Design of a lipase-nano particle biocatalysts and its use in the kinetic resolution of medicament precursors

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ContentslistsavailableatScienceDirect

Biochemical

Engineering

Journal

j ou rn a l h o m epa g e :w w w . e l s e v i e r . c o m / l o c a t e /b e j

Regular

article

Design

of

a

lipase-nano

particle

biocatalysts

and

its

use

in

the

kinetic

resolution

of

medicament

precursors

Rayanne

M. Bezerra

a

_,

_Davino

_M.

_Andrade

_Neto

b

_,

_Wesley

_S.

_Galvão

b

_,

_Nathalia

_S.

_Rios

a

_,

Ana

Caroline

L. de

M. Carvalho

c

_,

_Marcio

_A.

_Correa

e

_,

_Felipe

_Bohn

e

_,

Roberto

Fernandez-Lafuente

d

_,

_Pierre

_B.A.

_Fechine

b

_,

_Marcos

_C.

_de

_Mattos

c

_,

José

C.S.

dos

Santos

f,∗

_,

_Luciana

_R.B.

_Gonc¸

_alves

a,∗

a_Departamento_de_Engenharia_Química,_Universidade_Federal_do_Ceará,_Campus_do_Pici,_Bloco_709,_60455-760_Fortaleza,_CE,_Brazil

b_Departamento_de_Química_Analítica_e_{Físico-Química,}_Centro_de_Ciências,_Universidade_Federal_do_Ceará,_Av._Mister_Hull_s/n,_Pici,_60455-760,_Fortaleza,

CE,CP12200,Brazil

c_Departamento_de_Química_Orgânica_e_Inorgânica,_Centro_de_Ciências,_Universidade_Federal_do_Ceará,_Av._Mister_Hull_s/n,_Pici,_60455-760,_Fortaleza,_CE,_CP

12200Brazil

d_Departamento_de_{Biocatalisis,}_ICP-CSIC,_Campus_UAM-CSIC,_Cantoblanco,₂₈₀₄₉_Madrid,_Spain

e_Departamento_de_Física,_Universidade_Federal_do_Rio_Grande_do_Norte,_59078-900_Natal,_RN,_Brazil

f_Instituto_de_Engenharias_e_{Desenvolvimento}_{Sustentável,}_Universidade_da_Integrac¸_ão_{Internacional}_da_Lusofonia_{Afro-Brasileira,}_62785000_Acarape,_CE,

Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received17January2017

Receivedinrevisedform15May2017

Accepted31May2017

Availableonline2June2017

Key-words: Biocatalyst Superparamagneticnanoparticles 3-Amino-propyltriethoxysilane Branched-polyethylenimine Kineticresolution

a

b

s

t

r

a

c

t

Superparamagneticironoxidenanoparticles(Fe3O4)werepreparedbytheco-precipitationmethod andfunctionalizedwith3-amino-propyltriethoxysilane(APTES)orbranched-polyethylenimine(PEI). Afterthat,twoparallelsmethodstoimmobilizethelipasefromThermomyceslanuginosus(TLL)were performed:theﬁrstonebyionicexchangeandthesecondonebycovalentattachmentafterthe function-alizationofthesupportwithglutaraldehyde(GA).X-raypowderdiffraction,magnetometryandinfrared spectroscopyanalysiswereusedtocharacterizetheTLLpreparations.Theseanalysesshowedthatall samplespresentedsuperparamagneticpropertiesevenaftertheimmobilizationprocedure.TheSPMN (superparamagneticnanoparticle)@APTEScovalentpreparationhadaround450minofhalf-lifetimeat pH7.0and70◦Cwhilethatofthefreeenzymewas46min.Thesebiocatalystswereevaluatedinthekinetic resolutionofrac-1-methyl-2-(2,6-dimethylphenoxy)ethylacetateindifferentco-solvents(acetonitrile, isopropanol,ethyletherandtetrahydrofuran).Thebestresultswerefortheenzyme/substrateratioof 2:1,inthepresenceoftheethyletherorTHF(20%v/vboth)at30◦Cduring24hwiththeSPMN@PEI-TLL biocatalyst.Theconversionattainedwas50%andtheenantiomericexcessoftheproductwas99%.The newSPMNsupportareanexcellentstrategytoeasyrecoveryofthebiocatalystbyapplyingamagnetic ﬁeld.

1. Introduction

Lipasesareoneofthemostusedenzymesinbiocatalytic

pro-cesses,withgreatpotentialinlipidtechnologyandinthesynthesis

of enantiomerically pure intermediates [1–3], at academic and

industriallevel[4].Theseenzymesareusedinawidediversityof

reactionmedium,exhibitingexcellentstabilityandactivityinthe

∗ Correspondingauthors.

E-mailaddresses:jcs@unilab.edu.br(J.C.S.dosSantos),lrg@ufc.br,

lrgufc@gmail.com(L.R.B.Gonc¸alves).

presenceoforganicsolvents,thatmaybeusedtomakeeasierthe

solubilityofthehydrophobicsubstratetobemodiﬁed[2,3].

Microbiallipaseshavehighproductioncostsandwhenusedin

itssolubleform,itmightbeunstableincertainreactionconditions

[5].Theseproblemsmightbeovercomebyenzyme

immobiliza-tionbeforeitsindustrialimplementation,whichisasimplewayto

separatetheenzymefromthereactionmediaandtoreuseit[5].

Ifproperlyperformed,immobilizationmaytuninglipase

proper-ties,asactivity,selectivity,speciﬁcity,resistancetoinhibitorsand

stabilization[6].

One interesting support that may be used to immobilize

enzymesaremagneticnanoparticles,which havesuitable

prop-ertiesthatalloweffectivelyimmobilization,becauseoftheirhigh

http://dx.doi.org/10.1016/j.bej.2017.05.024

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speciﬁcsurface area, besidesbeingeasily separated from

reac-tionmediumbytheuseofamagnet[7].Moreover,themagnetic

nanoparticlesimmobilizeenzymemoleculesonthesurface,which

prevents internal diffusional limitations [7]. In addition, other

advantages of nanoparticlesassupports for theimmobilization

ofenzymesare biocompatibility, non-toxicityandpossibility of

superparamagnetism(SPM)[7].Whenmagneticnanoparticlesare

synthesized below the critical volume, they can present SPM

properties [8]. In this sense,the superparamagnetic

nanoparti-cles(SPMN)areofgreatinterest,sincethesenanoparticlespresent

magneticresponseonlywhentheyareinpresenceofanexternal

magneticﬁeld,ceasingthemagnetismassoonasthemagneticﬁeld

isremoved[8](seeFig.S1).However,theymayhavesome

draw-backs,asthelackofprotectiveeffectsoftheimmobilizationinside

aporoussystemtointeractionwithexternalinterfaces,butthis

issuemaybesolvedviaappropriatestrategies[9].

Inthiscontext,theimmobilizationofThermomyceslanuginosus

lipase(TLL)onsuperparamagneticironoxidenanoparticles(Fe3O4)

coatedwithpolymerfunctionalizingbranched-polyethylenimine

(PEI)or3-amino-propyltriethoxysilane(APTES)wasinvestigated.

TLL is thermo stable withhighcatalytic efﬁciency, strict

enan-tioselectivityandenantiospecifcity,broadspeciﬁcity,singlechain

proteinconsistingof269aminoacidswithamolecularweightof

31.7kDa, isoelectricpoint of4.4 anda sizeof 35Å×45Å×50Å

[10].Thisenzymehasbeenappliedinmanydifferentindustrial

areas,suchasintheproductionofbiodiesel,detergent,cosmetic,

andotherorganicchemicals[10].

TLLhasanisoelectricpointof4.4,therefore,atpH9theprotein

surfacewillhaveastronganioniccharacter.However,itis

impor-tanttoremarkthation-exchangeadsorptionofproteinsisfavored

iftheproteinandthesupporthaveanoppositenetchargebutthis

isnotcompulsory.Someauthorsreport[11,12]thatthepositiveor

negativeregionsthatareavailableonproteinsurfacearecapable

ofadsorbonthesupportviamultipointinteraction.Forexample,

morethan80%oftheproteinscontainedincrudeextractscould

beimmobilizedonPEI[11]coatedagarose.Moreover,alarge

per-centageofproteinscouldbeimmobilizedonmixedcation/anion

exchangers,infact,itwaspossibletoimmobilizeproteinsthatwere

notimmobilizedinanyofthe“pure”ionexchangers[12].

PEIhasionizablegroups,mainlyonitsmainchain[13].This

cationic polymer has several applications in biological,

indus-trial and pharmaceutical ﬁelds [10,13]. PEI has been ascribed

tobeabletostabilizeproteinsviadifferent factors,inaddition

hasbeenobservedthattheincreasedlengthenedpolymerchain

resultsinincreasingtheimmobilizationamountofenzyme[11].

APTESis asilanecouplingreagentthatisextensivelyemployed

forbiomolecule immobilizationtodevelopbiosensors[14].This

reagentisusedtocreateanaminolayeronthesupport,thatmaybe

usedforbiomoleculeimmobilization[15].Bothstructuresarequite

different,whilePEIcoatingformsanionicbedwheretheenzyme

maybeincluded[11],APTESonlypromotestheformationofaﬂat

surfacewheretheenzymemayinteract.

Inordertointroduceafunctionabletocovalentlyreactwith

theenzyme[14],theresultantsurfacewasnexttreatedwith

glu-taraldehyde[16].Thosealdehydegroupsinthesupportmayreact

withaminegroupsoftheproteintoimmobilizetheenzyme[16,17].

Furthermore,thisreagenthasagreatpotentialinpreparing

bio-catalystswithanintensemultipointcovalentattachment,andmay

alsointroduceinterorintracrosslinkingthatcanproducepositive

effectsonenzymestability[16,18].

Being aware of the fact that biocatalysis offers an

alterna-tive to improve the conventional chemical synthesis of chiral

enantiomerically pure drugs [1], we investigated the new TLL

biocatalysts in the resolution of racemic

Mexiletine[1-(2,6-dimethylphenoxy)propan-2-amine] via hydrolytic reactions. An

elegant strategy is tocombine a biocatalytic step with a

con-ventional chemical route, known as chemoenzymaticsynthetic

routes [2,3,9]. This enables the production of enantiomerically

enriched chiral alcohols, which have highadded valueand are

used as building blocks in the synthesis of various

biologi-cally active compounds. Speciﬁcally, the chiral alcohol

1-(2,6-dimethylphenoxy)propan-2-ol,whensubmittedtootherreactions

withexchangeofthehydroxylgroupbyanaminogroup,leadsto

theformationofenantiomericallypuredrug

(R)-Mexiletine[(2R)-1-(2,6-dimethylphenoxy)propan-2-amine].Mexiletineinitsracemic

form is an antiarrhythmic agent, but in enantiomerically

(R)-enrichedformisacompoundabletoblockthesodiumchannels

andhasitsactivityenhanced[19].

2. Materialsandmethods

2.1. Materials

The commercial TLL extract (15.83mg of protein per mL)

was obtained from Novozymes (Spain). 6 BCL agarose, 25%

(v/v) glutaraldehyde solution, cetyl trimethyl ammonium

bro-mide(CTAB)andp-nitrophenylbutyrate(p-NPB)werepurchased

from Sigma ChemicalCo (St.Louis, MO, USA). The commercial

preparationofTLLcovalentlyimmobilizedonimmobead-150(TLL,

250U/g),branched-polyethylenimine(MW10,000)and

3-amino-propyltriethoxysilane(>98%)werepurchasedfromSigma-Aldrich

(St.Louis,MO,USA).FeCl3·6H2OandFeSO4·7H2Oweresuppliedby

Sigma-AldrichandVetecQuímica,respectively.Allothersreagents

andsolventsusedwereofanalyticalgrade.

2.2. SynthesisofFe3O4andfunctionalizationwithAPTES

Intheco-precipitationrouteassisted byultrasound,metallic

salts containingFe2+_/Fe3+ _were_dissolved _and _mixed _in _Milli-Q

waterin themolarratioof1:2toformthespinelphaseFe3O4.

Brieﬂy,5.82mmolofFeCl2·4H2Oand 10.57mmolofFeCl3·6H2O

were dissolvedin a 50mL of Milli-Qwater. Theaqueous

mix-tureremainedundervigorousultrasoundstirringwhen10mLof

a solutionof NH4OH wasaddeddrop wise toformthe

precip-itate,accordingwiththeproportion1:8(Fen+_/ammonium)._The

precipitatewaswashedseveraltimeswithMilli-Qwateruntilthe

residualsolutionbecameneutral,followedbydryingthemagnetic

nanoparticles.

Inthefunctionalizationprocedure,100mgofmagnetiteFe3O4

weresuspendedinabeakerusing20mLofethanoland20mLof

toluene.Afterthat,100␮LofAPTESwasadded.Thereactionsystem

wassubjectedtovigorousultrasonicstirringfor15minat50◦C.

Finally,thenanocompositeformedwaswashedseveraltimes,dried

andstoredinadesiccator.

2.3. SynthesisofFe3O4andfunctionalizationwithPEI

Synthesis and functionalization of Fe3O4 SPMNs were

per-formed in two-steps, using a probe ultrasound (Ultrasonique

Desruptor)withfrequencyof20kHzandpowerof750W.Initially,

twosolutionswereprepared.Theﬁrstwasanironsaltssolution

(SolutionA)andthesecondone,aPEIaqueoussolution(Solution

B).SolutionAwascomposedof1.16gofFeSO4·7H2Oand1.85gof

FeCl3·6H2Odissolvedin15mLofdeionizedwater,whereas,

solu-tionBwasconsistedof1.0gofPEIin4.0mLofdeionizedwater.

Firstly,thesolutionAwassonicatedfor4min,untilitreaches

thetemperatureof60◦C.Then,7.0mLofconcentratedNH4OHwere

added,undersonication,usingaburette.Thereafter,thecolorof

solutionAchangedfromorangetoblack,evidencingtheformation

ofFe3O4SPMNs.After4min,solutionBwasaddedtothereaction

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ToremovetheexcessofNH4OHandunbounded

functionaliz-ingagent,theresultantSPMNswerewashedseveraltimeswith

distilledwater and precipitatedwithacetone.Then, theSPMNs

weredispersedinwaterandcentrifugedfor10minand3000rpm

toremove theweaklyfunctionalizedSPMNs.Finally,SPMN@PEI

weredriedunderthevacuum.

Thesynthesisofnon-functionalizedFe3O4(labeledSPMN)were

performedaccordingtotheprocedurealreadypublishedbyour

group[20].Theprocedureusedinthereferenceissimilartothe

oneforSPMN@PEI.

2.4. ActivationofSPMN@APTESandSPMN@PEIwith

glutaraldehyde(GA)

SPMN@APTESandSPMN@PEIwereactivatedwith

glutaralde-hydeaccordingtoXieetal.,2009[21],withmodiﬁcations.Inthis

procedure, 25␮Lof glutaraldehyde areplaced in direct contact

with10mgofdrysuperparamagneticsupport.Themixturewas

keptunderagitationfor2hat25◦C.Finally,supportswerewashed

3timeswithphosphatebuffer(25mMandpH7)toremovethe

excessofglutaraldehyde.Thesupportwasnamed

SPMN@APTES-GAandSPMN@PEI-GA.

2.5. Characterizationofthesupportsandbiocatalysts

2.5.1. X-raypowderdiffraction(XRPD)

Thestructuralanalysisandveriﬁcationofsingle-phasenature

ofthesampleswerestudiedusingXRPD.Themeasurementswere

carriedoutusingRigakuX-raydiffractometerequippedwithCoKa

radiationtube(␭=1.7889Å)operatedwithvoltageof30kVand

currentof15mA.Thephaseidentiﬁcationanalysiswasperformed

bycomparingpowderdiffractogramswithstandardpatternsfrom

theInternationalcentrefordiffractiondata(ICDD).Rietveld

reﬁne-mentprocedure[22]wasperformedtoalldiffractionpatternsusing

theDBWS2.25[23].Thecrystallitesizesofnanoparticleswere

cal-culatedfromtheXRPDdatausingScherrer’sequation[24].

2.5.2. Magneticcharacterization

The magnetic characterization at room temperature was

obtainedusingavibratingsamplemagnetometer(VSM)Lakeshore

7400,withmaximummagneticﬁeldamplitudeof17kOe.TheVSM

hasbeenpreviouslycalibratedusingapureNisample.Thus,after

measuringthemassofeachsample,therespectivemagnetization

isgiveninemu/g.

2.5.3. FTIRanalysis

Spectroscopydataintheinfraredregion(FTIR)wereobtained

onaPerkinElmerSpectrometerFTIR.Forthesemeasurements,the

sampleswerepreviouslydilutedinKBrandthenthespectrawere

collectedintherangeof400−4000cm−1.

2.6. Preparationofglyoxyl-agarosebeads

Thepreparationoftheglyoxyl-agarosesupportwasperformed

accordingtotheproceduredescribedby[25],withsome

modiﬁ-cations.Initially,10gofagarosesupportwereaddedto2.86mL

ofwater,4.76mLofNaOH1.7M,containing135.7mgofsodium

borohydrideand,aftercoolinginawater-icebath,3.43mLof

glyci-dolwasslowlyadded.Thismixturewasmaintainedunderstirring

for15hatroomtemperature.Then,thesupportwaswashedwith

distilledwater,dried,suspendedin100mLofdistilledwaterand,

afterthat, was added 16.1mg of sodiumperiodate to form 75

micro-equivalentofaldehyde groupsactivatedpergofsupport.

Themixturewaskeptundermagneticstirringfor2hatroom

tem-peratureand,afterall,theglyoxyl-agarosesupportwaswashed

withdistilledwater,driedvacuumandstorageat4◦C[26].

2.7. Immobilizationprocedure

2.7.1. CovalentimmobilizationoflipaseonSPMN@APTES-GAor

SPMN@PEI-GA

TheTLLwasimmobilizedonSPMN@APTES-GAor

SPMN@PEI-GAinbatchmode,producingthebiocatalysts

SPMN@APTES-GA-TLLorSPMN@PEI-GA-TLL.Forthispurpose,thesupport(100mg)

wassuspendedin10mLof25mMsodiumphosphatebufferatpH

7solutioncontainingthelipase(enzymeload:3mg/gsupport)and

0.01%(v/v)CTABatpH7.0[27].Theimmobilizationprocesswas

carriedoutat25◦Cfor1hunderconstantagitation.Themagnetic

particlescontainingimmobilizedTLLwereremovedbymagnetic

separationandwashedwith25mMsodiumphosphatebufferat

pH7.0.TheamountofTLLimmobilizedonthepreviouslyactivated

supportswasdeterminedbymeasuringtheinitialandﬁnal

concen-trationofTLLinthesupernatantoftheimmobilizationsuspension.

TheimmobilizationproceduresinSPMNwereschematizedinFig.1.

2.7.2. IonicimmobilizationoflipaseonSPMN@APTESor

SPMN@PEI

The immobilizationprocedurewas performedby contacting

100mgofSPMN@APTESorSPMN@PEIwith10mLofenzyme

solu-tion(enzymeload:3mg/g support)in 5mMsodiumphosphate

buffer at pH 7.0 (immobilization by means of ionic exchange)

in batch mode, producing the biocatalyst SPMN@APTES-TLL or

SPMN@PEI-TLL. Theimmobilization processwassimilar tothat

describedintheprevioussection(Fig.1).

2.7.3. Lipaseimmobilizationonglyoxyl-agarosesupport

ImmobilizationofTLLonglyoxyl-agarosewascarriedoutby

adding1gofglyoxyl-agarosesupportto10mLofenzymesolution

(enzymeload:3mg/gsupport)in25mMsodiumcarbonatebuffer

atpH10.0containing0.01%(v/v)CTAB.Thelipasesmaybe

hyperac-tivatedduringbiocatalystpreparationbyusingconditionsinwhich

theenzymestructureisopenandabletobefurtherstabilizedinthis

conformation.Inthesecases,theopenconformationhastypically

beenproducedbyusingdetergents,asCTAB;theseamphipathic

moleculesstabilizetheopenconformationofthelipases.

The monitoring of the immobilization was carried out by

withdrawingsamplesofthesupernatantandsuspension,and

mea-suringitscatalyticactivity,asdescribedinSection2.8.Then,the

enzyme-supportcovalentreactionwhenusingglyoxylsupport,a

solutionofsodiumborohydride(1mg/mL)atpH10wasprepared,

them the immobilized enzyme was added and the suspension

wassubmittedtogentlestirring during30min[28].This

treat-ment reducesreversibleSchiff´ıs basesto verystable secondary

aminobondsandunreactedaldehydesgroupstofullyinerthydroxy

groups[28].Finally,thereducedderivativeswereﬁltered,washed

withabundantdistilledwaterandstoredat4◦C.

2.8. Determinationofenzymeactivityandproteinconcentration

TheactivitiesofthesolubleandimmobilizedTLLwere

deter-minedaccordingtothemethodologydescribed intheliterature

[29],withsomemodiﬁcations.Inthisprocedure,TLLactivitywas

determinedbythemeasuringtheincrementintheconcentrationof

thep-nitrophenolreleasedduringthehydrolysisofp-nitrophenyl

butyrate(p-NPB) asa substrate,50mMin acetonitrile,at pH7

(phosphatebuffer25mM)and25◦C ( undertheseconditions

10.052mol−1cm−1)[2,3].Thisproducthasayellowcolorationthat

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Fig.1.TheimmobilizationproceduresofTLLbyionicexchangeorbycovalentattachmentinSPMNrecoveredwithPEIorAPTES.

Proteinconcentrationwasmeasuredusing Bradfordmethod

[30]andbovineserumalbuminwasusedasthereference.

2.9. Immobilizationparameters

Inordertoevaluatetheefﬁciencyofimmobilizedenzymes,the

immobilizationparameterswerecalculatedaccordingtoSilvaetal.

[17].Theimmobilizationyield(IY)wasdeﬁnedastheratiobetween

theactivityofenzymesretainedonthesupport(At_i−At_f)and

ini-tialactivity(At_i).Thetheoreticalactivity(At_T)wascalculatedusing

theimmobilizationyield(IY)andtheenzymeload.Therecovery

activity(At_R)wasdeterminedastheratiobetweenthebiocatalyst

activity(At_B)andthetheoreticalactivity(At_T)[17].

2.10. SDS-PAGEelectrophoresis

SDS-PAGEwasconductedusinganelectrophoresisunit(model

Mini-PROTEAN3Cell,USA)with12%polyacrylamidegels,

accord-ingtotheliterature[31].10mgofbiocatalystwereputincontact

with100_␮Lofrupturebufferboiledduring10mintoremovethe

enzymefromthesupport.Therupturebufferwasprepared

accord-ing tomethodology reportedby [18]. TheSDS-PAGE gelswere

stainedbytheCoomassiebrilliantbluemethod,usinglow

molec-ularweightmarker(SDSMarker−GEHealthcareLifeSciences)as

standard.ThemolecularweightwascalculatedusingGelAnalyzer,

afreewaresoftware.

2.11. ThermalandpHinactivation

InordertocomparethethermalandpHstabilitiesoftheTLL

biocatalysts,100mgofthepreparedbiocatalystweresuspendedin

5mLof25mMsodiumacetateatpH5,25mMsodiumphosphate

atpH7or25mMsodiumcarbonateatpH9atdifferent

temper-atures(60–70◦C).Periodically,theactivitiesofthesampleswere

measuredusingp-NPBassubstrate.Half-lifetime(t1/2)foreach

immobilizedderivativewascalculatedaccordingtotheSadanaand

Henleymodel[32]usingMicrocalOriginversion8.1.

2.12. Solventstability

Toevaluatethesolventstabilityoftheproducedbiocatalysts,

100mgofthepreparedbiocatalystwereputincontactwithethyl

ether/aqueous buffersystem (20% v/v) at pH 7 and 30◦C. The

samples wereperiodically withdrawnand theiractivities were

measured,half-lifestimewerecalculatedfromthemethodology

describedinthesection2.11.

2.13. Operationalstability

The operational stability of biocatalysts was performed by

hydrolysisof0.4mMp-NPBin25mMsodiumphosphatebufferat

pH7.0and25◦C.Consecutivecyclesofp-NPBhydrolysisreaction

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bio-catalystwithmagnet.Then,therelativeactivityofthebiocatalyst

ineachcyclewascalculated,takingas100%theactivityintheﬁrst

cycleofhydrolysisreaction.

2.14. Tributyrinhydrolysis

Hydrolysisoftributyrincatalyzedbydifferentpreparationsof

immobilizedTLL wereperformedatpH7.0, 25◦C and followed

usinganautomatictitrator(MettlerToledo−T50).A50mMNaOH

solutionwasusedasatitratingreagent.Atributyrin-gumarabic

emulsionwaspreparedaccordingtothemethodologydescribedby

[33],withsomemodiﬁcations.Agumarabicemulsion6g/L,

con-tainingsodiumchloride(17.9g/L),monobasicsodiumphosphate

(0.41g/L)and135mLofglycerinwasprepared.Themixturewas

transferredinto250mLmeasuringﬂaskanddistilledwaterwas

addedtomakeupthevolume.Then,300␮Loftributyrinwasadded

into30mLofemulsion.Thesubstrateemulsionwasputunder

stir-ringat11000rpmfor1minand,following,sonicationfor1min.

Afterall,immobilized TLLwasaddedintotributyrin-gumarabic

emulsionandthehydrolysisreactionwascarriedoutat37◦Cfor

10min,undermechanicalstirring.Oneunitoflipaseactivity(U)is

theamountofenzymecapabletohydrolyze1␮moloftributyrinat

assayconditions.

2.15. Preparationoftheracemicsubstrates

2.15.1. Synthesisofrac-1-(2,6-dimethylphenoxy)propan-2-ol

A mass of 341mg of sodium borohydride NaBH4 was

slowly added to a solution containing 805mg of

1-(2,6-dimethylphenoxy)propan-2-onein44mLofmethanolat4◦C.The

reactionmixturewasstirredat4◦Cfor3h.Afterthattime,the

solventwasevaporatedunderreducedpressure.Theresulting

sus-pension was added to 10mL of H2O and extracted with ethyl

acetate(EtOAc)(3×50mL).Organic phaseswerecombinedand

driedoversodiumsulfate(Na2SO4)anhydride,ﬁlteredandthe

sol-ventwasevaporatedunderreducedpressure.Theresultingcrude

waspuriﬁedbyﬂashchromatography(20–80%EtOAc/hexane)to

affordrac-1-(2,6-dimethylphenoxy)propan-2-olasayellowoilin

90%yield.

2.15.2. Synthesisofrac-1-methyl-2-(2,6-dimethylphenoxy)ethyl

acetate

Avolumeof831␮Lofaceticanhydride(CH3CO)2(8.45mmol)

and 62.2mg of 4-dimethylaminopiridine (DMAP) were added

to 17mL of dichloromethane containing 305mg of

rac-1-(2,6-dimethylphenoxy)propan-2-ol (1.7mmol). The solutionreaction

wasstirredatroomtemperatureduring4hand,afterthattime,

thesolventwasevaporatedunderreducedpressure.Posteriorly,

theresultingcrudewaspuriﬁedbyﬂashchromatographyonsilica

gel(20–80%EtOAc/hexane)toaffordthedesired

rac-1-methyl-2-(2,6-dimethylphenoxy)ethylacetateasayellowoilin92%yield.

2.16. Kineticenzymaticresolutionof

rac-1-methyl-2-(2,6-dimethylphenoxy)ethylacetatevia

hydrolysisreactionusingTLLimmobilized

A mass of 20mg of the prepared biocatalyst was added to

a solutionof 10mg rac-1-methyl-2-(2,6-dimethylphenoxy)ethyl

acetate(ratio2:1inweightwithrespecttotheacetate)in100mM

sodiumphosphatebufferpH7.0/co-solvent(80/20v/v),and

sub-mittedtostirringat250rpmand30◦C.After24h,thereactionwas

stoppedbyﬁltration.Then,theproductwasextractedwithEtOAc

(3_×10mL)andtheorganicphaseswerecombinedanddriedwith

anhydroussodiumsulfate. Subsequently,theorganicphasewas

ﬁlteredandthesolventwasevaporatedunderreducedpressure.

Thereactionmediawasfinallypurifiedbyflashchromatography

onsilicagel(20–80%EtOAc/hexane).

2.17. Procedureforcalculatingenantiomericexcess,conversion

andenantiomericratio

The efﬁciency of kinetic resolution wasevaluated based on

the optical purity of the compounds, expressed in terms of

enantiomeric excess of thesubstrate (e.e.s)and product (e.e.p),

respectivelygivenby:

e·e·S= A_AS−BS

S+BS×100 (1)

and

e·e·P= A_AP−BP

P+BP ×100 (2)

whereAdenotethemajorityenantiomerandBdenotetheminority

enantiomerrepresentedbythechromatographicpeakareas.The

indexSdenotessubstrateandP,product.

Theconversion(c)andenantiospeciﬁcity(E)wererespectively

determinedby: c= _e e·e·S ·e·S+e·e·P (3) and E= ln [1−c(1+e.e.P)] ln [1−c(1−e.e.P)] (4) 2.18. Analysis

The1_H,13_C_nuclear_magnetic_resonance_(NMR),_and_distortion

lessenhancementbypolarizationtransfer(DEPT)wereobtained

usingaBrukerSpectrometer,modelAvanceDPX300,operatingat

frequenciesof300MHzforhydrogenandfrequenciesof75MHzfor

carbon,respectively.Thechemicalshiftsaregivenindelta(␦)

val-uesandthecouplingconstants(J)inHertz(Hz).Themeasurement

oftheopticalrotationwasdoneinaPerkin-Elmer241polarimeter.

Gaschromatograph(GC)analysiswerecarriedoutinaShimadzu

chromatographmodelGC2010,withaﬂameionizationdetector

usingachiralcolumnCP-chirasil-dex(25m×0.25mm×0.25␮m,

0.5barN2)forthefollowingofthereactiontimecourses:100◦C;

0.5◦C/min130◦C (hold 15min); 5.0◦C/min140◦C (hold 5min).

Retentiontimeswere:(S)-acetate71.8min;(R)-acetate76.6min;

(S)-alcohol62.9min;(R)-alcohol62.3min.Theenzymatic

hydroly-siswasperformedinashaker(CientecCT-712model).

rac-1-(2,6-dimethylphenoxy)propan-2-ol: yellow oil Rf

(20%EtOAc/Hexane): Rf 0,36. 1H NMR (CDCl3, 300MHz): ␦ (ppm)1,26 (d, 3H); 4,21 (m,1H); 3.73(dd,J 12Hz, 3Hz, 1Hc); 3,64(dd,J18Hz, 6Hz, 1H);2,27 (s,2H); 7.00 (d,J7,29Hz, 2H); 6,92 (dd, J2,19Hz, 1H).13C-BB NMR (75MHz,CDCl3):␦(ppm) 16,52(CH3);18,81(CH3);67,33(CH);77,18(CH2);124,28(CH); 129,18(CH);130,97(C);155,41(C).

rac-1-methyl-2-(2,6-dimethylphenoxy)ethylacetate:yellowoil

Rf(20%EtOAc/Hexane):0,63.1HNRM(CDCl3,300MHz):␦(ppm) 2,11(s,3H);1,41(d,J7,95Hz,3H);5,26(m,1H);3,82(d,J4,83Hz, 2H);2,33 (s,Hz, 6H);7,01(d, J7,23Hz, 2H);6,93 (dd,J8,46Hz, 1H).13C-BBNRM(75MHz,CDCl3):␦(ppm)21,36(CH3);170,7(C); 16,24(CH3);69,86(CH);73,85(CH2);155,42(CH);16,73(CH3); 129,14(CH);130,90(C);124,14(CH).

(6)

Table1

StructuralparametersobtainedfromRietvieldreﬁnement.

Sample XRPD VSM

Latticeparameters(a)(Å) Rwp(%) S Averagecrystallitesize(nm) Ms(emu/g)

SPMN@APTES-GA-TLL 8.367(1) 15.7 0.93 15±0.31 63.4

SPMN@APTES-TLL 8.369(4) 18.9 0.92 15±0.29 65.5

SPMN@PEI-TLL 8.365(5) 15.2 0.91 11±0.18 60.5

SPMN@PEI-GA-TLL 8.366(2) 14.8 0.93 10±0.17 56.0

SPMN 8.358(2) 13.58 1.00 12.1±0.20 56.7

Fig.2.XRPDresultsfortheinvestigatedsamples.Here,thebluelinerepresentsthe relativedifferencebetweenexperimental(YObs,blackdots)andcalculated(YCalc, redline)intensitiesobtainedthroughthereﬁnement.(Forinterpretationofthe ref-erencestocolourinthisﬁgurelegend,thereaderisreferredtothewebversionof thisarticle.)

3. Resultsanddiscussion

3.1. Characterizationofbiocatalyst

3.1.1. XRPDanalysis

XRPDanalyses wereused toconﬁrm theformation of

crys-tallinephaseandtoverifythestructuralparametersofthemagnetic

nanoparticles.Fig.2 presentstheXRPDresultsobtainedforthe

investigatedsamples.In particular,theblue linerepresentsthe

relative differencebetween theexperimental(YObs, black dots)

Fig.3. Magnetizationcurveatroomtemperatureforthestudiedsamples.

and thecalculated (YCalc,red line)intensitiesobtainedthrough

thereﬁnement.BycomparisonwiththeInorganicCrystal

Struc-tureDatabase(ICSD)andthereﬁnement,itwaspossibletoconﬁrm

theformationofasinglephaseofFe3O4(ICSD/PDF-08-4611)forall

samples.Theindexationofthepeakwiththediffractionpatterns

alsoshowedtheformationofinversespinelferritewithspatial

groupFd3M.ThevaluesobtainedfromRietveldmethodwere

sum-marizedinTable1.

Thereﬁnementconvergedsatisfactorilytoweightedproﬁle

R-factor(Rwp)andS(∼1)values[34].Theaveragecrystallitesizewas

calculatefrom Scherrer´ısequation,which relatesthe crystallite

sizewiththehalf-widthofthediffractionpeak[35].Thesamples

presentedvaluesbelowof15nm,indicatingthepresenceofSPM

phenomena[36].Itispossibletoobservethatthesamplescovered

withPEIobtainedsmall crystallitesize.During co-precipitation

procedure,aprocessofnucleationfollowedofthegrowthof

crys-taloccurs.TheinsertionofPEIrightaftertheprecipitationshould

preventthecontinuityofthecrystalgrowth,providingmagnetic

nanoparticleswithreducedsize.

3.1.2. Magneticmeasurement

The VSM analysis was performed to investigate the

mag-neticpropertiesofthesamples.Fig.3presentsthemagnetization

curves at room temperature obtained for the produced

sam-ples. In particular, the values of saturation magnetization(MS)

fortheSPMN@PEI-GA-TLL,SPMN@PEI-TLL,SPMN@APTES-GA-TLL

andSPMN@APTES-TLLandSPMNwere56.0,60.5,63.40,65.5and

56.7emu/g,respectively.TheincreaseofMSvaluesindicatesthe

decreaseoftotalmassofthefunctionalizedagentanchoredonthe

nanoparticlesurfacefortheimmobilizedsamples.Ingeneral,the

magneticcurvesshowagreateramountofmassforthesamples

coveredwithPEI.Ashighertheamountofanchoredmass,greater

thenumber of amino groups availablefor enzyme

immobiliza-tion.However,thesampleSPMN,whichwerenotfunctionalizedor

(7)

Fig.4.FT-IRspectrumofthesamples:superparamagneticnanoparticle(SPMN)

andTLLpreparations,SPMNfunctionalizedwith3-amino-propyltriethoxysilane

(SPMN@APTES-TLL), SPMN functionalized with 3-amino-propyltriethoxysilane

and Glutaraldehyde (SPMN@APTES-GA-TLL), SPMN functionalized with

branched-polyethylenimine (SPMN@PEI-TLL), SPMN functionalized with

branched-polyethylenimineandGlutaraldehyde(SPMN@PEI-GA-TLL).

intheMSvaluesafterfunctionalizationwerealreadyreportedin

theliterature[37,38]and isduetotheincreasingof

crystalliza-tionofthenanoparticle surface,which is aconsequence ofthe

bondbetweenFeatomsatthesurfaceandaminegroupsofPEI

andAPTES[37,39].TheroomtemperatureM_Svaluesobtainedfor

allsamplesaresmallerthantheonereportedbulkFe3O4,which

isof70emu/g[40].Thisresultconﬁrmsthesurfacemodiﬁcation

processofmagnetite.Moreover,accordingtothemagneticcurves

obtainedat300K,itisveriﬁedthatthecurvesdonotpresent

hys-teresis,i.e., coerciveﬁeld and remanentmagnetizationequalto

zero,asignatureoftheSPMbehaviorofthesamples.

3.1.3. FT-IRanalysis

TheFT-IRanalyseswereperformedtoinvestigatetheprocess

ofsurfacemodiﬁcationofSPMNsandlipaseimmobilization.The

spectrafor all samplesare shown in Fig.4 and Table 2 shows

themostimportant vibrationalmodes, usedfor supportingthe

discussion.Allsamplespresentcharacteristicbandsin585cm−1,

whichisassignedtotheFe Ostretching(FeO)fromFe3O4;and

at3413cm−1,thatcanbeattributedtotheO Hstretchingband

rel-ativetoHO-groupspresentatthemagnetitesurfaceorabsorbed

watermolecules[41].

Inallsamples,exceptfor SPMN,a vibrationalmodeat1050

and1625cm−1 relativetoC Nstretching(C N)andN H

bend-ing,respectively[13],wereobserved,whichisduetotheamine

groupspresentinPEI,APTESandTLL.Theseresultsconﬁrmthat

thepreparationofhybridmaterialscomposedofamagnetic

crys-tallinecore(Fe3O4)coatedwithanorganicsubstance(PEIorAPTES

andTLL).

3.2. Immobilizationparameters

Table3showstheimmobilizationparametersonthedifferent

supports.Proteinloadwas3mgproteinpergramofsupportand

theimmobilizationparameterswereevaluatedbythehydrolysis

reactionofp-NPB(0.4mM)after1hofimmobilization.Theyieldof

immobilizationwashigherforimmobilizationsbyionicexchange.

ThederivativeSPMN@APTES-GA-TLLpresentedthehighest

activ-ity(AtD)despitehavingthelowestyield ofimmobilization(IY)

(SeeTable3).Otherauthors[42]immobilizedTLLontomagnetic

nanoparticles(MNPs)functionalizedwithAPTESand

glutaralde-hyde,achievinganimmobilizationyieldof63±3.5%,whichseems

comparable with the obtained for SPMN@APTES-GA-TLL. It is

importanttomentionthatthecontacttimebetweenenzymeand

supportwasnotreportedbytheauthors[42].Inourcase,theyield

wouldbeimprovedifmorethan1hwereused.

DuetothepolymericbedformedbyPEI,a largeramountof

enzymemaybeimmobilizedin1husingthisactivationprotocol.

Thisfactmayexplainthehighercatalyticactivityofthe

SPMN@PEI-TLLcomparedtotheSPMN@APTES-TLL.However,inthecaseof

SPMN@PEI-GA-TLL,thecovalentbondsprobablydistortthe

struc-tureoftheenzyme,affectingitscatalyticactivity.Ontheotherhand,

glutaraldehydeis arelativelyhydrophobicmolecule,

glutaralde-hydegroupsarequitereactivewithotheramino,theenzymeamino

groups, andthat maygreatly affecttheglobal physical

proper-tiesoftheenzymesurface(e.g.,hydrophobicity),alsoalteringthe

enzyme catalytic properties.For themore, thesebonds

amino-glutaraldehydeshouldalwayscauseacertainincreaseofenzyme

rigidityinthatareaoftheproteinthatmayproduceornota

cer-tainstabilizationoftheenzyme.Theamino-glutaraldehydegroups

arequitereactivewithotheramino-glutaraldehydegroupsorwith

freeglutaraldehyde,thusbeingthebestwaytogeta

glutaralde-hydecrosslinkingandthedecreaseofimmobilizationyieldafter

activationwithglutaraldehydegroups.Thepolymericnatureofthe

PEIwillgivepoorrigiditytotheimmobilizedenzyme,andmay

introducesometensionontheirstructure.

3.3. SDS-PAGEanalysisofthesamples

SDS-PAGEanalyseswerecarriedoutforsamplesofimmobilized

TLLandarepresentedinFig.5.Thebandsdetectedareproteins

removedfromthesupportbythebreakingbufferunderboiling.

Thus,ifanenzymemoleculeiscovalentlyimmobilized,itisnot

releasedand,consequently,itisnotdetectedinSDS-PAGE[2,3].

TheproteinbandobservedintheSPMN@PEI-TLLismoreintense

than that in the SPMN@APTES-TLL, indicating greater amount

of proteinadsorbed bythe PEIstructure (Table S1).The bands

observedhave33kDa(molecularweightcalculatedusing

GelAn-alyzer,afreewaresoftware).Theseresultsareinagreementwith

themolecularweightfoundintheliteratureforthisprotein[10].

Theactivationofthesupports withglutaraldehyde maygive

threedifferentkindsofinteractionswiththeTLLlipase:

hydropho-bic, anionic exchange and covalent [16]. Thus, to ensure that

thecovalentimmobilizationhastakenplace,acationicdetergent

(CTAB)wasaddedtotheimmobilizationmediainordertoprevent

hydrophobicandionicinteractions.Furthermore,theCTAB

concen-trationemployediscapableofbreakingproteindimers,preventing

agglomeration.Thelackofproteinbandsonlanes3and6,Fig.5,

conﬁrmsthatTLLiscovalentlyboundedtoSPMN@APTES-GAand

SPMN@PEI-GA.Nevertheless,thesebiocatalystsalsohave

nonco-valentlyadsorbedenzyme,sinceproteinbandsweredetectedon

(8)

Table2

AssignmentsofthevibrationalmodesoftheFT-IRspectraforimmobilizedSPMNs.

Wavenumber(cm−1) Vibrationalmode Description

585 Fe-O Fe-ObondsintheFe3O4structure

1050 C N AminegroupsinPEI,APTESandTLL

1625 ıN H

3413 O H HO-groupsonFe3O4orabsorbedwatermolecules

:stretchingandı:bending Table3

Immobilizationparameters:Immobilizationyield(IY),theoreticalactivity(AtT),biocatalystactivity(AtB)andrecoveryactivity(AtR).

Biocatalyst IY(%) AtT(U/g) AtD(U/g) AtR(%)

SPMN@APTES-TLL 64.3±7.8 22.5 23.0±0.8 102.3

SPMN@PEI-TLL 69.1±5.9 24.2 45.5±4.4 188.2

SPMN@APTES-GA-TLL 45.3±4.4 15.9 59.3±4.9 373.4

SPMN@PEI-GA-TLL 50.6±2.4 17.7 6.2±0.6 35.1

Fig.5.SDS-PAGEanalysisofdifferentTLLpreparations.Lane1:molecularweight markers(valuesinkDa),Lane2:supernatantoftheSPMN@APTES-GA-TLL,Lane3: supernatantoftheSPMN@APTES-GA-TLLafterreactioncycles,Lane4:supernatant oftheSPMN@APTES-TLL,Lane5:supernatantoftheSPMN@PEI-GA-TLL,Lane6: supernatantoftheSPMN@PEI-GA-TLLafterreactioncycles,Lane7:supernatant oftheSPMN@PEI-TLL.(Freeware1Dgelelectrophoresisimageanalysissoftware GelAnalyzer).

3.4. ThermalstabilityatdifferentpHvalues

Inordertoevaluatethestabilityoftheimmobilizedenzyme,

theproducedbiocatalystswereincubated atdifferent

tempera-turesandpHvaluesandinthepresenceorabsenceofethylether.

Table4 showsthehalf-lives of thebiocatalystsatthedifferent

studiedconditions.

AccordingtoTable4,bothcovalentpreparationsaremore

sta-blethantheionicexchangedonesatpH’s5and7.Thisstabilization

maybeduetotheformationofseveralcovalentlinkagesbetween

theprimaryaminegroupsoftheenzymeandtheglutaraldehyde

groupsofthesupport,orjusttoapreventionoftheenzymerelease

duringinactivation[43].However,atpH9,theionicpreparations

aresigniﬁcantlymorestablethanthecovalentpreparations.This

mayduetodifferentinactivationwaysforthedifferent

prepara-tionsatdifferentpHvalues[44],oratthestrongeradsorptionof

theproteinatpH9(theenzymewillhaveahigheranionicnature

atthispHvalue).

TheresultsofTable4indicatethatSPMN@PEI-TLListhemost

stable ionicexchanged preparation, since its half-lifetime was

higherthanSPMN@APTES-TLL’shalf-lifetimeinallconditions

stud-ied.Thehigherthermalstabilitiesofthelipasesimmobilizedon

SPMN@PEIcomparedtotheotherionicpreparationcanbedueto

moreintensemultipointionexchangethatavoidsenzymerelease

fromthesupportevenathightemperature.

On theother hand, the SPMN@APTES supports allowed the

achievementofthemoststablecovalentpreparation.Forinstance,

thebiocatalystSPMN@APTES-GA-TLL is3-foldmorestablethan

SPMN@PEI-GA-TLLat70◦CandpH7.Moreover,inorderto

com-parethestabilityoftheTLL immobilizedonnanoparticleswith

otherTLL preparations,the biocatalystglyoxyl-agarose-TLL was

produced. Accordingto the results from Table 4, the

immobi-lizationonnanoparticlesbycovalentattachmentaremorestable

thanTLLcovalentimmobilizedonglyoxyl-agarosesupport,mainly

atpH7,sincetheSPMN@PEI-GA-TLL andSPMN@APTES-GA-TLL

increasedthestabilityby3.2and9.7-foldwithrespecttothatof

glyoxyl-agarose-TLL,respectively.Otherauthors,studiedmagnetic

nanoparticles functionalizedusing 3-marcaptopropyl

trimetoxi-sylane(MPTS) or1-(3-thrimetoxysilyl propyl) urea (TMSPU) to

immobilize TLL [45]. The immobilized enzyme was submitted

tothermalstability experiments(30–80◦C),half-livesof30min

wereobtainedat50and60◦CforMPTSandTMSPU,respectively.

SPMN@APTES-GA-TLLandSPMN@PEI-GA-TLL(Table4)aremore

stablesincehalf-livesat70◦Cwerehigherthan440and140min,

respectively.

Thesolventstabilitytestwascarriedoutonlyfor

SPMN@PEI-TLLandSPMN@PEI-GA-TLLinthepresenceoftheethyletherat

30◦C, sincethe bestresult ofthe kineticresolution of the

rac-1-methyl-2-(2,6-dimethylphenoxy)ethylacetatewasobtainedin

suchconditions,aswillbeshowninSection3.7.Covalent

prepa-ration remained more stable than the just ionically exchanged

enzyme.

3.5. Tributyrinhydrolysis

Inorder toanalysetheactivityof theproduced biocatalysts,

hydrolysesoftributyrincatalyzedbyTLLimmobilizedwere

per-formedandresultsareshowninTable4.AccordingtoTable4,all

theTLLimmobilizedonnanoparticleshadhighhydrolysisactivities

(>60U/g),whencomparedtoglyoxyl-agarose-TLL(about0.8U/g).

SPMN@APTES-TLL presented the highest hydrolytic activity

(about120U/g).Ontheotherhand,thisbiocatalysthasthe

low-estthermalstability(seeTable4)withrespecttotheothersTLL

nanoparticlesimmobilizedpreparations.Inaddition,themost

sta-ble biocatalyst SPMN@APTES-GA-TLL had the lowest hydrolytic

activity (see Table 4).The activity of thePEI ionic preparation

islowerthanthatoftheAPTESionicone(seeTable4), andthe

thermalstabilitytendencyrevealsthePEIionicpreparationtobe

morestablethanthatforAPTESionicpreparation.Thisbehavior

canbeexplainedbythenatureofPEIandAPTESmolecules,since

(9)

Table4

-EffectofdifferentincubationconditionsontheenzymestabilityofimmobilizedTLLlipasebiocatalysts.(pH5–65◦_C,_pH_7–70◦_C,_pH_9–60◦_C,_pH_7–30◦_C_in_the_organic

solvent)andhydrolyticactivityoftheimmobilizedTLLpreparations(substrate:tributyrin;reactionconditions:37◦C,pH7).

Typeofbiocatalyst Half-lifetime(min) Immobilizedenzymeactivity(U/g)

pH5 pH7 pH9 Ethylether20% SPMN@APTES-TLL 49.9 2.3 49.7 – 122.7±13.1 SPMN@PEI-TLL 97.0 4.5 63.0 3285.5 88.5±0.4 SPMN@APTES-GA-TLL 85.3 447.3 14.2 – 66.0±6.3 SPMN@PEI-GA-TLL 100.7 146.6 32.6 >4320 72.0±0.8 Glyoxyl-agarose-TLL 34.1 46.0 111.8 – 0.84±0.06

Fig.6.(A)Synthesisreactionofrac-1-methyl-2-(2,6-dimethylphenoxy)ethylacetate.(B)Hydrolysisreactionofrac-1-methyl-2-(2,6-dimethylphenoxy)ethylacetate.

Therefore, supports graftingwith branchedmolecules probably

interactmoreintensivelywiththeenzyme,duetobiggersurfaceof

contactenzyme-support.Thus,thissuperiorinteractioncan

pro-motechangesinthe3Dstructureoftheenzyme,decreasingthe

enzymaticactivityand,incontrast,increasingthestabilityofthe

biocatalyst.

Accordingtothebiocatalyst activitydata (AtD)measuredby

hydrolysis of p-NPB (0.4mM), see Table 3,

SPMN@APTES-GA-TLLhasthehighestcatalyticactivity,followedbySPMN@PEI-TLL

and SPMN@APTES-TLL. The biocatalyst SPMN@PEI-GA-TLL has

thelowest catalyticactivity.Comparedwiththeseresults, ionic

preparations showedhigher catalytic activity in the hydrolysis

oftributyrinthancovalentpreparations.Thissuggestsa certain

changeontheenzymespeciﬁcityuponimmobilizationfollowing

differentprotocols,ashasbeendescribedinotherpaper[46].

3.6. Synthesisofrac-1-methyl-2-(2,6-dimethylphenoxy)ethyl

acetate

The 1-(2,6-dimethylphenoxy)propan-2-one was reduced

using sodium borohydride in methanol (MeOH) to yield

rac-1-(2,6-dimethylphenoxy)propan-2-ol (90% yield after

ﬂash-chromatography). Next, the chemical acetylation

rac-1-(2,6-dimethylphenoxy)propan-2-ol by using acetic anhydride

(Ac2O), 4-dimethylaminopyridine (DMAP) in dichloromethane

(CH2Cl2)atroomtemperatureallowedthepreparationofthe

cor-responding rac-1-methyl-2-(2,6-dimethylphenoxy)ethyl acetate

with92%isolatedyieldafterﬂashchromatography,showninFig.6.

AppropriatechiralGCanalysesweredevelopedforboth

rac-1-(2,6-dimethylphenoxy)propan-2-ol and

rac-1-methyl-2-(2,6-dimethylphenoxy)ethyl acetate in order to achieve a reliable

methodtomeasuretheenantiomeric excessvalues oftheﬁnal

productfromthelipase-catalyzedresolution(Fig.7).

Fig.7. (A)GC chromatogramsof rac-1-(2,6-dimethylphenoxy)propan-2-oltR

62,3min.(R),tR 62,9min.(S). (B)GC chromatogramsof

rac-1-methyl-2-(2,6-dimethylphenoxy)ethylacetatetR71,8min.(S),tR76,6min.(R).

3.7. Hydrolysisofrac-1-methyl-2-(2,6-dimethylphenoxy)ethyl

acetateusinglipase

Thehydrolysisofrac-1-methyl-2-(2,6-dimethylphenoxy)ethyl

acetate was catalyzed by the biocatalysts

(10)

Table5

Enzymatickineticresolutioncarriedoutwithrac-1-methyl-2-(2,6-dimethylphenoxy)ethylacetate.

Entry Biocatalyst Co-solvent e.e.p(%) c(%) E

1 Glyoxyl-agarose-TLL Ethylether 99 5 209

2 SPMN@APTES-GA-TLL Ethylether 99 10 221

3 SPMN@PEI-GA-TLL Ethylether 99 34 327

4 SPMN@APTES-TLL Ethylether 99 16 238

5 SPMN@PEI-TLL Ethylether 99 50 1057

6 IMOBED150-TLL Ethylether 99 50 1057

7 SPMN@APTES-TLL – 99 3 205 8 SPMN@PEI-TLL – 99 6 211 9 SPMN@APTES-GA-TLL Acetonitrile 99 17 13 10 SPMN@PEI-GA-TLL Acetonitrile 99 17 242 11 SPMN@APTES-TLL Acetonitrile 99 15 237 12 SPMN@PEI-TLL Acetonitrile 99 34 328 13 SPMN@PEI-GA-TLL IPA 68 17 6 14 SPMN@PEI-TLL IPA 99 11 223 15 SPMN@PEI-TLL THF 99 50 1057

Glyoxyl-agarose-TLL.Theconﬁgurationsofthestereocenterswere

determinedbyopticalrotationusingapolarimeterandthevalues

obtainedwerecomparedwiththosedescribedintheliterature.The

valuesofopticalrotations:

(1R)-1-(2,6-dimethylphenoxy)propan-2-ol [␣]D20=+1.46 (c 8.0, CHCl3) e.e 99%, literature value

[␣]D20=+0.9(c5.5,CHCl3)e.e98%[47]and

(1S)-1-methyl-2-(2,6-dimethylphenoxy)ethylacetate[␣]D20=−10.8(c8.0,CHCl3),value

notdescribedintheliterature.Theconﬁgurationsofthe

stereocen-tersoftheproductandsubstraterespectedKazlauskaempirical

rule, being the alcohol obtained with R-conﬁguration and the

remainingsubstratewithS-conﬁguration.

Hydrolysisreactionswerealsoperformedwiththecommercial

IMMOBED150-TLLforcomparison.Theresultsoftheenzymatic

kineticresolutionofrac-1-methyl-2-(2,6-dimethylphenoxy)ethyl

acetate using the developed biocatalysts (SPMN@PEI-GA-TLL,

SPMN@APTES-GA-TLL, SPMN@PEI-TLL, SPMN@APTES-TLL and

glyoxyl-TLL)showedsatisfactoryresultswhencarriedoutinthe

presenceofethylether(at30◦Cduring24h),asshowninFig.6.

For theSPMN@PEI-TLL,theconversionvalues were50%,the

enantiomericexcessoftheproductwas>99%andthe

enantiospeci-ﬁcity was 1057 using tetrahydrofuran (THF) or ethyl ether as

co-solvents(Table5,entry5and15).Theseresultsweresameas

thoseobtainedwiththecommercialbiocatalystIMOBED150-TLL

(Table5,entry6).Theotherbiocatalystspresentconversionvalues

between3and34%andenantiomericexcessoftheproductof>99%,

withenantiospeciﬁcity>200,exceptingtheSPMN@PEI-GA-TLLin

thepresence of isopropanol (IPA) which had low enantiomeric

excessof theproduct (68%)and low enantiospeciﬁcity(6), and

SPMN@APTES-GA-TLLinpresenceofacetonitrilewhich hadlow

enantiospeciﬁcity(13).

Thehydrolysisreactioninanaqueousmediumoccurredonly

inthepresenceofthebiocatalystsimmobilizationbyionic

adsorp-tion(SPMN@APTES-TLLandSPMN@PEI-TLL).Thesereactionsledto

highvaluesofenantiomericexcessoftheproduct>99%,butwith

lowreactionratesthatdrovetolowconversionvalue(3–6%)and

enantiospeciﬁcityabove200,Table5entries7and8,respectively.

Alltheﬁvepreparedbiocatalystswereevaluatedinthekinetic

resolution of the rac-1-methyl-2-(2,6-dimethylphenoxy)ethyl

acetate (at 30◦C during 24h) in presence of four different

co-solvents (acetonitrile, isopropanol, ethyl ether and

tetrahy-drofuran)andwithoutco-solvent.However,forthecaseswhere

there wasno reaction, theresultswere not shown in Table 5.

TheSPMN@PEI-TLLwastheonlybiocatalystthatpresented

cat-alyticactivityintheﬁveconditionsofreaction(withandwithout

co-solvent). These results again show the great potential of

immobilizationintuningenzyme features,inthiscase notonly

speciﬁcity,butalsoresponsetochangesinthereactionconditions

[46].

Fig.8.Operationalstability(recycle)ofthebiocatalystsbyhydrolysisof-NPB

(50mM)at25◦_C.

3.8. Operationalstabilityoftheimmobilizedenzyme

Thereusabilityofthemostpromisingbiocatalysts,

SPMN@PEI-GA-TLLandSPMN@PEI-TLL(Table5,entries3and5,respectively)

wereevaluatedinthehydrolysisofp-NPBandintheenzymatic

kineticresolutionofrac-1-methyl-2-(2,6-dimethylphenoxy)ethyl

acetate.Itcanbeseen,forthekineticresolution,thatboththe

activ-ityandspeciﬁcityremainedhighinthesecondreactioncyclewhen

usingSPMN@PEI-TLL(Table6,entry1).Theobserveddecreaseof

itsactivitymaybecausedbyinactivationordesorptionofsome

enzymemoleculesfromthesupport,which havebeendetected,

seeFig.5,lane5.Theinactivationmaytakeplaceduringreactionor

maybecausedbyanincorrecteliminationoftheremaining

com-poundsduringthewashingsteps(betweencycles).Ontheother

hand,inthehydrolysisofp-NPB(Fig.8),theoperationalstabilityof

SPMN@PEI-GA-TLLwashigher,sinceitremainedactive(43%ofthe

initialactivity)after8cycles.Itcanbeobserved(Fig.8)thatthereisa

highlossofactivitybetweentheﬁrstandsecondcycles,whenusing

theSPMN@PEI-GA-TLL.Thisbehaviorconﬁrmstheresultsshown

inFig.5,lane5,indicatingtheexistenceofnoncovalentlyadsorbed

enzymemoleculesthatwasleachedbytheorganicsolventinthe

ﬁrstreactioncycle.Fromthesecondcycle,thelossofactivityis

notassharp,whichisconsistentwiththeformationofcovalent

boundsbetweentheenzymeandthesupport.Resultsobtainedby

(11)

functional-Table6

Reuseoftheimmobilizedenzymeinthepresenceofethyletherandbiocatalyst/substratemassratioof2:1at30◦_C_and₂₄_h_of_reaction_per_cycle.

Entry Biocatalyst cycles e.e.p(%) c(%) E

1 SPMN@PEI-TLL 1 99 50 1057

2 99 50 1057

3 99 26 280

2 SPMN@PEI-GA-TLL 1 99 34 327

2 99 4 207

izedwithAPTESandglutaraldehyde−MNPs-TLL)inthehydrolysis

ofp-nitrophenylpalmitate,showsthatbothSPMN@PEI-GA-TLLand

SPMN@PEI-TLLaremorestablebecausetheyretainedmorethan

20%ofitsinitialactivitywhileMNPs-TLLhadnoactivityafterthe

8thcycle.

Cyclesofreactionmaycausethedenaturationbecauseenzymes

areverysensitivetoenvironmentalchanges.Whenthereactionis

conductedintheabsenceofsolvent,theinitialreactionraterapidly

declined,causedby:i)theincreaseofrestrictionstosubstrate

dif-fusioninthesolidbiocatalyst,ii)thestrongenzymeinactivation

byundilutedsubstrate,iii)enzymeloss,amongothers.Insolvent

media,anapparentactivationeffectcanberelatedtotheincrease

ofsubstratediffusionasthesupportswells[48,49].Furthermore,

productmayaccumulateonthematrixsurface,whichcanproduce

additionaldiffusionalrestrictiontothereagents[48,49].Finally,

thebiocatalystsmayalsoundergoenzymelosswhenenzymeis

immobilizedinthesupportbyphysicaladsorption/hydrophobic

interactions [50]. Since theenzyme is weakly stabilized, it can

easilybeleachedbyorganicsolvents[9,48,49].Theformationof

thecovalentlinkagesbetweenthesupportandlipasesleadstoan

increaseintheconformationalstabilityoflipasesoveramulti-cycle

reuse,whencomparedtotheionicpreparations.Thistechnique

notonlybounds theenzymetothesupport,butalsopromotes

enzymerigidiﬁcation,avoidingleachingandkeepingtheenzyme

activeforlongerperiodsoftime,thisisimportantforthereuseof

thebiocatalyst[2,3,16,25].

4. Conclusions

TheFe3O4nanoparticleshavecrystallinestructureandpresents

superparamagneticbehavior.ThesamplescoveredwithPEIhavea

greateramountofmasscomparedtotheAPTESsamples.Thus,the

PEIsamplespresentweakermagneticpropertiesthattheAPTES

ones.However,ashighertheamountofanchoredmass,greater

theamountofaminogroupsavailableforenzymeimmobilization.

Therefore,thesamplesfunctionalizedwithPEIpresentedgreater

amountofimmobilizedenzymes.Theinteractionbetweenthe

sup-port and the lipase is stronger when theimmobilization takes

placebycovalentbondsratherthanbyionicadsorption.

There-fore,the strongattraction of covalentbonds and thebranched

structure of PEI maycause distortions in the protein structure

and affect the catalytic activity of the biocatalyst. In general,

theactivationofthesupportbyglutaraldehyde produced more

stable and resistant biocatalyst to the most severe conditions,

but with lower catalytic activity. The preparations presented

good stability without an expressive loss of catalytic activity,

which shows the potential application of the TLL immobilized

onsuperparamagnetic nanoparticles as heterogeneous

biocata-lysttoreplacechemicalcatalysts.Thisnewstrategytoobtainof

medicamentprecursorsusinganewbiocatalystfromSPMN

sup-portsmatrixes can beconsidered environmentally benign, low

cost, commercially available, stable, reusable and highly

enan-tioselective. The conversion attained in the kinetic resolution

of rac-1-methyl-2-(2,6-dimethylphenoxy)ethyl acetate was 50%

(maximumconversion)andthee.e.oftheproductwas99%,forthe

enzyme/substrateratioof2:1,inthepresenceoftheethylether

orTHF(20%both)at30◦Cduring24hwiththeSPMN@PEI-TLL

biocatalyst.

Acknowledgments

Wegratefully acknowledgetheﬁnancialsupportofBrazilian

AgenciesforScientiﬁcandTechnologicalDevelopment,Fundac¸ão

CearensedeApoioao DesenvolvimentoCientíﬁcoeTecnológico

(FUNCAP), Conselho Nacional de Desenvolvimento Cientíﬁco e

Tecnológico(CNPq),Coordenac¸ãodeAperfeic¸oamentodeEnsino

Superior(CAPES),andMINECOfromSpanishGovernment,(project

numberCTQ2013-41507-R)isgratefullyacknowledged.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in

theonlineversion,athttp://dx.doi.org/10.1016/j.bej.2017.05.024.

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