The Kinetic Resolution of Racemic Amines Using a Whole-Cell Biocatalyst Co-Expressing Amine

13 Pages·2017·2.3 MB·English

catalysts
Article
The Kinetic Resolution of Racemic Amines Using
a Whole-Cell Biocatalyst Co-Expressing Amine
Dehydrogenase and NADH Oxidase
HyunwooJeon1,†,SanghanYoon1,†,MdMurshidulAhsan2,SihyongSung1,Geon-HeeKim1,
UthayasuriyaSundaramoorthy1,Seung-KeunRhee2andHyungdonYun1,*
1 DepartmentofSystemsBiotechnology,KonkukUniversity,Seoul05029,Korea;
[email protected](H.J.);[email protected](S.Y.);[email protected](S.S.);
[email protected](G.-H.K.);[email protected](U.S.)
2 SchoolofBiotechnology,YeungnamUniversity,Kyongsan38541,Korea;
[email protected](M.M.A.);[email protected](S.-K.R.)
* Correspondence:[email protected];Tel.:[phone];Fax:[phone]
† HyunwooJeonandSanghanYoonhavecontributedequallytothiswork.
Received:9August2017;Accepted:23August2017;Published:25August2017
Abstract: Amine dehydrogenase (AmDH) possesses tremendous potential for the synthesis of
chiralaminesbecauseAmDHcatalyzestheasymmetricreductiveaminationofketonewithhigh
enatioselectivity. AlthoughareductiveapplicationofAmDHisfavoredinpractice,theoxidative
routeisinterestingaswellforthepreparationofchiralamines. Here,thekineticresolutionofracemic
amines using AmDH was first extensively studied, and the AmDH reaction was combined with
anNADHoxidase(Nox)toregenerateNAD+andtodrivethereactionforward. Whenthekinetic
resolutionwascarriedoutwith10mMrac-2-aminoheptaneand5mMrac-α-methylbenzylamine
(α-MBA)usingpurifiedenzymes,theenantiomericexcess(ee)valueswerelessthan26%duetothe
productinhibitionofAmDHbyketoneandtheinhibitionofNoxbythesubstrateamine. Theuse
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-aminoheptaneand20mMrac-α-MBAweresuccessfullyresolvedintothe(S)-formwith>99%
ee using whole cells. The present study demonstrates the potential of a whole-cell biocatalyst
co-expressingAmDHandNoxforthekineticresolutionofracemicamines.
Keywords:aminedehydrogenase;biocatalysts;chiralamine;kineticresolution;oxidativedeamination
1. Introduction
Recently, considerable efforts have been made to economically obtain enantiomerically pure
intermediatesfordrugsandagrochemicals. Amongthem,thechiralaminesareofgreatinterestto
thepharmaceuticalandfine-chemicalindustries,sinceenantiopureaminesareimportantbuilding
blocksforpharmaceuticalmanufacturing[1]. Giventhesignificanceofchiralamines,theirefficient
synthesisinenantiopureformhasbecomeanattractivechallengetoorganicchemistsandbiologists
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],ammonialyases[9],Pictet-Spenglerase[10],barberinebridgeenzyme[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
moredetails).
Catalysts2017,7,251;doi:10.3390/catal7090251 www.mdpi.com/journal/catalysts
Catalysts2017,7,251 2of13
Among them, as AmDH catalyzes the asymmetric reductive amination of ketones, and uses
cheap ammoniaas thereagentand generatesonly wateras byproduct, withhigh atomefficiency
andenantioselectivity[14],AmDHishighlyattractiveandpossessestremendouspotentialforthe
synthesisofchiralamines[13]. Untilnow,onlyoneNAD+-dependentAmDHfoundinnaturehas
beenreported,inStreptomycesvirginiae,butitshowedpoorenantioselectivityanditsDNAinformation
is not available [15]. Recently, AmDHs have been created through protein engineering using the
existingL-aminoaciddehydrogenase. First,AmDHwasengineeredbytheBommariusgroupusing
leucinedehydrogenase(LeuDH)fromBacillusstearothomophilustoacceptketonesubstratesthroughthe
introductionof2–4pointmutations[16]. Subsequently,asecondaminedehydrogenasewasdeveloped
basedonthescaffoldofphenylalaninedehydrogenase(PheDH)fromBacillusbadiusbythesamegroup
usingasimilarapproach[17]. Further,achimericenzymewasgeneratedbycombiningtheN-terminal
section (residues 1–149) of the second AmDH (PheDH variant) with residues 140–366 of the first
AmDH(LeuDHvariant),andthechimericenzymedisplayedgoodactivitytowardsvariousamines,
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-aminoaciddehydrogenasesfromRhodococcussp. M4,Exiguobacteriumsibiricum,andCaldalkalibacillus
thermarum[14,19,20]. Asystematicinvestigationofsubstrateacceptance,optimalreactionconditions,
andthechemo-andstereoselectivityofAmDHshasbeencarriedout[13,20]. Inaddition,AmDHs
havebeenusedineleganthydrogenborrowingdual-enzymecascadereactionsforthesynthesisof
chiralaminesfromalcoholswith“closed-loop”recyclingofthecofactor[19,21]. Duetotheimportance
ofthereductiveaminationofketonesbyAmDH,anextensiveanddetailedreviewonrelevantstudies
hasrecentlybeenpublished[3].
Althoughareductiveapplicationofdehydrogenasesisfavoredinpractice,theoxidativeroute
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,ortheconversionofhydroxyacids,aminoacids,andalcoholsintothecorresponding
ketocompounds[22]. Untilnow,allreportedAmDHsexhibit(R)-enantioselectivitybecausetheywere
generatedfromanL-aminoaciddehydrogenasescaffold. Therefore,(R)-aminesareproducedthrough
reductiveaminationbyAmDHsfromketones(forcertaincompounds,(S)-formsweregenerateddue
totheCahn–Ingold–Prelogpriorityrulesfornamingchiralcenters). Since(S)-specificAmDHisnot
available, (S)-amines cannot be synthesized via reductive amination. Although (S)-amines can be
obtainedthroughthekineticresolutionofracemicaminesusingoxidativedeaminationby(R)-specific
AmDH,thekineticresolutionofaminesusingAmDHhasnotbeenextensivelystudieduntilrecently.
Wedescribehereintheproductionof(S)-aminesthroughtheoxidativedeaminationofracemic
amines by AmDH with help of an NADH oxidase (Nox) (Figure 1). 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
flavoproteinwhichoxidizesNADHtoNAD+ viaacoupledreactionofmolecularoxygentoeither
waterinafour-electronreductionortohydrogenperoxideinatwo-electronreduction[22]. Dueto
thetoxicityofhydrogenperoxide[23],water-formingNoxfromLactobacillusbreviswasusedinthis
study[24]. AmongthereportedAmDHs,thechimericenzyme[18]generatedbythedomainshuffling
oftheLeuDHandPheDHvariantswasusedinthisstudy.
CCaattaallyyssttss 22001177,, 77,, 225511   33 ooff 1133 
Catalysts 2017, 7, 251   3 of 13 
NH NH O
RR1 NHRR222 AAmmDDHH RR1 NHRR222 ++ RR1 O RR2++ NNHH44++
1 2 1 2 1 2
NNAADD++ NNAADDHH
HH22OO NNOOXX 11//22 OO22  

FFiigguurree 11.. TThhee kkiinneettiicc rreessoolluuttiioonn ooff aammiinnee bbyy ((RR))‐-ssppeecciiffiicc AAmmDDHH iinn ccoommbbiinnaattiioonn wwiitthh NNAADDHH ooxxiiddaassee 
Figure 1. The kinetic resolution of amine by (R)‐specific AmDH in combination with NADH oxidase 
((NNooxx)).. ((RR))‐-aammiinnee iiss ooxxiiddiizzeedd iinnttoo tthhee ccoorrrreessppoonnddiinngg kkeettoonnee bbyy AAmmDDHH uuttiilliizziinngg NNAADD++.. AAddddiittiioonnaallllyy,, 
(Nox). (R)‐amine is oxidized into the corresponding ketone by AmDH utilizing NAD+. Additionally, 
NNAADD++ iiss rreeggeenneerraatteedd bbyy NNooxx,, aanndd eennaannttiioommeerriiccaallllyy ppuurree ((SS))‐-aammiinnee iiss oobbttaaiinneedd.. 
NAD+ is regenerated by Nox, and enantiomerically pure (S)‐amine is obtained. 
22.. RReessuullttss aanndd DDiissccuussssiioonn 
2. Results and Discussion 
22..11.. SSuubbssttrraattee SSppeecciiffiicciittyy aanndd KKiinneettiicc PPaarraammeetteerrss ooff AAmmDDHH 
2.1. Substrate Specificity and Kinetic Parameters of AmDH 
TThhee AAmmDDHH ggeennee wwaass cclloonneedd iinnttoo aa ppEETT2244mmaa vveeccttoorr wwiitthh aa CC-‐tteerrmmiinnaall HHiiss66-‐ttaagg,, eeffffeeccttiivveellyy 
The AmDH gene was cloned into a pET24ma vector with a C‐terminal His6‐tag, effectively 
eexxpprreesssseedd inint htheeE sEcshcehriecrhiciahicao lcioBliL 2B1Lc2e1l lc(eDllE (3D),Ea3n)d, saunbds esquubesneqtluyepnutrlyifi peduroinfieadN oi-nN aT ANaif‐fiNnTitAy caoflfuinmitny. 
expressed in the Escherichia coli BL21 cell (DE3), and subsequently purified on a Ni‐NTA affinity 
Acoslummennt. iAonse mdeanbtoivoen,etdh eabmoavien, itnhtee rmesatino finthteisresstut doyf tihsitsh setuodxiyd iast itvhee doexaimdaintiavteio dneaomfaimnaintieosnb oyf AammiDneHs. 
column. As mentioned above, the main interest of this study is the oxidative deamination of amines 
Tbyh eAomxiDdaHti.v Tehdee aomxiidnaattiivoen daectaimviitnyaotfioAnm aDctHivtitoyw oafr dAsmvaDrHio utoswamaridnes svwaraisoeuxsa amminineedsi nwoarsd eexratmoeinxpedlo irne 
by AmDH. The oxidative deamination activity of AmDH towards various amines was examined in 
tohredearp tpol iecxapbliolirtey tohfe tahpepelinczaybmilietyt ooft thhee keinnzeytimcer etsoo tlhuet ikoinneotficv raersiooulustiaomn ionfe vsa(rFioiguusr aem2)in. eTsh (eFiegnuzryem 2)e. 
order to explore the applicability of the enzyme to the kinetic resolution of various amines (Figure 2). 
sThhoew eendzycomnes isdheorawbelde accotnivsiitdyetroawblaer dascttihveityte sttoewdaarmdsi ntehse. Ttehseteedn zaymmineessh. oTwheed egnozoydmaec tsihvoitwyetodw gaorodds 
The enzyme showed considerable activity towards the tested amines. The enzyme showed good 
(aRct)i-vαi-tmy ettohwylabrednsz y(Rla)m‐αi‐nmee(tαh-yMlbBeAnz,yal2a)maninde (S(α)-‐pMhBenAy, laal2a)n iannodl (a(S9)),‐pbhuetngyalvaelanneingolilg i(bal9e),a cbtiuvti tygafvoer 
activity  towards  (R)‐α‐methylbenzylamine  (α‐MBA,  a2)  and  (S)‐phenylalaninol  (a9),  but  gave 
(nSe)g-αli-gMibBleA  (aac3t)ivaintyd (fRo)r- ph(Se)n‐αyl‐aMlaBnAin o(la(3a)1 0a).nIdt su(Rgg)‐epshteednythlaaltatnhienoeln a(nat1io0s).e leIct tivsuitgygoefstthede enthzaytm  ethies 
negligible  activity  for  (S)‐α‐MBA  (a3)  and  (R)‐phenylalaninol  (a10).  It  suggested  that  the 
henigahn,taionsdeltehcetirveistuy ltoifs tchoen esniszteynmtew iist hhtihgaht, raenpdo rttheed rperseuvlito iuss clyon[1s8is]t.eTnht eweinthzy tmhaet’s rreepaocrtitvedit yptroewviaorudsslya 
enantioselectivity of the enzyme is high, and the result is consistent with that reported previously 
b[1r8o]a. dThraen egnezyomfseu’sb sretraactteivsiatyn dtohwigahrdesn aa bnrtiooasde lreacntigvei toyf isnudbisctartaetetsh eanadp phliigcha beinliatnytoiofstehleecetnivziytym iendinictahtee 
[18]. The enzyme’s reactivity towards a broad range of substrates and high enantioselectivity indicate 
pthreo daupcptliiocnaboiflicthy ioraf lthame einnezsy.mAef tienr tthhea tp,rroadc-uαc-tMioBnA of( ac1h)iraanld amraicn-2e-sa. mAifnteorh tehpatta, nreac(‐aα1‐2M)wBAer (eas1e)l aenctde draacs‐
the applicability of the enzyme in the production of chiral amines. After that, rac‐α‐MBA (a1) and rac‐
r2e‐apmreisneonhtaetpivtaenaer o(am1a2t)i cwaenrde asleiplehcatetidc aams irneepsrefosernftuarttihveer asrtuomdya.tTico adnedte ramlipinheattihce akminienteics pfoarr afmurettheersr 
2‐aminoheptane (a12) were selected as representative aromatic and aliphatic amines for further 
ostfutdhye. Tenoz dyemteer,mtihnee itnhiet ikailnreattice poafrtahmeeetenrzsy omf tehwe eanszaynmaely, ztheed inwitiitahl rvaatrei oouf sthceo ennczeynmtrea twioanss aonfalay1zeodr 
study. To determine the kinetic parameters of the enzyme, the initial rate of the enzyme was analyzed 
aw1i2th( 0v–a2r5iomusM co)nincenthtreaptiroensse nofc ea1o ofr1 am12M (0–N2A5 Dm+M()F iing uthree p3raesnedncFei gouf r1e mSM2). NTAhDe+k (iFniegtuicrepsa 3r aamnde tSe2r)s. 
with various concentrations of a1 or a12 (0–25 mM) in the presence of 1 mM NAD+ (Figures 3 and S2). 
w(3aoToT±.1ffhh9e  2AA0ee9r .e  w2mmkk(5d±iieDDnn)er0eemetHH.ett 3ii8rM  cc9ff.m  1oo)pp7rraimaan   naa(rre±dM11aad0  mmww1.2ba1eeee5an.ttrr8)eesdee 8errm  ss33d0(  M..±ww.99o9990 n6eea  .rr((5nt(±±ee3h±d00  )dde.. 0331meeL.991tt0eeii)).4nn8  rrmm38mme−) wMM1(iim±,nne0  reeaaaie.ndd5nnvs3−  pddebb)r1e   aa00–,mcss..Btr99eeiieu66vddns  re−  ((pkoo1l±±,yenn 00pr.c..  e00tlttosihh44vptee33.ee  ))LLlTc  ymmtiih.nniviieeeTnnewwK−−hl11y,,meee  .rraa Keeavvssnmeeppdrree––acckBBnttcuuiiadvvtrreevkkkllc  ayyppalt..ull  ooTTvetthhas..  leeTTou  KKfhheAeesmm    KKmfaaonnmmDrdd  aaaH  kknn1ccdd2aaftto    vvwkkrccaaaaeattll  uur1vveeeaawssll8uu  eff.1ooeer7essrr    
a12 were 8.17 (±0.25) mM and 11.88 (±0.53) min−1, respectively. 
NNHH22 NNHH22 NNHH22 NNHH22
aa11 aa22 aa33 FF aa44
NNHH22 NNHH22 NNHH22 NNHH22
F
F
FF aa55 aa66 aa77 aa88
FF FF NNHH22 NNHH22
NH NH
NH2 NH2
2 2
OH OH
aa99 OH aa1100 OH aa1111 aa1122   
(a)
(a)
Figure2.Cont.
CatCalaytsatlsys2t0s1 270,177,,2 75,1 251   4o4f 1o3f 13 
Catalysts 2017, 7, 251   4 of 13 
300
300
275
275
)
)%
%
(
ctivity (activity 252050
e ave 121020
elativRelati1081000800
R
60
60
40
40
20
20
0
0
a1 a2 a3 a4 a5 a6 a7 a8 a9 a10 a11 a12
a1 a2 a3 a4 a5 a6 a7 a8 a9 a10 a11 a12
Substrate
Substrate  

(b)
(b)
Figure 2. (a) Substrate spectrum; (b) Substrate specificity of AmDH. Oxidative deamination was 
Figure 2. (a) Substrate spectrum; (b) Substrate specificity of AmDH. Oxidative deamination was 
Figure2.(a)Substratespectrum;(b)SubstratespecificityofAmDH.Oxidativedeaminationwascarried
carried out in 100 mM glycine buffer (pH 10.0) containing AmDH (0.15 mg/mL), 5 mM substrate, and 
carried out in 100 mM glycine buffer (pH 10.0) containing AmDH (0.15 mg/mL), 5 mM substrate, and 
outin100mMglycinebuffer(pH10.0)containingAmDH(0.15mg/mL),5mMsubstrate,and1mM
1 mM NAD+ at 25 °C. One hundred percent (100%) corresponds to 44 mU/mg. 
N1 AmDM+ NatA2D5+◦ Cat. 2O5n °eCh. uOnnder ehdunpderrceedn pte(1rc0e0n%t )(1c0o0rr%es) pcoonrrdessptoon44dsm toU /44m mg.U/mg. 
0.06
0.06
)
)n
minmi
M/M/

n rate (on rate (0.004.04 2--2aM-m-aBMimAnBo iAn(hao e(1hap)e1tap)ntaen (ea (1a21)2)
actioeacti0.002.02
eR
R
0.00
0.00
0 5 10 15 20 25
0 5 10 15 20 25
Concentration (mM)
Concentration (mM)  

FigFuigreur3e. 3.M Micihchaaeelilsi–s–MMeenntetenn pplloott ooff tthhee rreeaaccttiioonn rraattee ddeeppeennddiningg onon thteh esusbusbtrsatrtea tceocnocnencetrnattriaotnio. nT.he 
Figure 3. Michaelis–Menten plot of the reaction rate depending on the substrate concentration. The 
Threeraecaticotinosn wswereer ceacrarrieride douotu itni n10100 0mmMM TrTirsi/sH/CHlC (lp(Hp H8.58). 5c)ocnotnaitnaiinnign sgusbusbtrsatrtae t(e0(–03–03 m0mMM), )A,AmmDDHH (0.3 
reactions were carried out in 100 mM Tris/HCl (pH 8.5) containing substrate (0–30 mM), AmDH (0.3 
(0.3mmg/gm/Lm fLorf oar1a21, 20,.405.4 m5gm/mg/Lm foLrf ao1r)a, 1a)n,da n1d m1Mm NMANDA+ Dat+ 2a5t °2C5. ◦C.
mg/mL for a12, 0.45 mg/mL for a1), and 1 mM NAD+ at 25 °C. 
2.22..2K.i KneintiectRice Rsoelsuotliuotnioonf oAf mAimneisneUs sUinsginagP au PriufireidfieAdm ADmHD/HN/oNxoCxo CupoulipnlginSgy sStyesmtem 
2.2. Kinetic Resolution of Amines Using a Purified AmDH/Nox Coupling System 
InItnh etkhien ektiicneretisco luretsioonluotfioanm ionfe sabmyinAems DbHy ,tAhmerDegHe,n ethraet iorengoefnceorfaatciotonr NofA Dco+faisctoofri mNpAoDrta+ nicse ,of 
In  the  kinetic  resolution  of  amines  by  AmDH,  the  regeneration  of  cofactor  NAD+  is  of 
asitmhpeorretaacntcioen, arse qtuheir erseaactsitoonic hreioqmuieretrsi ca asmtooicuhnitomofeNtrAic Da+m(oFuignut roef 1N).AIDn+a d(Fdigituiorne t1o). rIend uacdidnigtiothne to 
importance, as the reaction requires a stoichiometric amount of NAD+ (Figure 1). In addition to 
corsetdoufctihneg ethnez ycmosat toicf trheea cetniozny,mNatAicD r+eaccotfioanct,o NrAreDg+e ncoefraacttioorn rseigmenpelirfiaetisopnr soidmupcltifiiseos lpatrioodnu,cpt riesvoelanttison, 
reducing the cost of the enzymatic reaction, NAD+ cofactor regeneration simplifies product isolation, 
propbrelevmenstosf pprroodbulecmtisn hoifb iptiroondbuyctt hienchoibfaitcitoonr, abnyd tchaen fcaovfaocratobrl,y adnridv ectahne rfeaavcotrioanblfyo rdwraivrde .tFhoer NreAacDti+on 
prevents problems of product inhibition by the cofactor, and can favorably drive the reaction 
forward. For NAD+ regeneration, Nox from Lactobacillus brevis was used [24]. The reduced byproduct 
forward. For NAD+ regeneration, Nox from Lactobacillus brevis was used [24]. The reduced byproduct 
ofo Nf Noxo ixs  iHs H2O2O, a,n adn dth tihs irse raecaticotino nis  icso cnosnidsiedreerde dir rirerveevresribsilbe,l ew, whihchic hm meaenasn tsh tahta itt  iits  itsh teh de rdivriivnign gfo frocrec teo t o
Catalysts2017,7,251 5of13
Catalysts 2017, 7, 251   5 of 13 
regeneration,NoxfromLactobacillusbreviswasused[24]. ThereducedbyproductofNoxisH O,and
move the reaction forward in a coupled AmDH/Nox reaction [25]. In the coupled enzyme 2reaction, 
thisreactionisconsideredirreversible,whichmeansthatitisthedrivingforcetomovethereaction
especially when the optimum pH of the two enzymes is different, the pH of the reaction should be 
forwardinacoupledAmDH/Noxreaction[25]. Inthecoupledenzymereaction,especiallywhenthe
carefully determined to balance the two enzyme activities. Therefore, the pH dependence of the 
optimumpHofthetwoenzymesisdifferent,thepHofthereactionshouldbecarefullydetermined
activities of AmDH and Nox was examined (Figure 4). The AmDH and Nox showed the highest 
tobalancethetwoenzymeactivities. Therefore,thepHdependenceoftheactivitiesofAmDHand
activity at pH 10.0 and 7.5, respectively. The results are in good agreement with previously reported 
Noxwasexamined(Figure4). TheAmDHandNoxshowedthehighestactivityatpH10.0and7.5,
results [18,26]. The specific activity of AmDH and Nox was 48 mU/mg and 95 U/mg at the optimum 
respectively. Theresultsareingoodagreementwithpreviouslyreportedresults[18,26]. Thespecific
pH, respectively. 
activityofAmDHandNoxwas48mU/mgand95U/mgattheoptimumpH,respectively.
100
80
)
%
(
y
vit 60
cti
a
e AmDH, Tris/HCl buffer
v
ati 40 AmDH, Glycine buffer
el Nox, Tris/HCl buffer
R Nox, Glycine buffer
20
0
7 8 9 10 11
pH

FiFgiugruere4 .4E. Efffefcetcot fopf HpHo nonth tehea catcivtiivtiytyo foAf AmmDDHHa nadndN Noxo.xF. Forort htheeA AmmDDHHa catcitvivitiytya sassasya,yt,h tehere raecatciotinosns 
wwereerep eprefrofromrmededi nin1 01000m mMMT rTisr/isH/HCCllb buufffefrer( p(pHH7 .70.–09–9.0.0))o orr1 01000m mMMg lgylycicnineeb buufffefrer( p(pHH9 .90.–01–11.10.)0) 
cocnotnatianiinningg2 020m mMMa 1a,11, 1m mMMN NADA+D,+a, nadndA AmmDDHH( 0(.01.515m mg/gm/mLL).).F Foorrt htheeN Nooxxa actcitvivitiytya sassasay,y0, .02.2m mMM 
NNAADDHHa nadnd0 .00.101m mg/gm/mLLo offN Nooxxw weerereu usesedd.. 
Next, the kinetic resolution of 10 mM rac‐2‐aminoheptane (a12) was carried out in 100 mM 
Next, the kinetic resolution of 10 mM rac-2-aminoheptane (a12) was carried out in 100 mM
TrTirsi/sH/HCCllb buufffeferr( p(pHH8 .80.–09–.90.)0)c ocnotnatianiinnigng1 1m mMMN NAADD+w+ witihthA AmmDDHH(1 (1m mg/gm/mLL))a nanddN Noxox( 1(1m mg/gm/mLL).). 
The enantiomeric excess (ee) value of a12 was 22%, 26%, and 19% at pH 8.0, 8.5, and 9.0, respectively 
Theenantiomericexcess(ee)valueofa12was22%,26%,and19%atpH8.0,8.5,and9.0,respectively
(Figure 5). Also, when the kinetic resolution was carried out with 5 mM rac‐α‐MBA (a1) at the 
(Figure5).Also,whenthekineticresolutionwascarriedoutwith5mMrac-α-MBA(a1)atthecondition
condition described above at pH 8.5 for 24 h, the ee value of a1 was only 20%. The results suggest that 
described above at pH 8.5 for 24 h, the ee value of a1 was only 20%. The results suggest that the
the activities of AmDH and Nox were affected by the substrate and/or product. Several amino acid 
activities of AmDH and Nox were affected by the substrate and/or product. Several amino acid
ddehehyyddroroggenenaasessesh haavveeb beeeenno obbsseerrvveedd ttoo ssuuffffeerr ffrroomm ssttrroonngg pprroodduucctt iinnhhiibbiittiioonn,, oofftteenn bbyy ththeeirir nnaatitvivee L‐
amino acid product [20]. In addition, in the asymmetric synthesis of amines from ketones by AmDH, 
L-aminoacidproduct[20]. Inaddition,intheasymmetricsynthesisofaminesfromketonesbyAmDH,
product inhibition has been previously suggested by the use of a biphasic reaction system to 
productinhibitionhasbeenpreviouslysuggestedbytheuseofabiphasicreactionsystemtoostensibly
ostensibly overcome inhibition in AmDH‐catalyzed reactions [27]. 
overcomeinhibitioninAmDH-catalyzedreactions[27].
Catalysts 2017, 7, 251   6 of 13 
Catalysts2017,7,251 6of13
Catalysts 2017, 7, 251   6 of 13 
30
30
25
25
20
20
)
%
)
%( 15
( e 15
ee
e pH 8.0
10 ppHH 88..05
10 ppHH 89..50
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 
FiguFriegu5r.eT 5h.e Tkhien ektiincertiecs orelusotiluontioonf o10f 1m0 MmMa1 2a.12R. eRaecaticotinonc ocnodnidtiitoionns:s:1 1m mMMN NAADD++,, 110000 mmMM TTrriiss//HHCCl l
buffer (pH 8.0–9.0), 1 mg/mL of AmDH, and 1 mg/mL of Nox. 
buffbeurf(fpeHr (p8H.0 –89.0.0–)9,.10)m, 1g m/mg/LmLof oAf AmmDDHH,a, nandd1 1m mgg//mmLL ooff NNooxx.. 
Therefore, the product inhibition of AmDH by ketones (2‐aminoheptanone or acetophenone) 
Therefore, the product inhibition of AmDH by ketones (2‐aminoheptanone or acetophenone) 
wTahse reexfaomrei,ntehde ipnr otdhue cptrinesheinbcitei oonf o1f0A mmMD Ha1b yankde toan12e s((F2i-gaumrein 6oah)e. pItna nthoen eporresaecnecteo pohfe 2n omneM) w2a‐s
was examined in the presence of 10 mM a1 and a12 (Figure 6a). In the presence of 2 mM 2‐
examaminiendohinepthtaenpornees eanncde aocfe1to0pmheMnoan1ea, nthdea e1n2z(yFmigeu srheo6wa)e.dI notnhlye p60re%s eanncde o49f%2 mofM its2 -oarmigiinnoahl eapcttiavnitoyn. e
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 
andacetophenone,theenzymeshowedonly60%and49%ofitsoriginalactivity. Theseresultsindicate
These results indicate that the continuous removal of ketone in the reaction mixture could improve 
the degree of conversion. Also, the activity inhibition of Nox by the substrate and product was 
thatthecontinuousremovalofketoneinthereactionmixturecouldimprovethedegreeofconversion.
the degree of conversion. Also, the activity inhibition of Nox by the substrate and product was 
examined (Figure 6b). The ketones did not significantly affect the activity of Nox, but the activity was 
Also,theactivityinhibitionofNoxbythesubstrateandproductwasexamined(Figure6b).Theketones
examined (Figure 6b). The ketones did not significantly affect the activity of Nox, but the activity was 
dramatically reduced in the presence of amine (5 mM). The relative activities of Nox in the presence 
diddnroatmsiagtniciafilclya nrteldyuacfefedc itnt htheea cptrievsietyncoef oNf oaxm,binuet (t5h emaMct)i.v Tithyew realsatdivraem acattiivciatlileys roefd Nuocexd inin ththe epprerseesnencec e
of a1 and a12 were only 38% and 74% of its original activity, respectively. The unexpected poor 
ofamofi na1e (a5nmd Ma1)2. Twheerree loantliyv e3a8c%ti vaintide s7o4f%N oofx iitns tohreigpinreasl eanccteivoitfya, 1reasnpdecat1iv2ewlye. rTehoen luyn3e8x%peacntedd 7p4o%oro f
conversion of the coupled AmDH/Nox reaction is partly due to product inhibition of AmDH by 
itsocroignivnearlsaiocnti voift yt,hree scpoeucptilvedel yA.mThDeHu/nNeoxxp ercetaecdtiopno oisr cpoanrvtleyr sdiuone otof tphreocdouucpt liendhiAbimtioDnH o/fN AomxDreHac tbiyo n
ketone and activity inhibition of Nox by the amine. 
ispakrettloynde uaendto apctriovdituyc itnihnihbiitbioitnio onf oNfoAx mbyD tHheb aymkienteo. neandactivityinhibitionofNoxbytheamine.
100
100
2-heptanone
80 2a-cheetopptahneonnoene
%) 80 acetophenone
)
%(
(y
activity e activit 6600
ative elativ 4400
elR
R
20
20
0
0
0 2 4 6 8 10
0 2 4 6 8 10
Concentration (mM)
Concentration (mM)  
(a)  
(a)
Figure6.Cont.
Catalysts2017,7,251 7of13
Catalysts 2017, 7, 251   7 of 13 
120
100
)
%
( 80
y
vit
cti 60
a
e
v
ati
el 40
R
20
0
CON a12 2-heptanone a1 acetophenone

(b)
Figure 6. Substrate and product inhibition of AmDH and Nox; (a) Effect of ketone concentration on 
Figure6.SubstrateandproductinhibitionofAmDHandNox;(a)Effectofketoneconcentrationon
AmDAmHDaHct iavcittiyv.itRy.e Racetaicotniocno cnodnidtiiotinosn:s:1 10000 mmMM TTrriiss//HHCCl lbbuufffefre r(p(HpH 8.58).,5 1), m1Mm MNANDA+, D10+ ,m1M0 ma1M2 (ao1r 2
a1), 2‐heptanone (or acetophenone) (0–25 mM), 0.3 mg/mL of AmDH; (b) Effect of amine and ketone 
(ora1),2-heptanone(oracetophenone)(0–25mM),0.3mg/mLofAmDH;(b)Effectofamineand
on Nox activity. Reaction conditions: 0.2 mM NADH, 5 mM amine or ketone, 100 mM Tris/HCl buffer 
ketoneonNoxactivity.Reactionconditions:0.2mMNADH,5mMamineorketone,100mMTris/HCl
(pH 8.5), and 0.05 mg/mL of Nox. (CON: control, the Nox activity in the absence of amine and ketone.) 
buffer(pH8.5),and0.05mg/mLofNox. (CON:control,theNoxactivityintheabsenceofamine
andketone.)
2.3. Kinetic Resolution of Amines Using E. coli Co‐Expressing AmDH and Nox 
The immobilization of enzymes on a solid support system improves catalyst stability, recycling, 
2.3. KineticResolutionofAminesUsingE.coliCo-ExpressingAmDHandNox
and longevity under industrially relevant process conditions [28]. For example, polyethyleneimine‐
acTthiveateimd magoabroilsiez abteiaodns woferee nuzsyedm teos noonn‐coavasloelnidtly sauttpapcho rpthossypshteomrylaimtedp rvoevrseisoncsa otfa ltyhset cosftaacbtoilristy ,
recyNclAinDg+,, FaAnDd+, laonndg PevLiPt,y crueantdinegr ainn dasussotcriiaatliloyn/dreislesovcainattiopnr eoqcuesilsibrciounmd iotfi opnhsos[p2h8o].rylaFteodr aenxda nmopnl‐e,
polypehtohsyplhenoreyimlatiende -accotfiavcatotersd  oang asruopsepobret a[d2s9]w. Aerne iunscerdeastoe nino ntu-cronvoavleern tnlyumabttearc hwapsh oosbpsherovreydla tined 
verscioomnspoarfitshoen ctoof ancotno‐rismNmAoDbi+li,zFeAd Den+z,yamndesP oLrP ,ncorne‐aitminmgoabniliazsesdo ccioaftaiocnto/rds.i sHsoocwiaetvieorn, einq uthiliisb rsituumdyo, f
phossupbhsotrraytlea taendda pnrdodnuocnt- pinhhoisbpithioonrsy loaft eednzcyomfaecst owrseroen thsue pmpaojortr [h2u9]r.dAlens fionrc rceaarsryeiinngt ouurnt othvee refnfuicmienbte r
waskoinbesetircv reedsoinluctioomn poaf rriasocenmtoic naomni-nimesm uosibnigli zAemdDenHz yamnde sNoorxn. oTnh-eimremfooreb, irliezceodmcboifnaacntot rEss.cHheoriwcheiva ecro,lii n
thiscsetlulsd cyo,‐seuxbpsrtersastiengan AdmpDroHd uacntdi nNhoibxi twioenrse oufseendz aysm beisocwatearleystthse (mvidaejo irnhfrua)r.d Tlehse fuorsec aorfr ywihnogleo‐ucetltlhs e
efficiinesntteakdin oetfi cisroelsaoteludt ieonnzyomfreasc einm bicioalmogiinceasl upsroincgesAsems DhaHs aan sdimNpolxe .aTnhde reecfoonroem,rieccaol madbvinaanntatgEes,c haesr tihchei a
coliccoesllts focor -ceexllp lryessissi anngdA pmurDifHicaatinodn Nis orexdwuceered u[3s0e]d. Aasnobtihoecra atadlvyasntsta(gveid oefi na fwrah).oTleh‐ceeull sreeaocftiwonh osyles-tceemll s
is that cofactor‐dependent enzymatic reactions can be effectively performed by using endogenous 
insteadofisolatedenzymesinbiologicalprocesseshasasimpleandeconomicaladvantage,asthecost
cofactors without adding costly cofactors [30]. Sometimes, a whole cell reaction is less susceptible to 
forcelllysisandpurificationisreduced[30]. Anotheradvantageofawhole-cellreactionsystemisthat
enzyme inhibition by substrate and product due to the diffusion barrier of the cell membrane [31]. 
cofactor-dependentenzymaticreactionscanbeeffectivelyperformedbyusingendogenouscofactors
In order to develop a recombinant E. coli system expressing both enzymes in a single cell, the 
withoutaddingcostlycofactors[30]. Sometimes,awholecellreactionislesssusceptibletoenzyme
AmDH and Nox genes were cloned into pET24ma and pETDuet vectors, respectively. After 
inhibitionbysubstrateandproductduetothediffusionbarrierofthecellmembrane[31].
overnight induction at 20 °C, the cells were harvested, washed twice with 100 mM Tris/HCl buffer 
InordertodeveloparecombinantE.colisystemexpressingbothenzymesinasinglecell,the
(pH 8.5), and used for the whole‐cell reaction. When kinetic resolutions of 10 mM a12 and 5 mM a1 
AmDHandNoxgeneswereclonedintopET24maandpETDuetvectors,respectively. Afterovernight
were carried out in the 100 mM Tris/HCl buffer (pH 8.5) with whole‐cell (20 mg dry cell weight 
inductionat20◦C,thecellswereharvested,washedtwicewith100mMTris/HClbuffer(pH8.5),
(DCW)/mL) without adding cofactor NAD+ for 24 h, both of the amines were successfully resolved 
and used for the whole-cell reaction. When kinetic resolutions of 10 mM a12 and 5 mM a1 were
into the (S)‐form with a high ee (>99%). These results clearly showed that the use of whole‐cell was 
carriedoutinthe100mMTris/HClbuffer(pH8.5)withwhole-cell(20mgdrycellweight(DCW)/mL)
much more efficient than the reaction using purified enzymes that gave 26% ee (Figure 6b). The Shell 
withgorouutpa dredpionrgtecdo tfhaactt oinr NthAe aDs+ymfomre2tr4ich s,ybnoththesoisf otfh cehairmali anmesinwe ebrye AsumcDceHs,s ftuhell ylyroepshoillvizeedd iwnhtoolteh‐e
(S)-fcoerllm gawveit hbeattehri gcohnevee(r>si9o9n%s b).otThh iens ethree asbusletsnccele aanrdly psrhesoewnceed otfh caot‐stohleveunste hoefpwtanheo lteh-acne lllywopahsilmizuedch 
morleyseaftfie c[i2e0n]t. tThhaen utshee orfe awcthioolne‐ucesliln agpppuarreifinteldy erendzuycmese sthteh astubgsatvraete2/6p%rodeeuc(tF iignuhribeit6iobn)., aTnhde/oSrh ietl l
grouinpcrreeapsoerst ethdet shtaatbiinlittyh oefa tshyem enmzeytmriecss. yWnhtheens tihseo rfecahcitriaolnas mwiinthe 2b0y, 3A0m, 4D0,H an,tdh 5e0l ymoMph ai1li2z ewderwe hcaorleri-ecde ll
gavoeubte attt eprHc o8n.5v uerssiniogn wshboolteh‐cienll t(h25e mabgs eDnCcWe a/mndL)p froers 2e4n che, tohfec eoe- vsoalluveesn tofh (eSp)‐taa1n2e wthearen >l9y9o%p,h 8il7iz%e,d 
lysa te [20]. The use of whole-cell apparently reduces the substrate/product inhibition, and/or it
Catalysts2017,7,251 8of13
Catalysts 2017, 7, 251   8 of 13 
increasesthestabilityoftheenzymes. Whenthereactionswith20,30,40,and50mMa12werecarried
outatpH8.5usingwhole-cell(25mgDCW/mL)for24h,theeevaluesof(S)-a12were>99%,87%,
48%, and 30%, respectively (Figure 7). At initial substrate concentrations of 20, 40, and 50 mM, the 
48%,and30%,respectively(Figure7). Atinitialsubstrateconcentrationsof20,40,and50mM,the
produced 2‐heptanone, determined by the consumed a12, was 14.0, 12.9, and 11.6 mM, respectively. 
produced2-heptanone,determinedbytheconsumeda12,was14.0,12.9,and11.6mM,respectively.
When 2‐heptanone is accumulated to some extent (11–14 mM), the reaction does not seem to proceed 
When2-heptanoneisaccumulatedtosomeextent(11–14mM),thereactiondoesnotseemtoproceed
any further. The main reason for the lower ee at higher substrate concentrations seems to be the 
any further. The main reason for the lower ee at higher substrate concentrations seems to be the
inhibition of AmDH by 2‐heptanone. 
inhibitionofAmDHby2-heptanone.
100
80
20 mM
30 mM
) 60 40 mM
%
50 mM
(
e
e
40
20
0
0 5 10 15 20 25
Time (h)

FigFuigreur7e. R7e. aRcteioanctpiorno fiplersoofifleths eokfi ntehteic kreinsoeltuict iornesaotludtififoenre natt cdoinffceernetnrat ticoonnscoefnat1ra2t.iRoneasc toiof nac1o2n. dRiteiaocntsi:on 
a12co(0n–d5i0tiomnMs: ),a11020 (m0–M50T rmisM/H), C1l0b0u mffeMr( pTHris8/.H5)C,wl hboulfef-ecre l(lp(H25 8m.5g),d wryhcoellel‐wceelilg h(2t5( DmCgW d)/rym cLe).ll weight 
(DCW)/mL). 
Thenextstepwastoincreasethefinalproduct’sconcentration,whichcouldbeaccomplished
The next step was to increase the final product’s concentration, which could be accomplished by 
byenhancingthebiocatalystconcentrationsinthereactionmedium. Comparedtoa12,thekinetic
enhancing the biocatalyst concentrations in the reaction medium. Compared to a12, the kinetic 
resolution of a1 is much more difficult due to the poor reactivity of AmDH for a1, a more severe
resolution of a1 is much more difficult due to the poor reactivity of AmDH for a1, a more severe 
productinhibitionbythecorrespondingketone,acetophenone,andastrongerinhibitionofNoxby
product inhibition by the corresponding ketone, acetophenone, and a stronger inhibition of Nox by 
a1 (Figure 6). After the concentrations of a1 and a12 were set at 20 mM and 50 mM, respectively,
a1 (Figure 6). After the concentrations of a1 and a12 were set at 20 mM and 50 mM, respectively, the 
the reaction was performed to determine the amount of whole-cell catalyst required to obtain the
reaction was performed to determine the amount of whole‐cell catalyst required to obtain the 
opticallypure(S)-amine(Figure8). Theminimumamountsofwhole-cellrequiredtoresolve50mM
optically pure (S)‐amine (Figure 8). The minimum amounts of whole‐cell required to resolve 50 mM 
a12and20mMa1into(S)-form(>99%ee)were60and100mgofDCW/mL,respectively. Next,the
a12 and 20 mM a1 into (S)‐form (>99% ee) were 60 and 100 mg of DCW/mL, respectively. Next, the 
reactionprofilesforthekineticresolutionofa1anda12weredetermined(Figure9). Inthe50mMa12
reaction profiles for the kinetic resolution of a1 and a12 were determined (Figure 9). In the 50 mM 
reaction,40mgDCW/mLofwhole-cellgaveonly77%eefor24h(Figure8),but60mgDCW/mLof
a12 reaction, 40 mg DCW/mL of whole‐cell gave only 77% ee for 24 h (Figure 8), but 60 mg DCW/mL 
whole-cellgave>99%eein1h(Figure9). Also,theeevalueofa1was95%in24hofreactionwith
of whole‐cell gave >99% ee in 1 h (Figure 9). Also, the ee value of a1 was 95% in 24 h of reaction with 
80mgDCW/mLofwhole-cell;however,theeevaluebecame>99%after5hofreactionwith100mg
80 mg DCW/mL of whole‐cell; however, the ee value became >99% after 5 h of reaction with 100 mg 
DCW/mLofwhole-cell. Thisresultisconsistentwiththeobservationthattheenzymeactivityof
DCW/mL of whole‐cell. This result is consistent with the observation that the enzyme activity of 
AmDHisinhibitedbytheketoneproduct.
AmDH is inhibited by the ketone product.
Catalysts 2017, 7, 251   9 of 13 
CaCtaatlyalsytsst2s0 21071,77,, 72,5 2151   99o fo1f 313 
100
100
a12
80 a1a21
80 a1
60
%) 60
%) (e
(ee 40
e
40
20
20
0
0 0 20 40 60 80 100
0 20 40 60 80 100
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. 
FiFgiRugeruaerce8t .i8oT.n hT cehoeen fedffeifcteitcootn fost:f h 1teh00ea  mmamoMuo unTntritos fo/Hfw wChlho bloeul-efc‐fecelerl l(lop onHnt  h8the.5ek) ,ki n5ine0t emitcicMr er seaos1ol2ul u(toitoiron 2n0o  ofmf5 M500m  am1MM), aa a1n12d2o  worrh2 2o00lme m‐cMeMlla  a1(11.0. –
RRea1ec0at0cito minongc  oDconCndWditiit/omionnLs:s).:  110000 mmMM TTrriiss//HHCCll bbuuffffeerr (p(pHH 8.85.)5,) 5,05 0mmMM a1a21 (2or(o 2r02 m0Mm Ma1)a, 1a)n,da nwdhwolhe‐ocleel-lc (e1l0l–
(1100–01 0m0gm DgCDWC/Wm/Lm). L).
100
100
80
a12
80
a1a21
a1
) 60
%
%) (e 60
(ee
e 40
40
20
20
0
0 0 2 4 6 8 10
0 2 4 6 8 10
Time (h)

Time (h)

FigFuirgeu9r.eR 9e. aRcetiaocntiporno fiplreosfiolefst hoef kthinee ktiicnreetsioc lruetsioonluotifo5n0 omf M50a m12Mo ra2102 morM 20a 1m.SMix aty1.m Siilxlitgyr mamilsli(g6r0ammsg )(60 
anFdimg1ug0r)0 ea m9n.gd R D1eCa0c0Wt imo/nmg  pLDrooCffWiwle/hsm ooLlfe  -tochefe l lwkwihnaoeslteiuc‐cs reeedlls ofwolurattsih ouensr eoedaf c 5tf0ioo rmn tMohfe 5 ar01e2ma coMtri o2an01  2mofoM r5 20a0 1mm. SMMix taay11 2,m roeilsrlp ig2er0ca tmimvesM l(y 6.a01 , 
mrge)s paencdti v1e0l0y . mg DCW/mL of whole‐cell was used for the reaction of 50 mM a12 or 20 mM a1, 
respectively. 
Toinvestigatetheutilityofawhole-cellsystemexpressingAmDHandNox,thekineticresolutions
To investigate the utility of a whole‐cell system expressing AmDH and Nox, the kinetic 
ofvariousrac-amines(10mM)wereperformed(Table1). a5,a7,anda8weresuccessfullyresolved
resoTluo tiionnvse ostfi gvaatreio uthse r auc‐tailmityin eosf  (1a0  wmhMol)e w‐ceerlle  psyesrftoemrm eedxp (Treasbslien g1) .A am5, Da7H, a nandd a 8N woexr,e  tshuec ckeisnsefutilcl y 
into (S)-form with a high ee (>99%). However, the kinetic resolutions of a4, a6, and a11 gave 66%,
rerseosloulvtieodn sin otfo v (aSr)i‐ofoursm ra wc‐iatmh ian hesig (h1 0e em (>M99) %w)e.r He powerefovremr, etdhe ( Tkainbeleti 1c )r. eas5o,l au7ti, oannsd o af8 a w4, ear6e,  sauncdc eas1s1f uglalyv e 
28%,and33%ee,respectively. Amongthefluorinatedamines(a4,a5,anda6),a4isthemostreactive
re6s6o%lv, e2d8 %in,t oa n(Sd) ‐3fo3%rm e we, irtehs ap ehcitgihve eley (. >A99m%o)n. gH tohwe efvluero,r tihnea tkeidn eatmic irneesso l(ua4ti,o an5s,  oafn ad4 ,a a66),,  aan4 dis a 1th1e g mavoes t 
substrate,butonlya5wasconvertedintoanopticallypureform. Also,althougha11wasthemost
66re%a,c t2i8v%e ,s uabnsdt r3a3te%,  beue,t  roenslpye ac5ti vwealys .c Aonmvoenrtge dth ine tfol uaonr ionpattiecdal laym piunrees  f(oar4m, a. 5A, lsaon,d a lat6h)o, uag4h i sa 1th1 ew maso tsht e 
reactivesubstrateinthetestedsubstrates(Figure2),theeevalueofa11wasonly33%. Ingeneral,the
remacotsitv ree saucbtisvter astueb, sbturat toen ilny  tah5e  wteasste cdo nsuvbesrttreadt eins t(oF iagnu orep 2ti)c, atlhlye  epeu vrael ufoer omf .a A11ls wo,a asl tohnolyu g3h3% a1. 1In w gaesn tehrea l, 
morereactivesubstratesareexpectedtohavehigherconversionratesintoproduct. However,itis
most reactive substrate in the tested substrates (Figure 2), the ee value of a11 was only 33%. In general, 
the more reactive substrates are expected to have higher conversion rates into product. However, it 
likelythattherewasnoclearcorrelationbetweenfinalconversionandsubstratereactivitybecauseof
thise  lmikoerlye  trheaact ttihveer esu wbastsr natoe sc laeraer  ceoxrpreecltaetdio nto b heatwvee ehnig fhinear lc coonnvveerrssiioonn r aanteds  siunbtos tprartoed ruecatc. tHivoitwy ebveecra,u ist e 
is likely that there was no clear correlation between final conversion and substrate reactivity because
Catalysts2017,7,251 10of13
anunclearcorrelationbetweenthereactivityandproductinhibitionofAmDHbythecorresponding
ketone. Inthecaseofa4,a6,anda11,thereactionswerecarriedoutatalowerconcentration(5mM)
withthesamecondition,andtheeevalueswere77%,78%,and51%,respectively(Table1). Although
theeevalueincreasedmorethanwhen10mMsubstratewasused,theopticallypureformcouldnot
beobtained.
Table1.KineticresolutionofvariousaminesusingAmDH/Noxa.
Substrate Conc.(mM) Conv.(%) eeS(%)
a4 10 40 66
a4 5 44 77
a5 10 50 >99
a6b 10 22 28
a6b 5 44 78
a7 10 50 >99
a8 10 50 >99
a11 10 25 33
a11 5 34 51
aThereactionconditions: 5mLofreactionvolume,120mgDCW/mLofwhole-cell,Tris/HClbuffer(100mM,
pH8.5),and5or10mMracemicsubstrate(seeFigure2)fora24hreactionat37◦C.Conversionandeewere
determinedbyCrownpakCR(+)column;bConversionandeeweredeterminedbyC18Symmetrycolumnafter
GITCderivatization.
3. MaterialsandMethods
3.1. Chemicals
Allamines(a1–a12),GITC(2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosylisothiocynate),cofactors
(NADHandNAD+),andisopropyl-β-D-thiogalactopyranosidewereobtainedfromSigma-Aldrich
Yongin,Korea.
3.2. EnzymeExpressionandPurification
TheAmDHandNoxgenes[18,24]weresynthesizedbyBioneerCorporation(Daejeon,Korea),and
eachgenewastheninsertedintoIPTG-induciblepET24mavectors. Theplasmidwasthenintroduced
intoE.coli(BL21)cellsandthetransformantsweregrownat37◦Cin1LLB-containingkanamycin
(100µg/mL)[32]. WhentheOD reached0.5,IPTGwasaddedtoafinalconcentrationof0.5mM.
600
Afterovernightinductionat20◦C,thecellswereharvestedandwashedtwicewith100mMTris/HCl
buffer(pH8.5). Aftercentrifugation,thecellpelletswereresuspendedin10mLvolumeof20mM
Tris/HClbuffer(pH8.5). Thesuspensionwasthensubjectedtoultrasonicdisruption. Thedisruption
periodwas5swith10sintervalsinanicebathforadurationof30min. TheC-terminalHis6-tagged
enzymewaspurifiedat4◦ConaNi-NTAagaroseresinobtainedfromQiagen(Hilden,Germany).
Theelutedsolutioncontainingpurifiedproteinwasdialyzedagainst50mMpotassiumphosphate
buffer(pH8.5)andconcentratedusinganAmiconPM-10ultrafiltrationunit. Glycerolwasaddedto
thepurifiedenzymesolution(25%)anditwasstoredat−20◦Cforfurtherstudy.
For the co-expression of AmDH and Nox, the AmDH gene was amplified and subsequently
insertedintothepETDuet-1(Novagen,Madison,WI,USA).Theplasmidwasthenintroducedinto
the E. coli (BL21) bearing pET24ma:Nox. Then, enzymes were overexpressed in the presence of
kanamycin(100µg/mL)andampicillin(100µg/mL)asdescribeabove. Afterharvesting,thecells
werewashedtwicewith100mMTris/HClbuffer(pH8.5)andtheywereimmediatelyusedforthe
whole-cellreaction.
3.3. EnzymeAssay
The activities of AmDH and Nox were determined using an NADH-dependent (340 nm,
λ =6220M−1cm−1) spectrophotometric assay. Activity is calculated from the stoichiometric
340