Enantioselective acetylation of (R,S)-atenolol: The use of Candidarugosa lipases immobilized onto magnetic chitosan nanoparticles inenzyme-catalyzed biotransformation

ContentslistsavailableatScienceDirect

Journal

of

Molecular

Catalysis

B:

Enzymatic

jo u r n al h om ep ag e :w w w . e l s e v i e r . c o m / l o c a t e / m o l c a t b

Enantioselective

acetylation

of

(R,S)-atenolol:

The

use

of

Candida

rugosa

lipases

immobilized

onto

magnetic

chitosan

nanoparticles

in

enzyme-catalyzed

biotransformation

Adam

Sikora

_,

_Dorota

_{Chełminiak-Dudkiewicz}

_,

_Tomasz

_Siódmiak

_,

Agata

Tarczykowska

_,

_Wiktor

_Dariusz

_Sroka

_,

_Marta

_{Ziegler-Borowska}

_,

Michał

Piotr

Marszałł

a_{DepartmentofMedicinalChemistry,CollegiumMedicuminBydgoszcz,FacultyofPharmacy,NicolausCopernicusUniversityinToru´n,Dr.A.Jurasza2,}

85-089Bydgoszcz,Poland

b_{ChairofChemistryandPhotochemistryofPolymers,FacultyofChemistry,NicolausCopernicusUniversityinToru´n,Gagarina7,87-100Toru´n,Poland}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received17August2016 Receivedinrevisedform 15September2016 Accepted19September2016 Availableonline20September2016 Keywords: Enantioselectiveacetylation (R,S)-atenolol Magneticnanoparticles Lipase

a

b

s

t

r

a

c

t

Thispaperdescribestheenzymeimmobilizationprotocolaswellastheenzymaticmethodforthedirect resolutionof(R,S)-atenolol.Theusedmagneticenzymecarrierspossessontheirsurfacenew-synthetized chitosanderivativeswithfreeaminegroupsdistancedbyethylorbutylchain.Additionallythe cat-alyticactivityoftwotypesofcommerciallyavailablelipasesfromCandidarugosaimmobilizedontotwo differentmagneticnanoparticleswerecompared.Thehighestvaluesofenantioselectivity(E=66.9), enan-tiomericexcessofproduct(eep=94.1%)andconversion(c=41.84%)wereobtainedbyusinglipasefrom

CandidarugosaOFimmobilizedontoFe3O4-CS-EtNH2.Thestudyconfirmedthatevenafter5reaction

cyclestheimmobilizedlipasemaintainitshighcatalyticactivity.

©2016ElsevierB.V.Allrightsreserved.

1. Introduction

Nowadays,theuseofbiotechnologyisanalternativeapproach, which offersmore environmentallyand economicallyattractive waystoobtainbioactivecompounds[1–3].Lipases(E.C.3.1.1.3)are ubiquitousenzymes,widelyusedinseveralindustrialapplications becauseoftheirability tocatalyseenantioselective biotransfor-mation[2,4].Inrecentyears,theseenzymeshavebeenusefulin organicsynthesisand kineticresolution ofracemiccompounds, whicharewelldescribedinnumerouspapers[1,5–11].However, lipasesasproteinsareextremelysensitivetoreactionmediaaswell astemperature,whichmayinfluencetheirenantioselectivityand catalyticactivity.Additionally,theutilizationofanativeenzyme fromareactionmixtureis anotherseriousissue,particularlyin industrial processes, because of the difficulty in its separation, recyclingandreuse,whichisdirectlyrelatedtohightotalcostof biotransformation[12,13].Thus,theapplicationoflipasesinthe

∗ Correspondingauthorat:CollegiumMedicuminBydgoszcz,Jurasza2,85-089 Bydgoszcz,Poland.

E-mailaddress:mmars@cm.umk.pl(M.P.Marszałł).

industryislimited.Nevertheless,theprocessofimmobilizationof enzymeonthesurfaceofnanoparticlescouldprovidemany addi-tionaladvantagesovertheuseofnativelipases,becauseitdoes notonlyallowtoreusetheboundedenzymeinanothercatalytic systembutalsoincreasesthecatalyticactivityandoperational sta-bilityofbiocatalysts[14,15],andthereforemanyimmobilization techniqueshavebeeninvestigated.

Magneticnanoparticlesarepromisingmaterialsformany appli-cations:biomedical,catalytic,analyticalandindustrial[14,16–18]. Theimmobilizationofenzymesontoa supportgivesthe oppor-tunity for its reusability [19–21]. The application of magnetic nanoparticles as enzyme carriers allows to easily separate the boundedbiocatalystfromreactionmixturebyattractingwithan externalmagneticfield.Itgivesthepossibilitytoreusetheenzyme inanothercatalyticsystem,sincetheremovalofenzymesfrom thereactionmixtureiseasy,itallowsforsimpleproduct purifica-tion[12,13,22,23].Additionally,magneticsupportsbasedonFe3O4

areusuallynontoxic[16].Duetothesimplicityoftheirsynthesis andmodificationofcommonlylargesurfaces,magnetite(Fe3O4)

nanoparticlesareverypopularandhavegainedagreatattention inmaterialsscience.

http://dx.doi.org/10.1016/j.molcatb.2016.09.017

44 A.Sikoraetal./JournalofMolecularCatalysisB:Enzymatic134(2016)43–50

Atenolol chemically knownas 2-(4-(2-hydroxy-3-(propan-2-ylamino)propoxy)phenyl)acetamideisoneofthemostimportant ␤-adrenolyticdrugwidelyusedintreatmentofhypertensionand cardiovasculardisorders[24].Duetothefact,␤-blockerspossess asymmetriccarbonatomintheirstructure,theyarepresentedin twoenantiomericforms[25,26].Itwasreportedbymanystudies, onlytheS-enantiomersofthesedrugspossessthedesired ther-apeuticeffect,whereastheadministrationoftheracematemay causedangeroussideeffectssuchasbronchoconstrictionor dia-betes [6,27,28]. Nevertheless, ␤-blockers are still commercially availabledrugsmainlyusedinmedicineasracemates.

Themainaimofthestudypresented hereinwastoperform thekineticresolutionof(R,S)-atenololwiththeuseoftwolipases fromCandidarugosa(OFandMY)immobilizedontotwotypesof notcommerciallyavailablemagnetic nanoparticles,which were denovosynthesised.Additionally,thereusabilityofimmobilized enzymewasinvestigated,andthehighcatalyticactivityofenzyme afterfivereactioncycleswasconfirmed.Whatismore,thenew analyticalmethodwiththeuseofchiralstationaryphasesandUPLC systemcoupledwithmassspectrometrywassuggestedinorderto determinethequalityandquantityoftheatenololenantiomersand itsderivatives.

2. Materialsandmethods 2.1. Chemicals

(R,S)-atenolol, (R)-atenolol, toluene, isopropenyl acetate, 2-propanol, acetonitrile, Iron(II) chloride tetrahydrate, iron(III) chloride hexahydrate, chitosan (low molecular weight), glutaraldehyde, epichlorohydrine, sodium periodate, ethylenediamine, acetic acid, sodium hydroxide, EDC (N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride), sulfo-NHS (N-hydroxy-sulfosuccinimide sodium salt), glycine, acetic acid, ethanol, diethylamine, 1,4-diaminobutane, acetyl chloride were purchased from Sigma-Aldrich Co. (Stainhaim, Germany).Sodiumsulphateanhydrous,sodiumsulphate decahy-drate,molecularsieves4Å,ethanolwerepurchased fromPOCH S.A.(Gliwice,Poland). Lipases OF and MY fromCandida rugosa wereagiftfromMeitoSangyoCo.,Ltd.(Japan).Waterusedinthe study wasprepared using a Milli-Q Water Purification System (Millipore,Bedford,MA,USA).Allincubationswereperformedat adjustedtemperatureandnumberofrotation.

2.2. Instrumentation

TheRefrigeratedCentriVapConcentratorwaspurchasedfrom Labconco,theInkubator1000andUnimax1010werepurchased fromHeidolph,theFT-IRSpectrometermodelSpectrumTwowas purchased fromPerkin Elmer, theShimadzu UPLC–MS/MS sys-tem(Japan)equippedwithsolventdeliverytwopumpsLC-30AD combinedwithgradientsystems,degassermodelDGU-20A5,an autosamplermodel SIL-30AC,a column oven model CTO-20AC, UVdetectormodelSPD-M20Aandtriplequadrupolemass spec-trometer detector LCMS-8030. Lux Cellulose-2 (LC-2) column withcellulosetris(3-chloro-4-methylphenylcarbamate)stationary phase and Guard Cartridge Systemmodel KJO-4282 were pur-chasedfromPhenomenexCo.

2.3. Chromatographicconditions

The most appropriate chromatographic conditions for the resolution of racemic atenolol and its acetylated forms were optimizedwithLuxCellulose-2(4.6mm_×250mm_×3_␮m)HPLC column,which was chosenfor the chromatographicseparation inpolar/organicphasemode(Fig.1).Themobilephaseconsisted

ofacetonitrile/2-propanol/diethylaminein ratio98/2/0.1(v/v/v). Thechromatographicprocesswasoperatedat30◦C.Thedetection wasmadeusingtriplequadrupolemass spectrometerin multi-plereactionmonitoringmode(MRM).ThetransitionsofMRMfor atenololwere 267.20>256.05; 267.20>190.05; 267.20>116.10; whereasforatenololacetatewere309.2>158.10;309.20>145.15; 309.20>116.10.In ordertodetermineoptical purityand enan-tioselectivityofenantioselectiveacetylation,theequationswere usedbasingonpeakareasfromchromatogramachievedin chro-matographicseparationof(R,S)-atenololanditsacetylatedforms. Thepercentageenantiomericexcessesofthesubstrate(ees)and

product(eep),conversion(c)aswellasenantioselectivity(E)were

calculated,asbelow[11]: ees=|R_R−₊S|_S ×100% eep=|R_R−₊S|_S ×100% c= ees ees+eep×100% E= ln













whereRwasvalues ofpeakareasfor (R)-atenololanditsester, whereasSwasvaluesofpeakareasfor(S)-atenololanditsester.

2.4. Synthesisofmagneticchitosannanoparticles: Fe3O4-CS-EtNH2andFe3O4-CS-BuNH2

Chitosan(0.2g)wasaddedinto20mLof1%aceticacid solu-tion and mechanically stirred at roomtemperature for 20min. Afterthat,iron(II) chloridetetrahydrate(0.74g,3.75mmol) and iron(III)chloridehexahydrate(2.02g,7.5mmol)wereadded(1:2 molarratio)andtheresultingsolutionwaschemicallyprecipitated atroomtemperaturebyaddingdropwise30%solutionofNaOH (7mL).Theblackmixturewasformed,separatedbyfiltrationand washedbydeionizedwaterforfivetimes.Next,10mLof bicar-bonatebufferpH−10and10mLof5%glutaraldehydesolution wereaddedandthecomposedmixturewasmechanicallystirred atroomtemperaturefor1h.Inordertodifferentiatethesynthesis betweentheFe3O4-CS-EtNH2andFe3O4-CS-BuNH2nanoparticles,

the20mLofaqueoussolutionofethylenediamine(2.4g,40mmol) or1,4-diaminobutane(3.53g,40mmol),respectively,wasadded andthemixtureswerestirredatroomtemperaturefor2h.The obtainedmagneticmaterialswererecoveredfromthesuspension byapplyingamagnet,washedfivetimeswithdeionizedwaterand driedundervacuumat50◦Cfor24h(Fig.2).

2.5. Lipaseimmobilizationontochitosanmagneticnanoparticles withtheuseofEDCandsulfo-NHS

ThecovalentcouplingofCandida rugosalipase ontothe sur-face of chitosan magnetic nanoparticles was performed bythe formationofanamidebondbetweenthecarboxylgroupoflipase andtheprimaryaminegroupof thenanoparticle.The prepara-tionprocedurewasperformedaccordingtothepreviouslyreported methodology[16,19],withfewmodifications(Fig.3).Inbrief,the 36.5mgoflipaseOFandMYweresuspendedseparatelyin1.0mL of50mMphosphatebuffer(pH 6.4).Afterthat,2mg ofEDC in 50␮L of phosphate bufferwasadded to each tubewith lipase suspension.Thesolutionswereincubatedat21◦Candshakenfor 1h. Afterthattime, 2.4mgofsulfo-NHSwasdissolvedin50␮L ofphosphatebuffer(50mM,pH=6.4)and addedtoeachofthe

Fig.1. Chromatogramofracemicatenololanditsestersafter240hofkineticresolutionof(R,S)-atenololwiththeuseofCandidarugosalipaseOFimmobilizedonFe3O4

-CS-EtNH2:(R)-enantiomerofatenololacetate(tR=7.678),(S)-enantiomerofatenololacetate(tR=8478),(S)-atenolol(tR=17.398),(R)-atenolol(tR=19.754).Chromatographic

conditions:LuxCellulose-2(4.6×250mm×3␮m)column,mobilephase:acetonitrile/2-propanol/diethylamine(98/2/0.1v/v/v),F=1mL/min,t=30◦_C.

tubecontaininglipaseandEDC.Thesolutionswerealsoincubated at21◦Cfor1handshaken.Further,50mgofchitosanmagnetic nanoparticles(Fe3O4-CS-EtNH2andFe3O4-CS-BuNH2)wereplaced

intoseparate2mLcentrifugetubesandsonicatedfor10minwith 50mM phosphate buffer (pH 6.4). Then, all prepared solutions withlipase-EDC-sulfo-NHScomplexweretransferredintoseparate centrifugetubesalongwiththepreviouslyrinsedchitosan mag-neticnanoparticles.Next,theresultingmixtureswereincubated at21◦Cfor2handshakenat600rpminathermomixer.Finally, lipase-immobilizedchitosannanoparticleswererinsedthreetimes with0.5mLof50mMphosphatebuffer(pH6.4)andweredried overnightat30◦C.Theresultedlipase-immobilizedmagnetic sup-portswereusedintheenantioselectiveacetylation.Theamountof

Fig.2.TEMimageofchitosanmagneticnanoparticles−Fe3O4-CS-EtNH2.

immobilizedlipaseadsorbedontothemagneticnanoparticleswas determinedbymeasuringtheinitialconcentrationoflipaseand itsfinalconcentrationinsupernatantafterimmobilizationusing theBradfordproteinassaymethod.Acalibrationcurvewas con-structedwithlipaseOFandMYsuspendedin50mMphosphate bufferofknownconcentration(1–9mg/mL)andthenwasusedin thecalculationofproteinintheinitialsolutionandsupernatant.

2.6. Chemicalacetylationof(R,S)-atenolol

The (R,S)-atenolol wasacetylated according to the reported methodologywithfewmodifications.Briefly,(R,S)-atenolol(0.02g; 0.075mmol) was refluxed with dichloromethane (20mL) and acetylchloride(8_␮L;0.076mmol)wasveryslowlyadded.After that,thereactionmixturewasincubatedat30◦Cfor2h,and fur-thersuccessivelywashedwithequalvolumesofsaturatedaqueous sodiumbicarbonateandbrine.Theorganiclayerwascollectedand evaporatedtodryness undervacuumtoafford atenololacetate. Finally,resultedderivateofatenololwasusedasstandardinorder toestablishoptimalchromatographicmethodwhichallowedto determinequantitativelyracemicatenololanditsacetylatedform.

2.7. Enzymaticacetylationof(R,S)-atenolol

Enzymaticacetylationof(R,S)-atenololwasperformedin25mL flasks.Thereactionwascarriedoutin10mLofreactionmedium. Thereactionmixturewascomposedoftoluene,racemicatenolol (3.0mg, 0.01mM)and isopropenylacetate (2␮L, 0.018mM)as acetyldonor(Fig.4).Theenzymaticacetylationwasstartedbythe directadditionofimmobilized lipase.Thereactionmixturewas shakenat250rpmat35◦C.Theprocessofenantioselective acety-lationwasmonitoredwiththeuseofchiralstationaryphases.The sampleswerewithdrawnatestablishedtimepoints(0,24,48,72, 96,120,144,168,192,214,240h)andafterthatwereevaporated andredissolvedinacetonitrileandafterfiltrationinjectedonthe UPLC–MS/MSsystem.

46 A.Sikoraetal./JournalofMolecularCatalysisB:Enzymatic134(2016)43–50

Fig.3. ImmobilizationoflipasesfromCandidarugosausingEDCandsulfo-NHSontothesurfaceofchitosanmagneticnanoparticles.

3. Resultsanddiscussion

3.1. Characterizationofthenativeandimmobilizedlipasefrom Candidarugosaaswellasmagneticchitosannanoparticles: Fe3O4-CS-EtNH2andFe3O4-CS-BuNH2

Inpresentedstudy,themagneticchitosannanoparticleswith aminegroupswereemployedfortheimmobilizationoflipaseusing couplingreactionbetweentheaminogroupsofthemagnetic sup-portandcarboxylgroupsofthebiocatalysts.Thestructureofthe preparednanoparticles,nativelipasesfromCandidarugosa(OFand MY),andimmobilizedbiocatalystsontonanoparticleswere char-acterizedwiththeAttenuatedTotalReflectanceFourierTransform Infrared(ATR-FTIR) − Spectrum TwoTM _{(Perkin Elmer). Spectra}

wererecordedonovertheregionfrom4000to400cm−1(Fig.5). TheC NandN Hcharacteristicsvibrationpeaksatabout1640and 1540cm−1wereobservedforbothmagneticchitosannanoparticles (Fe3O4-CS-EtNH2andFe3O4-CS-BuNH2).Thepeaksat1410cm−1

andat860cm−1aswellas766cm−1increasedwithN H stretch-ingandN Hwaggingvibrationsinprimaryaminegroups.These peaks were not observed in nanoparticles coated with pure

Fig.4.Enantioselectiveacetylationofracemicatenololwiththeuseofimmobilized lipaseasbiocatalyst.

chitosan[29].Thesignalat571cm−1 wasassignedtotheFe O group of magnetite. On pure lipase spectrum the characteris-tic bandfrom carboxylicgroups at 1649,1540cm−1 and 1646, 1543cm−1forCandidarugosalipaseOFandMY,respectively,were

Fig.5.FTIRspectraofthenativelipasesfromCandidarugosa(MYandOF),chitosanmagneticnanoparticles(Fe3O4-CS-Et(NH2)andFe3O4-CS-Bu(NH2)),andimmobilized

lipasesontothemagneticnanoparticles.

observed.Afterlipaseimmobilizationthespectrumshowedonly twoweakpeaksat1632,1651cm−1 (CandidarugosalipaseOF), and1639,1563cm−1(CandidarugosalipaseMY)correspondingto thecharacteristicamidebondformationwhichprovesthe cova-lentlipasebonding,whichwaspreviouslyreportedbynumerous studies[19,30,31].

3.2. ApplicationofimmobilizedCandidarugosalipaseonto chitosannanoparticles

TwocommerciallyavailablelipasesfromCandidarugosaOFand MYwerecoupledtosurfaceoftwotypesofchitosannanoparticles andfurtherusedinenantioselectiveacetylationof(R,S)-atenolol. Duetothedissimilarnanomaterialsanddifferenttypeoflipases, bothenzymesshoweddifferenteffectsonthekineticresolution ofracemicatenolol.Asitisshowninthetablestheapplicationof twoenzymespreparationshowedacceptableparametersof per-formedreaction.However,theuseoflipasefromCandidarugosa OF (Table 1)allowed to obtainthe product with higher enan-tiomericexcesscompared totheresultwiththeuseofCandida rugosaMY lipase(Table2), both onFe3O4-CS-EtNH2 aswellas

Fe3O4-CS-BuNH2.Ontheotherside,theimmobilizationoflipases

onto Fe3O4-CS-EtNH2 as theenzymesupport resultedin better

enzyme activitywhich is described byconversion. The conver-sionofenantioselectiveacetylationofracemicatenololishigher for both lipases immobilized on Fe3O4-CS-EtNH2 compared to

Fe3O4-CS-BuNH2.Ontheotherhand,theenantiomericexcesses

of productswere similar while thesame type of enzyme cou-pledwithdifferentchitosanmagneticnanoparticlesisconsidered. Themaindifferenceofeepvaluewasobserved,whiletheresults

Table1

Enzymaticparametersincludingenantioselectivity(E),enantiomericexcessesof bothsubstrate(ees)andproduct(eep)andconversion(c)ofenantioselective

acety-lationof(R,S)-atenololwiththeuseoflipasefromCandidarugosaOF.

Reaction

time[h]

E ees% eep% c%

Candidarugosalipase

OFimmobilizedonto Fe3O4-CS-EtNH2 24 48.76 18.14 95.22 16 48 37.01 19.69 93.66 17.37 72 34.78 23.54 93.02 20.2 96 27.13 25.2 91 21.69 120 45.48 30.64 94.24 24.54 144 43.5 33.99 93.79 26.6 168 58.51 39.74 95.07 29.48 192 53.68 42.11 94.52 30.82 216 65.17 52.41 94.95 35.57 240 66.9 67.71 94.1 41.84

Candidarugosalipase

OFimmobilizedonto Fe3O4-CS-BuNH2 24 20.06 10.5 89.52 10.49 48 22.84 10.87 90.71 10.7 72 28.82 10.33 92.6 10.03 96 36.32 10.66 94.07 10.18 120 32.16 10.91 93.31 10.47 144 30.46 11.09 92.94 10.66 168 29.29 11.29 92.65 10.86 192 27.96 11.53 92.3 11.11 216 33.55 11.89 93.52 11.28 240 34.04 13.39 93.52 12.53

obtainedwiththeuseoflipasefromCandidarugosaOFandMYwere

compared. Nevertheless, after240hof thereaction thehighest

valuesofenantioselectivity(E=66.9),conversion(c=41.84%)and

bothenantiomericexcessesofsubstrate(ees=67.71%)and

prod-uct(eep=94.10%)wereobservedwiththeuseofCandidarugosaOF

48 A.Sikoraetal./JournalofMolecularCatalysisB:Enzymatic134(2016)43–50

Table2

Enzymaticparametersincludingenantioselectivity(E),enantiomericexcessesof

bothsubstrate(ees)andproduct(eep)andconversion(c)ofenantioselective

acety-lationof(R,S)-atenololwiththeuseoflipasefromCandidarugosaMY.

Reaction

time[h]

E ees% eep% c%

Candidarugosalipase

MYimmobilizedonto Fe3O4-CS-EtNH2 24 18.7 8.74 88.98 8.94 48 13.87 10.22 85.23 10.71 72 12.91 9.97 84.25 10.59 96 12.34 10.06 83.58 10.74 120 20.51 11.82 89.61 11.66 144 23.44 15.25 90.56 14.41 168 20.61 17.21 89.14 16.18 192 19.47 22.92 87.93 20.68 216 23.23 30.77 89.02 25.69 240 34.96 45.5 91.42 33.23

Candidarugosalipase

MYimmobilizedonto Fe3O4-CS-BuNH2 24 8.81 2.18 79.22 2.67 48 13.17 2.92 85.5 3.3 72 15.41 3.47 87.41 3.82 96 19.85 3.94 90.04 4.2 120 16.66 4.11 87.87 4.45 144 18.24 4.2 89.26 4.49 168 13.72 4.3 85.86 4.77 192 15.06 4.94 86.96 5.38 216 18.07 5.3 88.98 5.62 240 22.11 5.68 90.87 5.88

3.3. Effectofreactiontemperatureonkineticresolutionof

(R,S)-atenolol

Thetemperatureplaysmainroleinenantioselective

biotrans-formationofchiralcompoundswiththeuseofenzymesascatalysts.

Theinfluenceoftemperature(25–45◦C)ontheenantioselective

acetylationof(R,S)-atenololwiththeuseofCandidarugosalipase

OFandMYimmobilizedontoFe3O4-CS-EtNH2aswellasFe3O4

-CS-BuNH2wasinvestigated(Fig.6).Generally,whenthetemperature

reached45◦Cthevaluesofconversion,enantiomericexcessesof substrateandproductaswellasenantioselectivityofallperformed reactionswerethelowest.Theenantiomericexcessesofproduct werethehighestforallreactionsthermostatedat25◦C,however theirvaluesarenotsignificantlyhighercomparedtothereaction performedat35◦C. Additionally,theenantioselectivity of men-tionedreactionwasthehighestat35◦C(E=66.9),whereasat25◦C

and45◦CthevalueswereE=34.0andE=12.9,respectively.The applicationofCandidarugosaMYlipaseimmobilizedontoFe3O4

-CS-BuNH2inreactionperformedat25◦Cresultedinobtainingthe

conversionvaluealmosttwotimesgreatercomparingtothe reac-tionincubatedat35◦C aswellas45◦C.Nevertheless, themost significant changes were observed in case of lipase from Can-didarugosaOFimmobilizedontoFe3O4-CS-EtNH2.Theconversion

increasedrapidlyfrom7.63%to41.84%withtheincreaseof tem-peraturefrom25◦Cto35◦C,however,withfurthertemperature riseupto45◦Ctheconversionvaluedecreasedto5.74%whichwas probablyrelatedwiththepartiallyenzymedestruction,duetothe hightemperature.

3.4. Influenceofreusedimmobilizedlipasesonkineticresolution of(R,S)-atenolol

Theabilitytoreusetheimmobilizedenzymeinthenextcatalytic systemisextremelyimportantfromeconomicalpointofview.In ordertoinvestigatetheefficiencyofimmobilizedlipasesOFand MYthesamenanoparticleswerereusedafterthespecifiedwashing procedure.Afterthefirstandfurtherreactioncyclesofthe enan-tioselectiveacetylationof(R,S)-atenololtheimmobilizedenzyme wasrecoveredfromthereactionmixturewiththeuseofmagnet andwashed3timeswithtoluene.Next,thelipase-coatedmagnetic nanoparticlesweredriedovernightinordertoremoveorganic sol-vent,andfurtherwereplacedintonewreactionsystem.Thestudy wasrepeateduptofivereactioncycleswiththesame immobi-lizedlipase.Thereactionparametersoftheperformedreactions weredetermined.Duringthefivecyclesofthekineticresolution of(R,S)-atenololslightchangesoftheenantioselectivityexpressed asenantiomericexcessofproductwereobserved.However,the obtainedresultsdemonstratedthattheperformedimmobilization procedureisefficientandallowedtomaintainthestability and catalyticactivityofusedlipasesafter5reactioncycles(Fig.7).

4. Conclusions

The process of enzyme immobilization allows to reuse the boundedenzymeinanothercatalyticsystem,whichisextremely importantfromeconomicalpointofview.Additionally,itincreases

0 10 20 30 40 50 60 70 80 90 100

E ees % eep % c % E ees % eep % c % E ees % eep % c %

25 °C 35 °C 45 °C

Fe3O4-CS-EtNH2-OF Fe3O4-CS-EtNH2-MY Fe3O4-CS-BuNH2-OF Fe3O4-CS-BuNH2-MY

Fig.6. Enzymaticparametersincludingenantioselectivity(E),enantiomericexcessesofbothsubstrate(ees)andproduct(eep)aswellasconversion(c)ofenantioselective

96 96 91 91 96 94 91 ₉₀ 95 93 ₉₀ 90 94 92 ₉₀ 90 94 92 ₉₀ 90 0 10 20 30 40 50 60 70 80 90 100 1 2 3 4 5 Enan iotmeric excess of p roduct (% ) Number of cycles

Candida rugosa lipase OF immobilized onto Fe3O4-CS-EtNH2 Candida rugosa lipase OF immobilized onto Fe3O4-CS-BuNH2 Candida rugosa lipase MY immobilized onto Fe3O4-CS-EtNH2 Candida rugosa lipase MY immobilized onto Fe3O4-CS-BuNH2

Fig.7.InfluenceofreusedimmobilizedlipasesfromCandidarugosaontomagneticnanoparticlesonthekineticresolutionof(R,S)-atenolol.

thecatalyticactivityandoperationalstabilityofbiocatalysts,and thereforemanyimmobilizationtechniqueshavebeeninvestigated [32–34].Theperformedstudyproved,thatthemagnetic nanopar-ticles,withnew-synthetizedchitosanderivativespossessingfree aminegroupontheirsurfacedistancedbyethylor butylgroup couldbeusedasenzymecarries.Thereportedresultsconfirmed thattwocommerciallyavailablelipasesfromCandidarugosaOF and MY immobilized onto new-synthesised chitosan magnetic nanoparticleshave differenteffects onthekineticresolution of (R,S)-atenolol.Amongalltestedcatalyticsystemstheone,which allowedtoachievethehighestenantioselectivity(E=66.9), enan-tiomericexcessofproduct(eep=94.1%)andconversion(c=41.84%)

wasthesystemcontaininglipasefromCandidarugosaOF immo-bilizedonto Fe3O4-CS-EtNH2. Additionally, thepresented study

demonstratedthat,theimmobilizedlipasecouldbefacilely trans-ferredfromonecatalyticsystemtoanotherallowingtoreusethe biocatalyst,whichisanimportantadvantagefromtheeconomic pointofview.Thus,thestudiedmagneticchitosannanoparticles mightbeofspecialimportanceforthefutureindustrialapplication ofthekineticresolutionof␤-adrenolyticdrugs.

Acknowledgment

TheauthorswishtoexpresstheirsincerethankstoMeitoSangyo Co.(Japan)forthesupplyoflipasesOFandMYfromCandidarugosa. The project was supported by a research grant from the NationalScienceCentre,2014/15/B/NZ7/00972.Additionally, this workwaspartiallysupportedbytheNationalScienceCentregrant 2014/15/D/NZ7/01805.

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