stronger and 50 inhibition mM, respectively, of Nox by thea1 reaction (Figure was6). After performed the concentrations to determine of thea1 and amount a12 were of whole-cell set at 20 mM catalyst and required50 mM, respectively, to obtain the the opticallyreaction pure was ( Sperformed)-amine (Figure to determine8). The minimum the amount amounts of whole of whole-cell‐cell catalyst required required to resolve to obtain 50 mM the a12opticallyand 20 mMpurea1 (S)into‐amine (S)-form (Figure (>99% 8). Theee) minimum were 60 and amounts 100 mg of of whole DCW/mL,‐cell required respectively. to resolve Next, 50 the mM reactiona12 and profiles 20 mM for a1 the into kinetic (S)‐form resolution (>99% ofee)a1 wereand 60a12 andwere 100 determined mg of DCW/mL, (Figure 9respectively.). In the 50 mM Next,a12 the reaction,reaction 40 profiles mg DCW/mL for the kinetic of whole-cell resolution gave of only a1 77%and a12ee for were 24 h determined (Figure8), but (Figure 60 mg 9). DCW/mL In the 50 ofmM whole-cella12 reaction, gave 40 >99% mg DCW/mLee in 1 h (Figure of whole9).‐cell Also, gave the onlyee value 77% ee of for a1 24 was h (Figure 95% in 8), 24 but h of 60 reaction mg DCW/mL with 80of mg whole DCW/mL‐cell gave of whole-cell; >99% ee in however,1 h (Figure the 9).ee Also,value the became ee value >99% of a1 after was 5 95% h of in reaction 24 h of with reaction 100 mg with DCW/mL80 mg DCW/mL of whole-cell. of whole This‐cell; result however, is consistent the ee value with became the observation >99% after that 5 h the of reaction enzyme with activity 100 of mg AmDHDCW/mL is inhibited of whole by‐cell. the ketone This result product. is consistent with the observation that the enzyme activity of AmDH is inhibited by the ketone product.

Catalysts 2017, 7, 251 9 of 13 CatalystsCatalysts2017 2017, 7,, 7 251, 251 99 of of 13 13

100 100 a12 80 a12a1 80 a1

60 60 (%) (%) ee 40 ee 40

20 20

0 0 0 20406080100 0 20406080100 Concentration (mg DCW/mL) Concentration (mg DCW/mL) Figure 8. The effect of the amount of whole‐cell on the kinetic resolution of 50 mM a12 or 20 mM a1. FigureFigureReaction 8. 8.The The conditions: effect effect of of the 100the amount mMamount Tris/HCl of of whole-cell whole buffer‐cell (pH on on the 8.5),the kinetic kinetic 50 mM resolution resolution a12 (or 20 of ofmM 50 50 mM a1mM), a12and a12or wholeor 20 20 mM mM‐cella1 a1(10–. . ReactionReaction100 mg conditions: DCW/mL).conditions: 100100 mMmM Tris/HCl buffer buffer (pH (pH 8.5), 8.5), 50 50 mM mM a12a12 (or(or 20 20 mM mM a1),a1 and), and whole whole-cell‐cell (10– (10–100100 mg mg DCW/mL). DCW/mL).

100 100

80 a12 80 a12a1 a1 60 60 (%) (%) ee

ee 40 40

20 20

0 0 0246810 0246810 Time (h) Time (h) FigureFigure 9. Reaction 9. Reaction profiles profiles of the of kineticthe kinetic resolution resolution of 50 of mM 50a12 mMor a12 20 mMor 20a1 mM. Sixty a1. milligrams Sixty milligrams (60 mg) (60 andFiguremg) 100 and mg9. Reaction DCW/mL100 mg profilesDCW/mL of whole-cell of theof wholekinetic was used‐cell resolution forwas the used reaction of 50for mM the of 50 a12reaction mM or 20a12 mMofor 50 20a1 mM mM. Sixty a12a1 ,milligrams respectively.or 20 mM (60 a1 , mg)respectively. and 100 mg DCW/mL of whole‐cell was used for the reaction of 50 mM a12 or 20 mM a1, respectively. To investigate the utility of a whole-cell system expressing AmDH and Nox, the kinetic resolutions To investigate the utility of a whole‐cell system expressing AmDH and Nox, the kinetic of various rac-amines (10 mM) were performed (Table1). a5, a7, and a8 were successfully resolved resolutionsTo investigate of various the rac utility‐amines of (10a wholemM) were‐cell performedsystem expressing (Table 1). AmDH a5, a7, and and a8 Nox, were thesuccessfully kinetic into (S)-form with a high ee (>99%). However, the kinetic resolutions of a4, a6, and a11 gave 66%, resolutionsresolved into of various (S)‐form rac with‐amines a high (10 ee mM) (>99%). were However, performed the (Table kinetic 1). resolutions a5, a7, and of a8 a4 were, a6, successfullyand a11 gave 28%, and 33% ee, respectively. Among the fluorinated amines (a4, a5, and a6), a4 is the most reactive resolved66%, 28%, into and (S) ‐33%form ee with, respectively. a high ee (>99%). Among However, the fluorinated the kinetic amines resolutions (a4, a5, ofand a4 ,a6 a6),, anda4 is a11 the gave most substrate, but only a5 was converted into an optically pure form. Also, although a11 was the most 66%,reactive 28%, substrate, and 33% butee, respectively.only a5 was converted Among the into fluorinated an optically amines pure form.(a4, a5 Also,, and although a6), a4 is a11 the was most the reactive substrate in the tested substrates (Figure2), the ee value of a11 was only 33%. In general, the reactivemost reactive substrate, substrate but only in thea5 wastested converted substrates into (Figure an optically 2), the eepure value form. of a11 Also, was although only 33%. a11 In was general, the more reactive substrates are expected to have higher conversion rates into product. However, it is mostthe morereactive reactive substrate substrates in the tested are expected substrates to have(Figure higher 2), the conversion ee value of rates a11 was into only product. 33%. However,In general, it likely that there was no clear correlation between final conversion and substrate reactivity because of theis likelymore thatreactive there substrates was no clear are correlationexpected to between have higher final conversion conversion rates and substrateinto product. reactivity However, because it is likely that there was no clear correlation between final conversion and substrate reactivity because

Catalysts 2017, 7, 251 10 of 13 an unclear correlation between the reactivity and product inhibition of AmDH by the corresponding ketone. In the case of a4, a6, and a11, the reactions were carried out at a lower concentration (5 mM) with the same condition, and the ee values were 77%, 78%, and 51%, respectively (Table1). Although the ee value increased more than when 10 mM substrate was used, the optically pure form could not be obtained.

Table 1. Kinetic resolution of various amines using AmDH/Nox a.

Substrate Conc. (mM) Conv. (%) eeS (%) a4 10 40 66 a4 5 44 77 a5 10 50 >99 a6 b 10 22 28 a6 b 5 44 78 a7 10 50 >99 a8 10 50 >99 a11 10 25 33 a11 5 34 51 a The reaction conditions: 5 mL of reaction volume, 120 mg DCW/mL of whole-cell, Tris/HCl buffer (100 mM, pH 8.5), and 5 or 10 mM racemic substrate (see Figure2) for a 24 h reaction at 37 ◦C. Conversion and ee were b determined by Crownpak CR(+) column; Conversion and ee were determined by C18 Symmetry column after GITC derivatization.

3. Materials and Methods

3.1. Chemicals

All amines (a1–a12), GITC (2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl isothiocynate), cofactors (NADH and NAD+), and isopropyl-β-D-thiogalactopyranoside were obtained from Sigma-Aldrich Yongin, Korea.

3.2. Enzyme Expression and Purification The AmDH and Nox genes [18,24] were synthesized by Bioneer Corporation (Daejeon, Korea), and each gene was then inserted into IPTG-inducible pET24ma vectors. The plasmid was then introduced into E. coli (BL21) cells and the transformants were grown at 37 ◦C in 1 L LB-containing kanamycin (100 µg/mL) [32]. When the OD600 reached 0.5, IPTG was added to a final concentration of 0.5 mM. After overnight induction at 20 ◦C, the cells were harvested and washed twice with 100 mM Tris/HCl buffer (pH 8.5). After centrifugation, the cell pellets were resuspended in 10 mL volume of 20 mM Tris/HCl buffer (pH 8.5). The suspension was then subjected to ultrasonic disruption. The disruption period was 5 s with 10 s intervals in an ice bath for a duration of 30 min. The C-terminal His6-tagged enzyme was purified at 4 ◦C on a Ni-NTA agarose resin obtained from Qiagen (Hilden, Germany). The eluted solution containing purified protein was dialyzed against 50 mM potassium phosphate buffer (pH 8.5) and concentrated using an Amicon PM-10 ultrafiltration unit. Glycerol was added to the purified enzyme solution (25%) and it was stored at −20 ◦C for further study. For the co-expression of AmDH and Nox, the AmDH gene was amplified and subsequently inserted into the pETDuet-1 (Novagen, Madison, WI, USA). The plasmid was then introduced into the E. coli (BL21) bearing pET24ma:Nox. Then, enzymes were overexpressed in the presence of kanamycin (100 µg/mL) and ampicillin (100 µg/mL) as describe above. After harvesting, the cells were washed twice with 100 mM Tris/HCl buffer (pH 8.5) and they were immediately used for the whole-cell reaction.

3.3. Enzyme Assay The activities of AmDH and Nox were determined using an NADH-dependent (340 nm, −1 −1 λ340 = 6220 M cm ) spectrophotometric assay. Activity is calculated from the stoichiometric Catalysts 2017, 7, 251 11 of 13 reduction of NAD+ or oxidation of NADH as measured by the change in absorbance over time. One unit of activity is deﬁned as the amount enzyme that catalyzes the formation of 1 µmol of NADH or formation of 1 µmol of NAD+ in 1 min. A standard AmDH activity assay was carried out in 100 mM glycine buffer (pH 10.0) containing AmDH (0.15 mg/mL), 5 mM substrate, and 1 mM NAD+ at 25 ◦C[18]. The standard solution for the assay of Nox contained 0.2 mM of NADH and an appropriate amount of the enzyme [24].

3.4. Analysis of Amines The conversion and ee analysis of amines were performed by HPLC using a Crownpak CR (Daicel Co., Osaka, Japan) column at 210 nm with elution with a perchloric acid solution of pH 1.5 (0.6 mL min−1)[33]. Each enantiomer was separated by these analytical conditions except a6 and a12. A quantitative chiral analysis of a6 and a12 was performed using a C18 symmetry column (Waters, Milford, MA, USA) with a Waters HPLC system at 254 nm after the derivatization of the sample with GITC [33]. The separation of each enantiomer of a6 and a12 was achieved through isocratic elution with a mixture of 50% methanol and 50% water (0.1% TFA) at a ﬂow rate of 1.0 mL min−1.

4. Conclusions To produce chiral amines, the stereoselective reductive amination of ketones by AmDH is favored in practice. To prepare chiral amines with opposite chirality, the oxidative deamination of racemic amines by AmDH is as important as reductive amination. Here, the kinetic resolution of racemic amines was performed using AmDH in combination with Nox, in which Nox was used to regenerate NAD+ and to drive the reaction forward. Compared to the puriﬁed enzyme system, the whole-cell system showed much better performance, probably due to the reduction of the inhibitory effect of substrate/product on the enzymes and the increased stability of the enzymes in the whole-cell system. By using the whole-cell system expressing AmDH and Nox, 50 mM 2-aminoheptane and 20 mM α-MBA were successfully resolved into (S)-form with >99% ee, respectively. The kinetic resolution of amines at a high concentration by AmDH can be carried out effectively by the following approach. First, inhibitory ketones in the reaction mixture should be removed by using ketone-extracting organic solvents (biphasic reaction) or ketone-converting enzymes (e.g., alcohol dehydrogenase) to convert them into non-inhibitory compounds [32]. Second, alkaliphilic enzymes, such as Nox from Brevibacterium sp. KU1309 [34], should be used for the coupling reaction, then the coupling reaction can be carried out at a pH that is close to the optimal pH (10.0) of AmDH. The further development of the kinetic resolution of amines using AmDH is currently being investigated with these approaches.

Supplementary Materials: The following are available online at www.mdpi.com/2073-4344/7/9/251/s1, Figure S1: SDS-PAGE analysis of purified AmDH (42.37 kDa), Figure S2: Lineweaver–Burk (double-reciprocal) plot of 1/v against 1/[S]title, Figure S3: SDS-PAGE analysis of recombinant E. coli expressing AmDH (42.37 kDa) and Nox (50.47 kDa). Table S1: Retention times of chiral amines in HPLC analysis. Acknowledgments: This paper was supported by Konkuk University in 2015 (#2015A0190097). Author Contributions: H.Y., H.J., and S.Y. conceived and designed the experiments; H.J., S.Y., and U.S. performed the experiments; G.-H.K. and S.Y. analyzed the data; S.S., M.M.A., and S.-K.R. contributed reagents/materials/analysis tools; H.J. and H.Y. wrote the paper. Conflicts of Interest: The authors declare no conflict of interest

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