Stabilization of enzymes on mixed polymer brush with aromatic groups

Abstract

Random co-polymer brushes with aromatic monomer ethylene glycol phenyl ether methacrylate (EGPMA) and zwitterionic sulfobetaine methacrylate (SBMA) to stabilize enzymes that are immobilized to the brush layer. It is shown herein that that the incorporation of 5% EGPMA results in the dramatic stabilization of immobilized lipase, which resulted in a 50°C increase in the enzyme's temperature optimum. By stabilizing the enzyme at elevated temperatures, a 50-fold increase in catalytic activity was observed relative to the unmodified enzyme. Importantly, the incorporation of EGPMA enables new types of interactions with the enzyme compared to prior brush surfaces that contribute to this stabilizing effect.

Description

[0001] - eemers a,ca.on camseene o..rovsona caono. ,,e awaresnevenaoonna wasc menacee wounaovoenr.nmeeno svuernomren unaesr cerranan n rumesrnenvenon. mmozes ennvzenmeosn reauneasoe moemoers m aaner caoms.osonsore caac enancemen o sa,m amozaon o enzmes on so suorsaseoenaorovencrease osensnc avn, an reusa,or acaonsn cemca anarmaceuca manuacurn, amano enzormemese,aon.norunae, manmmozaon maeras arenerenmmozaon aroac reau acvons oveeraama caacerormance.as neees an caacerormance.eresennvenn eecso enzmes an manans or enancesmoran nees as we aarenneooonwnrovsecso ssusree.ms an meos meenese e eresennvenonroves co-omerruses w aromac monomers, suc as suoeneeane mcoeacern eer meacrae , an zweronc monomers, suc as s sownerenaaeaenco,roor saaonze enzmesa aremmozeoerusaer. ommozease, wc resuen a oºncreasen rees eunzsmne’eramac sazaon sazne enzme a eevaeemeraures, a-oncreasesne cmaeraure omum. oserve reaveoe unmoe enzme.moran,encororaon oac acv ena waess n seawznes o eence.rac unonsu we asece o enzmsen cvoemnoanresoernocrorroursa sounr oacese araom coancr mueos suc as ,noerus suraces.s conemaeaencuson oo anromers, monomersn co-omerruses caneao a smarramac sazaon omac caac acv o a vare o enzmes.romacs an areaaas anncreasesn ruses as reaeso enzme sazaon.snvenons noeen exoren suc ocaass anosensnes.noes,e neeo sazaermcumar reevanoe crca an reresen oze enzmess n ars asesc a maor caenemaon. monomern eresennvenonroves a co-omerrusavn an aromac r comnaon w a non-aromac monomer.e co-omerrus accornoeos sa asezce c aann enazvem aen ennzeme aeraeco.eoe coe- cooomemrerrursus a.ccore cno-oomeerrsrus assecou cnnocouem an aona monomer or mo an c eaacaes aacmen o an enzmeoe cocroner m ceerruasn. e,menozameens oeaese,rs a asaenceeo esnzamaese c,anres an, casem, coaronc anrase, susn,orseraseroxase, aceconeserase,ucose rsn,romn, emerase,ase,roen,ransamnase, somerase,encn acase, reucase.n an avan nroreucase, unsecceroxenase, anmne anarccaase aeou,s emoomumene enzmesana ruosaase ,ana seuomonas suerraccerocoras meeeasease ,acus susase , anaseromnomonas s. -.n a,roaer smeomcesanunosusase , enzme suc as oxoreucases,ranseras nse,e enzme cane ae oransoca es,roases,ases,somerases,ases, an eer mense cse. arcan emomens oers asec,e aromac monomers eenecoen en eerr meaeacrae,re,een meacrae,enz meacrae,eeneco en eer meacrae,enaeneeneccoen eer meacrae,eraeeneco en eer meacrae,-a meaocraeen,- eaer mea mcrae,oeeneco meacrae,-nraceneme meacrae, srene, e eacrae,-reneme en acrae,enz acrae,eenecoen eeneer accroen eer acrae, en eer acrae,eraeenecoen eer acrae,enaeeane,reeneco acrae,oeenecoen eer acrae,-a acrae,-eaco acerna eeer reneme acrae,-nraceneme acrae, eenecoen eer acram,e-, ecno acernam eee,r aecnrzam acre,ameraee,eeneenecocoenen ee er aecrr acarmame,ee,nraeene coen eer acrame,oe eene a enecoen eer acrame,-a acrame,-a acrame,-reneme acrame an-nraceneme mc meraamcrea,m ee,eneeenceocoenen ee er mere maecracarmame,e,erne meeneacrcoameen,e enzer eeerac mreamacre,aemraee,eoneecoeneen eer meacrame,enaeenecoen mea co en eer meacrame,-a nraccreame,-a meacrame,-reneme meacrame or- rounn co ceem nre sasnn em meoa omcrena -sm m o. eaecrrso asamec,e non-aromac monomers seecerome meacrooxe rmeammonumn coorormeammonum core,- suoro acrae,-suoro acramre,-suoro meacrae,- meacrooxe me--suoroamme,on-suoro meacrame,- ac, acrame, meacrame,o umroxe, meacrc ac, acrc acrae,oeeneco acraemee,neco meacrae,oeeneco eamnoe meacrae,-eamonoeeene acco meacrame,- acrame, -eamnoe meacrame, nr-saoer,oa-eamnoe moronoe meacrae,--moronoe acrae,-cr-ame, -- acrame, an--mo moronoe n avanaeronoe meacrame. su ous emomens oers asec,e non-aromac monomers meoaceranae meacrae ,osorcone meacrae ,cosoxe ee non-aroma, ocr mooneomeren ceanco meacrae . acrae, suoeane e a zweronc monomer,ncun suoeane osorcone acr acarmame,e,os suoorecaonene me meacarcramame,e,o ascroorc seornnee, a accrae, ornne, acrosne, an ac ro n a secon asecerouamae. zweroncarom resennvenonroves aomerrus comrsn en eer mac coomer, suc as suoeane meacrae eeneco asec ca eacrae coomers.eomerrus accornoe secon rus accno cromnrose aeo seucon aos aecou comrses ao.un an aov aanouaeous emom.enn aaerocumarer avanaeous emomeneomerrus accornoe secon asec comrses aou zweron. nc aaromrac as coecomeers.reseen aromnvaenc mononroves aomerrus comrsn meacrae .n an avanaeous e omer cane eenecoen eero aou .n aarcuar avanmomeneomerrus comrses aou .eomerrus accorn aeous emomenerus comrses aou aacmen oeom oer asec canave a suor or so suraceor a eomerruesrr aucsco.rnoer asec canave aou.o aou mroomnoamcer m, oorn aomoer, aou o aou aromac monomer, aou o aou aromac e aromu aromac monomer. c ac monomerneomerrus accornoer asec cane eene eoeneenc eer meacrae ,en meacrae,enz meacrae,eraeenoen eer meacrae,reenecoen eer meacrae, oeecoen eer meacrae,enaeenecoen eer meacrae, meacraenee,-cro en eer meacrae,-a meacrae,-a een eneme meacrae an-nraceneme meacrae, srene, eer aceraceo,reenen eeer accoraeen, eener a accrraaee,,eernaze acerneae,coeenene eecroen enaeenecoen eer acrae,oeenecoen eer ac acrae, a acrae, -a acrae, rae,- nracen -reneme acrae, - a eme acrae, eenecoen eer acrame,en acrame,enz accrraame,eenecoen eer acrame,reenecoen eer acme,eraeenecoen eer acrame,enaeenecoen eer acrraammee,oeenecoen eer acrame,-a acrame,-a en ee,r m-eraecnremaeme, acrenam mee aancra-mner,aceennzeme mea accrraammee,, eeeenneeco en eer meacrame,reenecoen eer meacrame,eraco coen eer meacrame,enaeenecoen eer meacreamene oernae amne oure,c a-o serceneemn ee e resen mere m nvae ecnrac oar nma e, meac m roev ore, es-- a sna urraaccee-neram meeac e co mreamacre,am-ae. comose o an aou. o aou. moar o aromaocmerrus. suor comosen a o asec anerreeaseenrnavnenon mrooavr oes a ar soumraacce-rae cooo amouerrus m suoaror os aromac monomere or s muroaec.e-raaeac coaoesm aearc amcceonr on ano enzemeo asee ccoo canave an aona emomense aona monomer or moesc meamerrus.n ceran roxsuccnme meacrae -,-roxsuccnme acracre,aec ,- -socanaoe acrae,-socanaoe meacrae,-soc acrae,socanaoe meacrame, nroracec ac mea anaoe acrame,- nroracec ac acrame, nror crae, nroracec ac acrae, meacrae,mnoacec ac a acec ac meacrame,mnoacec ac meacr crae,mnoacec ac acrame, ormnoacec ac o an enna z am mne ae ov.anea ceoouos emmerormusen,s acona m meonaocmrera oer moe, waacc caanese araecsemen concenraon o aou.o aou , morereera a concenraon o n a surace-rae coomer accornoe asec canave an e aou.eoe .e enzme cane a , caronc anrase, cnzme covaen aace ase, aaneosaase,rsn, cmorsn,rom ocrome ,enzaee or aceconeserase.n an a n, susn,orseraseroxase, ,ana anarc vanaeous emomen,e enzmesana ruosaase ase ,seucaomoansaes suer,omucor meease ,acus susanunosusase , oraseromracceroasease,ermomces enzme cane oneromerou o oxnomonas s. -.oreenera,esomerases oreucases,ranserases,roases,ases,o a sca ss, u c o ror anceemases or aa. mecrosaeere.osmeurrreurs co ancecmoranoe varous asecs canerae e varous asecs canave an enzme, eaeomerrus accorno rus.er enzmes cannc suc asase, covaen aaceoeomer ,zomucor meeueas oeana, ruosaase ,ana anarccaase suerracceroasease,ermacous susase ,seuomonas romnomonas s. -.oreenermcesanunosusase , or aase oxoreucases,ranserases,roases,ases,asomereas eensz,meas ceasn ane ornaensroocmasese.rous o eaeoersc aruoenr, unaeernsann connne ocone wnveneo anc,co remeraenncen soruawnes m,an weoc:eoown .s a co seo ousraons sown an overvew ommozaon o on mxe o , merruses. acemac oerus suraces w cemca srucures rown on sca nan,o anarces..ase- secra o- omerruses srecn moes. c c nes reresen caracersceasor aromac- scaern.or eac comoness oeraeomeraer measure namc raus oeomer- son,e raus oe core2arce was suraceromeecnca recaesn =rae, acacorucne.nrroorrraorsa reareosnen o eerr soran reaare erroor oe se meanorree core scaarce. usraon oe resunrus suors ae uracon oe ne r conuaon o o .russ ar sa uee s or. es.r aasem soewranure-raemaence enn aacncvemen osn erormance on mxe suors.rrorars reresene sanar errormmoze anree on recaesor eac measuremenn =.eama oe mean oreeecnca acvaaromanea scaeoen corresonnoeemeraure-eenenmmoze enzme. curnov ervaeweeneowes an esurnover o anea.nzmac acve rer oeno aº oaneromaaonsneaseox o rrorars reresene sanar enroe ommoze urnncuaon aº. measuremenn =. e rror oe mean oreeecnca recaesor eac o curvesrom..rroeorrearcsa esomaaee va ororoaucaoonrm oanone croarrees eorrnorn areoncnueeraon areoo smao u anure a.romsa ae s o ces re oerve surea. s osneam aacsv so ownn mxeemac o muaonrs o surace exose uncon o concenraons exresse as aeamaor uses.cv as a w-e .or eaceama, coorae scan w e varous muans as we as o eacmmoze enzmew-e or muan,asase oneowes an es acv recanes corresono conons an rawaa are sownn..rosse acv.oe,eeamao weremmoze an w-e enzmea no measurae .s a -r sceWTsucaerom.a. cem mac an eamas ommoze - . a eamaasc co orresaoeneno e-aco, wmerc av seuraeorn coerm-uoosroonorreowss,a annce eacavg.emeraure ocoseurmvanso.ns.asneno waee numnesersn recareeseneer aesssooca uese vaoue os anveurasoerom unoe eac conon.umersnareneses reresen eoeracon,F,orrasn = oaseneeermnaon use sanarevaon ooeraconorve mnmzaon meo..roves a n eren sarnonsroro unsuervse .s aru sese os. arasec svoewrnn en ansear on rne aaem ve cocrs n asno aan o nsssor o.. on- an unmomonozre e on sue conon.rrorars reresene uncerann esmaon o rae ac ene e a.ene,..,aaac,..,aar,..c consans, asescre rucura anunconaeeroene ommozenzmewarz,..orrean . ucuaons oeoe an unoe saes measusre. aen rooo-m,e- ucuaons onvuaraecoresnnervas wenroen re an-suare unoe saes, resecve.rrorars reresene s mansneoe or measuremensor eac conon..ranarevaoneween aenvua rasn oves an enare verson o some oe ...s aomraer sruoswesn, m weaaseurre conac anes oa suracesunconaze w roc o – suraces.rrorear sses rseero meo, sowne smar orveec resene sanar error oe mean .ncas a rercaesn =. exraceroma. sown oma acvoptanemeraureoptommoze sae reon corr a as auncon oomerrus comoson.eaaonsne error oe meanoesronreeoeconptc aanoptvauesorree .rrorars reresene sanar .s anusraon so rwecaesn =. woere soven-exos n srucures oWTanosea-esne muans, ar e aromac resues are cooren rearranra-scae, anure em oarc a an reasues are e u csoeoreornue wasac,n anra-secae.maes wereeneraen . ose esor muans wereenerae .r musa a soen a on reaxaon, as exaneneeos secon. mmoze muanreeras sownemeraure-eenen acvos oree an os ares soownn.s. onr suors.e assocaeeamas reaeoese recae rorars reresene sanar error oe mean oreeecnca ane:n.r eac aees a m seea osur .mem aaen nees: sno =wn. -aee eecro.oarenses:e oo--aee.ane.:aaneer:. ueamaaeeer us- .ane:aer.man waserorme usn a oon .ns ae sxea ouowrora casn.nae anerexauor canne. or unoe saes o moee aesenaveme se o aonearo smuaon eos.oor coe rerese s a se-avon ranom co, asescrene an reacnr nsermar seuenceoson,reen ranra-scae an , reseca-scaeeas reresen ano uorooresnosons oee can.ven,e arnaceas reresene sare an enune oe oneonearo smua-uorooresancesruoneween ano ase reresenserac on w ses.e reonnreen erranra-scae aseosve oeono o ueno se saesa woue ecenc . anence a racon oesr ae, werease re reons arerra reresense .s auraon soaw wnou cemecaFReEnTaura.ounoe sae. rocoren asenauran.n aonoe cn curve o - , usnuanne m soumosae a .ames were aowoncenraon on, a cononsa measuremen eo euraeor mnueseore acv an.s w snser e ae a -mrc oau ecu sre eo. -wenermen reeaoonesracewoneenor mamcmrooscozcea-mea asureº e.nzemac errorars reresene sanar error oe meanorreee rca errorars corresono error cnca recaesn =.orzona eermnao noeraconorveeraonsn = oasene .nrosm a.. ..naoeenera sownesruon oonornenses o unoe saesrom scrm raeeseosrom eac eama, a wnow o acceornensesa a .o ase a sºe o oserv anraaonss s woaws snee rcee are.senºa.ven eexranmeae,s oonor a- raecoresn me seresc an are sownromraecores n acceornenses eacmeon are ov aove, wereonor an acceornensesor s arer saee sraaee ren eamas.reen ranra scae an re soe or unoe, re onsnanes c an corresononervas wen - a.ns a scemasce scovwen. a mue seuence anmen o w-e maWTaesne muans.oven-exose aromac resues are en eacenoas, aonn anure mu aaronm aarec s reoswnuees aorwee w-een s ceaune.nce.e reacemen amno acsor ao.suo aar oras sown oca asoron secra oaexauor an c roores.n eaco,e correconacorsor overan snas s uocuroaeore.n n eoarcmazon,ewo an waveen nms s an wascoe’s a asre ae maexm arae m aasorance o eac .s ara sowne overa reoneween emsson s re w a crce. eea an excaon secrum oo r eauorooreecrum oexauor was useoreermnaon oe overanera oua s.ease reon ouoroo n erser raus osar .res,s as a exanene meos. o surace-mmonzeusrroaeonns os a anunmmamoenzaa cona menarex oune ao soe w sureascere.e sazaon roenmmozaonn varousesncun osensn, amorance o oremea ocaass, an ase.on.s. annseausr oa aovno sonwn aro surace-exose aromac resues onacus sus urer sazaon o v macneracons,ese cane exoeoromoeemeraureeenenc.romaac-s-ascacnn.sos monreeremmoeercauuarrennseernacsovnsaveronounce .s anusraon sown-sac e. .s a scem n w aomeracone. an as aso sowac oerus suraces w cemca srucures o , , sourcerov.e ras aann onusn ara a corn s. caow anne mac ernoascoesooar snnernea m roeeceuceracn. uorescen oecs uo nm.n oecsnenerace on, wc exces oeacroun. nm an nmasers enaee e exce,eano emnaon .s a se oras sownme-seres ac exucsaon ouoroores. wemesneoe - an unoeow-on s anaesa ea anass.nass oransonseween saes. enarovanwree naesencaon o eweenoe, unoe oreace unoser saes aowsnnransons nass owemes enaesee vae saes, oservaon o ecenc. .s a se omaes armnn scne umnon an reon rae consans. nerranser .e acs sownnramoecuarrseresonance azoenaa -rece muaeness aoweenroucon o csene an- seccaenn wnez a z an ses.c oroona cemsres aowe se- orser raus: =. nm-.areuoroores s –exa anaeme –o . xermen.s werse a c soen oucmeaoese asn sc rememovaac os so-swancne eneec o aromac resue remova.mmozeroen sa.ree muan racons resusn aecrease o aromacs remove. sec s wereesne.nea surace exose ure aromacs remove.on waasoun saur reamceov exa oose-s aarocmanc rses ruemsovne a.ecrearsena sa o a muans wess aromacs an a s o suorreerenc erma conen. eoess aromac racon..s a see oesoeearac moans sow enc aeneernmneanaounr on cme-averaeoe saes o an areu onons.noe .s a s se, wereass ocaze. arameers. ee omaes an scemacs sown - eermnenamc unoeracon vram aauae enaecurn conons , suoreo er unon.ucuaon : caerone-easer re-on an sownown oncrease anass enaes measurn r oeoe an unoe saes.n ase mnos re anoc “sens”eoe sae va reucnucuaons. e unoe sae cou maeazne unoe saes.seasoeoessae r o acaon o ae enzmesrae aone unoeawa,eano ac. re-on. oeron.s ara sowna macroscoc acv correaes w -eermne oocaaessnsnn e cxormemeex en svnronemcen msa.enrsasreor enzomoem,meo usezaon w unoce u asnamcmmozaon suorsa enae s o nove coomerrusesmmoze enzmes wasnvesa ura-ooca caacerormance o ase onrusesoee w. as wasemonsrae mmoznacus sus rou mue non-covae romac moees, wc cannerac weasemmensenusra acaonns,neuracreomnesn.oaucs ausr seuaosnroaseenss a ana mcoerearae sasae w. eacaosrs an re asnoonrsmaeoarr enez ammoeu an ore suaraocne.-exeose aromac resues wc coue amonncreasene omaem mara,ncororaon o aromacseo an ne-moecue eraure o º, as we as a-o enancemenn acv. romon enzme reo sune asn re sveaaeznaee esnez sume’osrso ace a asno cmaamecca ca acervoene ssae. sacs enec an was-c mauoen wneernac aroonmsaocr r sesauezsa woenre. muea reesu osu,r seuseneesnereenm unoerance o- unexore enzme-suorneracons can enae uncare oor rscoreownusraoran unesor usn enzmesn sa,m amcov,zsaor anomna reu oon sa esn.zm,eosr a on socaon susorsaseoenaorovencrease osensn anoremeaon.norun n cemca anarmaceuca manuacurn, amano enzmes, reucn overaae, manmmozaon maeras areneren suc as aers or coom caacerormance.omexeeroeneous maeras, or enzmemmozaon erruses,ave sncanoena as cusomzae suorsunae, comrsn nov.ese snec maeras are exceona namc an cemca assem w moeese cemca moeesanerac non-covaen varansen se-nroucon oommence enzme surace.moran,omerrus suors enaee -sacn, an-ca non-covaenneracons,e-onn,rooc se-assem, ooca macroonneracons.eaer aromac-reaeneracons areounn exremoc ormanosecuar assemes,ncunoue-srane ,roensromneracons are asoemse,v annraceuaru-u ase searaon meus.ucemeraure varaons, aneo conrueoerma sazaonvenernsensvo rousnommoza canewce as sron as sares.encororaon o aromac w aromac or caon suors maromoe sazn-sacn an-caonneracons enroucononc su oraceo-eoxocase- ennzme resues, resecve. cemcaeeroeneous suor maerassrre saznneraconsnonamc anmmoze enzmes. e suc mecansmosv ces comex sazaon mecansmsor convenona caracerzaon meos, sne moeane caennosenane usn mcroscoasrove unue mecancsuerser resonance enerranser- suors w aroeruneaance o enzme-omcernsns,emonsrananamc ommoze enzmes sazneoe sae anoerracaonsmroveeerormance “caerone-e” eec.araonsnomer srucure ma can reon.e., va ammoze oer new ooruneso enance mmo enzzameoner oormaancce. meacrae omeruruss su susorsarosemoe a on zweronc suoeane .ese enancemens cane arueo sazn remarroaecernmea sazaon o ’s surace an .semonsraeeren,on omerrursaceso wns veween amouns o aromac eenecoen eer meacrae monomer canaurrouesr e enevaanceeeem aceravure. ann s caonras o wmm eoorszeo sa,z we ecnzsm recs wn ar roamnaomc re csooues, a ruses,eresence oe aromac comonen enaes-sacn a merneracons.ourersoaee reevance o aromacne nor-caon aromac resuesn ons sazaon encracons,emac o removn na,- was emoeo sue conormaonaorornaamonc o was sue. oan mecanscnssnoe comex unon an reons onammmcoze anaer.vera,e comne macroscoc an moecuar-sca s wnerusmorance oroeruneneracons e oservaons unerscoree suor an enzme surace re eween aromac moeesnemmozaonmmoze enzme suesorreservn an rescuneoe an acve sae o saus. raneoow,omerrus suors comrsn zweroncaromac erma sa omm coomers sncan enancee caacerormance an omaune moz seuor,ro an enzme w aromac conen.ecca, an oncreasen maxmum c moe aºncreasen omaemeraure an a- exose orure a aac acv comareoree enzme. muans w reuce manvove-sacronma anc coonre-nca weorne snaeraczeonso anveossvenr exeen a,r soumaecsnrouas sazaon - anass sowea ruses more eecvereserveenoeer suase. oammzoaonze o aansermaenauraon comareo oer suors. on eorues seae was conssen w reuce unonnecsormmoze as- ucuaons.nance reonnecs oransen unoe enzme as we exe a consra annea usnso suee ssaeeaa rmmoze on ruses co, wc ma exane enance reonneoe rea comareo aca ranom comex maerasomar sura-oo ecs.esennsusraeeoena o ooruneso exore neweero caroereso enzmes we aso oenn uncereecenene ocuor eneousomers.mae,ese new maerasrove ex aunnenseroes anow sne uesec o eonzranmseosrmnaaorsns. envronmens anus caaze oramamxamm coe enazna – ceomnn e oess an nsn sa ona .rroacerz aca z zawon o oneronc rusoesmr weu ars s creus osns ssuorseas surace oe enzmeen roc as conrme usn our-acen a worme. uez,on..,ovaenene,m.m.o,czawaornz,e.as.oaaar,.-.cevuceranez-ome.ounranmcs uon mercanemcaoce , - o a oe r na, oerasesa are more aooc areeer saze essroc mxeomerruses conann sonveno-eexoseene aromcaoc m reesaucres,a acec.onunerensnor ~, oas an unusua comoson o on areessroocanac s surace.e aromacacesoun roocases esorme aac resues, wc areresenn more nersan .ncez-orn,.,ez,..,cwarz,.. aar,.. an nesnuesormzneneraceeweenmmozenzmes an uroemrmoroe,oemerruses. eaerasneraces, - care a suraceroc o s asoueoeresence o numerous oservaonns, warsooc reesszeuesasne, m aarneneu,r asera sraae,zeuamanero.ucase onese w aromacrous wn ruses, wc ma seccan n monomers sacn an caonc-caon resues.ese a erac w aromac- oerneracons w ,er romacrous ma asooenaromoe moeesa surrou oocneracons we soven exose aac erorma n ’s acve se,oenamrovns overa sa an caac anoncees va -sese o moaoe r oers m ase r,c suarnascme-s.rae ranom coomerrus suors comose o omerzaon omac .a werereare va aomranser raca rusesncrome surace o sca nanoseres.n aono an ,e suee moe moarc meacrae o aow covaen aacmen o . oer wors s o =*moereso_a moes o monomere.., .n ncororaon o was conrme usnmoes_ moes_ moes_ . secrosco, wc reveae a ssemacncrease ouse ree areocmaanceco-ur serr-ercansnorm monerarencreasn conen ..aracerzaon oeomer-moearc w namc scaern sueseae sovaeomer corona was- es c.as, we veree wea o arusesrown ona sca wa nmc . sac conac anes o ~ºor a suraces.. ers anoun smar a xame –xe uorsonerura-ooca cv an rosse o s raesoru onureera ane asmmoze was assae measurnena rae o was moreermos auncon oemeraureeween-º.emmoze rus como ae.e., acve a eremerauresanree or aomer one sons,e acv ommoze a eevaeemeraureseene sronemeraur raconnerusaer.s cane seenn.a, wc sowse sazne e-eecenen acvroes oree anmmoze .neresn,e suors conann on ncrease. w conen uo anenecreaseor suor resuen aramac caac eecnacnac,mmozaon o one measure acv oree , aon emen o uo-o comareoe maxmum ºoº .. ona, w we anncreaseneemeraure omumoptrom acv aoveº, mmoze on aree so swe aronounceoss o caac overe enre measureemeraure r uor exencreasn acv saznaneure suorsawne.ouoa,.es n suors wereess a nuance sazn mecansm assoc on-monooncren suese vsuazaon,eerenaerormae w an omaracon o .or ease oemeraurerom ance.e.,urnover ommoze as auncon oemeraure- .a was reresene as aeama ..s ee monoonc conr eenenncrease o urnover wenmmoze on suors,n o acasv w aoev sncanower acv on,, an suors, aneoss reaonseweeº on suors.ourerusraee non-monoonc aunc en acv anrus comoson,eurnover oe enzme aº asoe oonm oa, conens sownn.c.ne conex o , was oserve or .e oum aaroaerc reane o o mae useore reave amoun o e enzme so nae o an aromac monomer weeenen uoneen o conseraueroe..e,mmo.ze.surer conemaeae coue enzmes.omo sexueereo aeneous o suors m-caonarnonon sueo orseraoorneaservee emnzem oe amcmvoze sm smcuua aae roonr aorea a suºco o wr ras se co mmenasou, sreem n an ons, a.e-e ou.enen en o cnaeaac, aecv suecroera oser omm om roeoaev aeze m acucv wasna acv.owever,e acv ommoze was reaneoronermeser ce eera r reen saec su nsonore com oosroouncs, noarmmeao .o vsuazee cumuave acv overme, r n was esmae neraneaan., wc comos soue nso arrea .r uonver e.ee e a re seeaansve nw ac sc uev nee r rs se coee rcno ecn c eru sorv uue rcs. a-oes a o cn coa amsas erre rev ormoeae ane oaererus conann orromon unreceene erormance a eevaeceem oe surauorerss. xaasme –emovnromacneraconseucesermaa s e one sa resusescre aove,e eecs o aromacneracons one saazzaon o was exore. sucneracons conrueo sazaon,e sn eec o soue reuce aromac resuesne enzme were remove. amouns oo seusrsac wea-esxeossee aneusrenn aromraeec reserueens were m vuaraaenson a wannceexeo rseave meonnevaneure.e muans wereesnae , w soven-accees or aromac surace resues muaeo reucee aromac sDSA-50%se a surace aromac resues remove urace area ,DSA-100%, w removeae,..e , anDburied-100%, w aure aromac resues a sraomac resues accessensee muoanes saaceaeDeSsA-5n0%o an weeDrSrAe-m10o0vn res suoveenn-e rxeuocsee reucazcn res-suaecs ancce asnse-canone unneorac%arom eon ssa weDburied.-1aoonwae, reesmonva o o wuereer nece r aromacneracons00%ena w ne unoe sae, wc cou oeca eeaomans, m asoeao an overaecreasen acv anor sa. anure wer o acv reveaea muans w reuce aromac conenexose ,e a snaaze e onomer suors wower aromac comosonan w-e ecca,WaTre ven omzemmozaon oese muans wasess sazn. ane omea muans were o sumaor saorze on suor.s w.esso ormmoze muansa reuceerma sa reaWTveommozena, a,osree acv aoveº, evenou some oe muans were more acve an saWTn ..ese oservaons suesea aromac resuesn ae anemaonrWTnromon saznneracons w -conann suorsW,Tsuornean roe aen-asa mcecnan ansms m-caa aonso areenev oess o ronveen,nncuen saezoarmona oon ommorozeoc ncWeTs. wenr areomrausc coaneern maa m caanroemoeeomroer coacnn reraco,n ws.cs co auso aaeursee sea-as csanesn erusaer aroune enzme’soe an unoe saes. em o oecuexame –onormaonaaes ommozenzmeeermne usnnenuence o-r mcrosco was useonvesaee mecanscass unernnne anme-avus cemsr onmmoze sa.n conras wraona enseme e conormaeraonna s maeercsna omcrso oenm smaoz,e en-zmes onro av moesecuuaen-a-mvoeecnuormaon on - exermensns wor comne muexe sne-moecueeocass.e w ,o measure conormaona canes onvua enzme moe azaon resouon.o,.,on,. a,.raccaueo snecues w nm-scae eos,- cuer,.ne-moecue oroe-nm soreuccuuer .aure - armer.ourna oanooecnoo, .or- exermene annamcs reore varan w a soven exose csene ane non- s, arevous azoenaanne was useez,..,ene,.., canonca amno ac- ncreasenaacerormance ommo cwarz,..aar,..ramac rusuors. aass, - ze.asess enaeere uaszaon onomer cemsroae se- e o oroona cc - ,. secca w -areexauor ano osone sucaae, w wecr was conrme - ..euoroores were erser raus o =.e nnm c,oseroxm~. nmneoe sae o reaveo aarnermar se0u resunn .eonor an acceor were asoar was ence, resunnare searaonsne unoe sae.s searaon smu easomnae moene ranom co unoe sae o va a se-avon wa us,,en an averaener-uorooresance o aroxmae. nm ..n su aecar ene creecerease-n o w case emxceaceen uauornaon, w-c re usnuoenn a. ssse wmaasc conecrrmeaese -enc a wsa asumncmoon ozenauran concenraon.. man sca waers, an -e e oxnerrumsenes comose o,, an onnormaon aou sazaon mecan s wereerorme aº anº,rovn conons, resecve. ea sms unererma sae anermaenaurn were coeceo enae sasccaem reroarouures, m aeaasruere nmuemne or ooraemceo-raevserae-uaneasee.., avoenraor-eacoceeorracsoonra amneanmamasc waeraremeeenrserae.e., araren unon an reon raes. comoson an were useoenoe om a oservaonsor eacrus n eac exermena reazaon. an unoe saeouaonsormmoze acceor an onornenses co .nese sorams,eouaon wow ouaon w acceor anoworresoneoe aaren unoe sae, wee or eacrus co nornenses corresoneoe aarenoe sae. reso moson,eouaons wereene usn a mnmumneraenens comosonn wea ass ca scouwanen ase. num.er oeomee-av-seraaee oseorvaeonrsacvoneFoero eaac nrus o oserva umer unue ceo a an ansa. es os r oeaonnnoraceon une cxonecoerma roenve uaneeraerme suor was ecca, uner non-enaurn conons aº aenaurn conons. oeracon one suor.e.,e,mmoomozeomer w exe =.e.es we o on an wasower.. an.F, aº,e suorreservee sam ., =. a., we - mm eeve oon as aºFreuce s oze on an suors exe conormaeosnea saaaF= su. unesee. a rerman a a. enareue. rn s,u resoercv ceom.osons conerre sncan soue , an acv measuremens conons.oa,ºs aoveemor soue sare unoe aº.eosuunsaoptoree was ~ºereore, remnar caracerzao se,.,canc,.oson,.urcaon an -oeran e n oe exraceuarase oacus sus, an exremeasc vaue nzme.uroeanourna oocemsr,- .us,e non-zero avos oFaºor mmoze on aree suor comosons cane arueommroaezerus-en szomwennnerac.onas. aencau asre conssen w e reenon o acv su e seons navemaº, was no orwresvenr,aFwaso swuer oonrse ae aon reane same soueorrsa rceoanv aeoo aooww oermeenraauures.emeraures, wcusraese seca naure os suor comos rn snurn ma exane acv an sa o mmooznen o snasz sunor as. oscuesserac aoonve a.nn enezem, aacua acavve a co erreevaaeone wmase orasuerrevee.wee.n- eermne o neresn, vsuansecon oe eamas reveaeae unoe sae uusaon o - mmoze one suor was more comac.e.,ess e uneoaene un-oe saeouaon on an suors.s suesea consrane com mmoze one suor was more conormaona sruon oonaroernoensee oseorr suera ucneso.es saee ornen ecaec co suurac aes,o soew oservene suorneeromoe a more consrane unoe sae..nae necsxame –omosonauneruseseucenon ancceeraeeon our suer uonrsersnanermea meceannasumrsna conromoones, anamercFnoorrmaonm wmaos eze on rome- raecores..ecca,ransonroaesewexrace an unoe saes wereeermneormmoze - , an useoeneoe eecvenec rae consans o unon an reon an o cacuaee conon.a usn aree-saeunfold foldr eac exermena wncreasn cone arov can moe. º,e aarenecreasenFaº, n correae w a ssemacecr-1easen reon rae.oa, wunfolnfoldwasreaesore rus edr wcr oaeasn coneno . rs semar on .rus. s-1an. - s-1. 1 an. s . .. an s-1.ecr se-a1s.e smuse ruam snaeeoc s,ua asn, ormnm aonaozeous ana-ss a oº. n reveae. a n suan.ce snersc eecaromoe er rFomo e rus onn unon-1an en. reo.n. s-1rs an,e rus-1romoe aerfold.. san oldrusnee unon.nec.s o smmoze suors.muaneous,e unf. . s-1comareoe . . s-1a-n as ev.en. aow s-e1r sower unorusnes. onmoran,erunseers caorreeaweee wne “ec aaearroenne-nce”aser reon ann enance caacerormance.o ars a rease oFa resue suor cane exane a sazeoroxema saon,eeavor o one ucuaonsneoe an unoe sae wee an aesaze unoe sae. mean-suareucuaons nn re aso measure racne roo- resen one sae ore oer..er-nauosros ooresa vnaceueusr on eacne srvaaes r weveeanee nuraonceen neacmccare,nse w o conen, wc wereuaave smar aoº anº.ncreasn conen, seuoesen sae ommoze ecrease ssemaca w resrcroen mo ancreaseroen-omer aromacneracons ma ruses won r.es was conssen we reuceunfoldoserve on an monoonca w sec coonce .eucuaons oe unoe sae vare non- rus.sren was remnnrsaceonn, o w reuce ore unoe sae one . conssen w eresence o ae aoremenone oservaonsrom. an aarenromoes a unue unoe saeocaazese usnsoe sae.e rus consrane s unoe sae reos a aas namc an more conormaona comosonsaromoeess resrc er eecve rae comareo oerrus oes’nsa ze arsomsa ocenranseone raae veor c uon onsoaones s e anaes. anu ucnuoaoens revea.ee ac crcuaca, roe w oas ena mo meoneosm aenr-resue’s n-sumaceronus a snoven-ca exoonsenerarcosonness, maen occureweenroans-sacn orsnes an arnnes-caon,nuencn aannes, an sazaon o caaca acve .or exame,eoe sae o meaneamrocs an eecveowerneree ener oeoe sae va sazn aromacneramoe ecreasn .onverse, wen unoe,revousureenaanne cons, couorm nuenwfold-sacnneracons w as we as s orrosnes uaernar ammonums o .nceemescae oom-caonneracons we o manueaseran enzme unon,er er se canucuaonss orers an se-asseme arounrevo uses couroee new exose resues ..ccuraesm usure aromacs.urez,.,eman,..ucerman, oes.ourna oaemonca oreoorenon annonmes:eonarovae ess,. uame,.ec o ane-oamnueanon on,e-omer aran,., oa meacraesnouo annamcs ooe n.acromoecues, - . ou wsn enzmeounn aaeor, sucneracons maacae reon eecverane namca rera unoe sae aone unonrocess, resunneocaze an eroas orce u ancoe sae oserveor suors.snurn ma reuce e unonrocess,ec oessn a sow ornecarae unoe sae.us,nn oe, caaca acve conmoarmauonne, wc w suor maeunne acoso oeromer comosons. as conssen we acceeraefoldreave oaer ,eras: c mreomacre,a aescorc, e ac,ene,,co′,′′,en′′- eenearm meeacreaeneramneouene,cne anrs-caroxe osne wereurcaser e , .ous, .-meacrooxe me--suoro-ammoomn orema wasurcaserom emcasoran,.eanoeu wmasroxe romsercenc.meormame wasurcaserom urcase enerae,.-corome enrcorosan acronneemcas orrsve,. cemcas were use w e wasurcaseromees wo-ncuse sca waersor sneou mouererurcaon. csanana,.wo-nc sc cueman wereurcaseromar urcase on waers w nave oxe coanor esomer were urcasenvrro rom omenae eer rxmaro ouoa sraee, erac-. am s,rane.enz- ccoocone-ma-se was urcaserom orema me was uracerearaon, .ous,. con oxe nanosuneconazaon, an caracerzaon: ncez- res were sneszeoown arevousescre meo. mznoron,mer.,counaovress,or.m.mo.,zcewarozc,aa.s.s aoamr,u.a.onaramenwaoror nzmeesur waecreeuncroonaozec w. a ss o nanoser vaassu,eoso-nn anrousouuesene,ue uns, n. vvor mnues.ereoson was comee, waers wer nouenex,soroanox an waer, an soren a v e rnse w urace-naeomerza acuumesccaor unurer use. ecvaorseenerae econs o mxe ruses were snesze meo.ez,.. ronranseromranseracaomerzaon aa ,ene,..,cwarz,..aar,..ramacncreasen acaersosrm,ance o-mmozeonaseosewcz,er. ea az.aeovne orsnoe-meaecrvrausonuaorcs. omerzaonneresence oeacoer.rcassessmen a an. a- ecansms.acromoecues, - wa, o.,eaenau,. raoasnzse owsa,a.s.acromoecu oese,-crae w . oncen neaxeansszevwes,an.,s aonn,m., auowns,.,eras,.usumo,.ranromuracesor “verone”:  unconazeeraersecncees o werer a.eanmour m, - .re,. o - n arorae rao o a mxure o:vve w a s, c meacrae. ,. m o-ascorc sc, an a sma coer wre. searae soc souon w coer an an was mae aenarae m, c oonann.. souon os w coerer rome, o , m o meano ccesx.onomer-cona eeassencenass reeaereeze-um-aw :,:,: an:.nen co soeru anonaan m-cxounres o an n raos:, eas conanne nanoseres an mono ann souon m wasranserreo m, wc was aoweo reacor amer mxure uner nroeno ana voume o concenraon o coer was m, an m aosve nroenressure os.ena ae.omerrus-coae n oar raos o a oer cemcas are summarzen wase w meano, anoseres were searae va cenruaon, seuena e resuna , waer, an meano, an soren aesccaor unurer use. -nsrumenr wces were caracerze va usn aermosercenccoe aareszer were ae sameoer.namc scaern measuremensnon uer a . anroer soonrme aer resusensonn.mere m soumosae or sne moecue mccarosocnoor, sarcocne oxseers woane. vvroeneroxe suurc acor, rnrsse were ceanenrana souon nroen, an exoseo-ozoneor.er e wos uonr,aure waer,re w erorme smar asescreorarces,un a camer omerzaons were ea o suraces was measure conac aneono w anner2amosere. w a cusom mer vae sessero meo m-uuansonesomne aenr. exr an uans w wereereesnneer ueseesss n oon a: orsoemaaca reseue. reumroacvea-accessDSeA-a50r%o,maDSA-100%were reaDbcueried-100%aanne muaons ., anose c resuesnroucon o eac muaon nonrouce maoanercore was useo vera reacemen oure aromac resues,ose rsurance oeoe sae.or avorae muaon.ure was no mua was aoweo seece mos enereca oeoseanercore, ae snces remova resuen aramacecrease aromac reacemen suesna was a crca sazn resue, an no non- srucures were aowe couo re coamx uesnsnaeeorosse remao revaax.aonerroosoecoa.-uncee m muuaanonse wserne, seeencscer, vea ceoneosn oormezsea monu.anse weenrees onr aou mcueannso are sow-nn eaxresson vecors nzmexresson,urcaon an e. e consruc oacus suasenas: moe verson o arevousescre a e was exresse recomnan w a oner,.,azen,..,acson, uonuconrooco.amm,..,ae- uornaeroensor- . sru.cue,..rearaon o se-seccaaee .asms - enconra c warac aerz-aeromn.naureroocos, - encon a sneaseorncororaon oaeexa unnsaurnaea anue azoenaannez a an amer so coon were co-ranso amno ac- conanewo muaons aosons an orncorormraeonn oo.zo a . resecve,oae se-secca w -areuoroores va ccn r csene, oones were seecerom a aarae conann m o amcn aneacons. o secnomcn.er overn row uo sauraonn non-nucn sanar m suemene w e aoremenone anocs, ces were cenrue, s meum remove, ces were resusenen serze uraure uernaan was conann auonucon mnmarow waer anranserreo cuureass -aranose was aeo ana con meum w anocs. ona, sere-ere º, w roaon a rm ancenaraon o. m.ncuaon was carre ouor a exresson, ces werearves ssroecerom n aumnumo.oown suseuenasrozen uneur vcaa coenruaon a rm aºurnmn an ao,raourr mesc.a aroen va ,, c a mesn wmer cer az roem osua eson anenra. en wa uanus caenrrre e, c oou ronca wnon a-m so couummnoso-aae, esae a re, anse va soncaon. cen n suseuen sames conann wereroecerom n a cases.er wruaono remove ceers, cesae wasoae one- coumn an eue rauer, wc consss ouer an mmazoe. nes, coumn an rereve ecraon oaso one ssem wereroece w aumnumooreven -nuce secr oe ar-azerou.racons conann wereene usn aanoro waoomeernsea o usnenmam oe cromaora ssem.useuen, rocosreu uesrn ex acano-ecaenon m soumosae a w uanne naur an concenraon were asseso-em-esan carreo-a, . exncon coecen o,-1cm-1.se va- an secrooomer, usn an ..,eanee,n. w.,as ccarwraerz o,u. w. a moaar,e. v.ersraomna ocrenvcroeuassenescre meos.ez, mmozeases erazaon onomerrusuoaacerormance o .an,.,amar,.. uer,. sme mres.oora eansansc,n- ooroona o ccoocne reae e o n.emcaommuncaons, - . an aoweo roa weeorerncuae w. o-mercaoeano an. m - esaemesno m s ao rouommeomeraure.oown aen, - was usn aea sn coumnsermosaseer ao w uannerocoreuer wc counerere we removeeresence o-mercaoeano, na conoceonwranonsu oer ex.c ma nnexe, se. an. an m,o resec-mveae.mee,rna o or reor,a weorne a aeoemeraure, - wasesaemes w aea sn coumnso remo room reeuoroore.emova oreeuoroore was as ve excess o an sna raos.erwars,aesesse va nanoro-s sazaon1cm m-1ea.suxrn5a-s1ocrma-1ncoers a, an ano n n ecenc o - was asses ,m, usn exncon coecens o.x5se- or overan reons.. resecve, we an correconacors .oro aen ecences were esmae as.or an -xre vsescoonrs o. wa en-coee eac ane muans was carre oun. co .e usn m o am ne wereransorme an sne coones were seecesoro---o cn as anoc.xresson o eacene wasnucem o rm.arve aacoranose an aoweo exress aºor w roaon a nzmesmnmo anzuaronca anon wcerve carreencao - . m o enaemmozaon,uressa ensz:mes wereuer excanen nzmomeza.o mnu waers cnocnussaen w o o an e meocmneer,rus-.co,aem nanosaer.es sou mon o us enac revovn ora saer. enzmes werencuaeor aº.ermmoz a enzmeoan onearces waseermne usn a massaanc aon, o eac enzme remannne suernaan ease one reave acv normazne acv oe enz oe reacon.eave acv was cacuae asence oarces.o ensurea amne nnon-ceov saueenrna aansoroee e anczvme w oas reem eonvzemeromnee mom soerumrusaer,earces were wase muemes wresuer conssn o emoseraaue a une enzmac acv oe suernaan was ne e. uoroax- s re-eenen acvroes were measure usn aoracenc aorncecrouoromeer.nec sa roes were carre ou usn arean concenraouna oon,r aene a anecmanmonnzeero enzmeaen re eaacer aosrsauo rreeascceonnce, measuremens.e connuous, was normazease one enzmeoan one m wc was mxe carre oun m soumosaeuer a , usncros reeres.eacons were uoroenc susrae. caraon ouorescence uns sorunurae as useo convere soeo u versusree resorun concenraon was ross was mea ns orouc concenraon. ona,eacroun susrae me sure an suracerom acv measuremens a eacemeraure.oremaseurranuemeraure-eenen acvroes,euer wasreeaeoeare nren an exerna waera. eormoere r-ounmo caecu onne a,z ama eun me:rs wereooeace overn usn a m near-aser. wnoworman, wc wan waer wasranserreno a cusom-uow ce w a , wc wasna soarsenna a me w soummmooszaonuer.earae, - rocore, wasuenommozaonuermcanee,ue.r, wmuaanone nau concenraon o x-12. ona a so , unaee w-e , was aeoe same eronncun a excess w reseco - , an a werenroucenoeow ce. uermaon sooruo aowmmozaono occur,eow ce was wase w m omae usn a cusommrossmae, an aoweo euraeor mn.ames were oncse- mcroscooanernaeeconuorescence aaraus usn a moecues were exce nmaese w ax waermmerson oecve.eonor easer wasrecenoearco oo,eamua an a m nmaserrsaaser. emcnrcarsm o. mm rausorovcseen scca suorrou ause sca macn o.e emsson oo onor ano us anc ause sca reracvenex- were searaeno secrasncman cannes uceoruorooresrom - ,roma.eonor sna wasurer ceane usnsn a a n nmcroc mrror emroc, ane acceor sna was ceane w a nmanassmaenreasser onor an acceorman cannes were ane usn anos eam semroc. esearc an caure usn annoron camera manane a − °r.maarne a mcovuess wonere w caos oecaeneor u esanc a conernaonn eexmcearaounre w ano ms o acusonme.reen reons oe surace.erosroc rrus comoson overeren revous worez,.. essn usn cusom soware asescren namcs uonuo,ene,..,cwarz,.. aar,..eucenzme ourna oemercannovaenmmozaoneasoa-cv rae-o. .ne moecue caraceermzacaoce , - ene,..cwarz,. mcaca , on o anomaousransorn an, ansorocm.naca cononsae.ra ., - raecores werencuene anassor a areaes ecores w anorma or overaeuorescen snas -raecores o or mo orre nroamn-esu wae-rae aeneaz oeeocrs m wenruee e oxrc uuneonroomurer anass. ooeace. eamas wereenerae u r or acceoruorooresransonarameers we sn aaaonsrom eacraecor,nec escre.ez,..,ree ensem,a.e., uscnw aarz,e.n.aroavar,.an. wramarecen scareeass aesrevous erormance ommoze ases erazaon onomerrusunaac araress, - ene,..,aaa ors. o c,..,aar,..cwarz,.. -anruc.urau acnuaounnc anoanass weaesro caerrnee ou om emsomaznennezr-mueosr.oorano, ase ononor an acceornenses.ac roo-suar esance waserormeor aaaonsa cor e-meanucuaon cacuaon aroen mo eaeccue unerw resone avenon saeoe or unoe.nervasn en mueransonseweenoe an unoe saes, awe ran ssaeer w eere erc c eeonncs ceae nnre a csrs searaeucuaon evensor cacuaon. eranssovererrnaeus caceuoaoown:n euaon:were reresens en an acceoruoroore, aner enoes sanceeweeneonor uoroores useu,..0erser raus caracerscorear o nacaoc ran,.esonancenerranser:eos an caons. ,er assocemaesr,- .orear ouorooresexauor ano 'rser raus*was anaca cacuae usneoown euaons: . w,eresseee oave-nraneraeweenonor’s emsson an acceor’s excaon secra exncon cDoecen o aocrcmeazeuorescence emsson secrum oeonor+,Ase moar orenaonacoreweenonoorr, ans w acacveeeonr, w,0serser raus, кse reave resecveners are sucenon anexeocesareeen ausoro,o arsesu romanon naer reracvenex oeranser meum an se D 2 oeesnrueo unn ooneer s-aueor ooore vsaancoenseoaro smuaon: va aonearo smua re unoe sae o was moee m on usn a se-avon ranom wa moe.noe was roeveoeus as aes ccrane o reorse oa ms o eua sze w a meanameer o,n accorance w se cans.venou nomaeaonn asm wneore acmo ssze accounnore voume oacone an saa consrans weremoseoe moe.e.,ew ooneease cxanno o occurnneweeneas, coe.neraconseweeneas were no consere.or eac smuauon see sa,mee sace or eweenewo amno acs wereuoroore sance were comue anoe s are se-seccaaee z an was cacuae as. as n am,soeraemnsn o.s.ornacmeens urseor ra.u nsmo wr ano esmae rae o “aseosves”, as consereo e smuaon aowe u wc coue ecenc . we unoe. conormaon so esmaea o aeosse ranom-co unoe mmoze,s o a c nnou co en r ozesmu men er wruser.esm. ceonmouosse ca oac vers aan varouus ammoeuns s oa, was. reumseesra ruesreu-eeenne sns aecmavc sraoezs reveaeancreasne aromac oeomer c aon o nerma an cemcaenaurn reosnueonns. enozwever, aer areso o nerus,nroucon o morencororaon c oomneanesna -awroza e muon ua. oroonsonrecsuon os oenreve unnraoueran u amno ac aowe se-secc ecenc asrox.ne moecue exermens reveanoan-reon u susn reanoeracon more ecenan oer suorsnermaenaurn conors aveaser reonnecscaerone eec an sower unonnecssaons, oe sae versus oermmozaon suors, an aeres aocaze zaon o wowucuaon wc couea unoe sae emova o aroma oacaon o reon. oma sazaon sc resues va muaonecrease sa ommozeroen, anneracons.a uor was seoower conen va remova o ^^ ^^-sacn moecue exercmroenscso.co enz somvaenc- aecxvose co anrreauere w aromoaec rerascuoens aeeermarnoee a sneenerac w aromac monomers va ^^ ^^-sacn. o ae mse uasne “aroeuas ooue enre acaon,eerms “a” an “an” are usene sense comonens or ses, unesnse”,e “a coneaesx a cearsr”, “ocnaee osr o moerrew”s oer. “aor euxraam”e, oe reerencencues aura o ces, eerm “a ce” er ceerm “anoncun mxuresereo. an o r” werever useerenncuese meann o “an”, “or” an “a or eomermn “aaoonu o” ore “a eemroens connece saerm”. , an morereera wnxm oa ae”v as useeren means wn,reera wn numerca rearneasn, amnouen os,e vraauens a enxamerceese n,n oar va e unuees osr o ranerew.se exress sece, a oe anemeraures o reacon s suc asoseor amouns o maeras,mes averae mo , raos o amouns, vauesor moecuar we weer numer oownoecuar we“n” or we averae moecuar we“w”, an oersne eerm “aron oe seccaon mae rea asreace e wor “aou” evenouncaeooue” c monara nro, exer neussmer acaeara wrameeres v saeueo,r amonune oro ranowe.nc scoercnca,o uness aace cams are aroxmaonsa ma vareenn uoneesreroeren anoe oane eresenscosure. e vereas, an no as an aemosm soue a souca aonea osee cooncsrrunee on euva oense noume scoe oe cams, eac numercaarameer ornar r er o reore sncan s an an escooou suwnn re asreanec annu roxaes. maeon nsu,meerc naum raenrceas a vnaueasra smeeeorrs senneo sreroa scoe o reore asrecse asosse.n numerca vaue,owev ecc exames are necessar resunrome sanarev er,neren conan ceran errors urermore, wen numer aonounner resecveesn measuremens. an comnaon oeca ranes o varn scoe are seoreren,s conemaea s useeren,see vaerumes “cnocmusve oe rece vaues mae use. an meosncuee reerencrsn”sneneo meanaeroucs, comosons essena o” wen use e comonens or ses,u no excun oers. “onssn oer comonens o oeneroucs, comosons an meos, sa mean excun oe rece comorn seenss o wo aun e nsose enxcau senraccea cnocne.amunsa,n a cso amnosaromna ccoenuscsan e ascsceena carrers. “onssn o” sa mean excun moreanrace eemens o oe ae ses. r comonens or secesn usereeenresn,neeer sme “ccomeo asmoounn”s,s anse wnee aso a enncomrass arouc comrsnenrec,rom a comnaon oe secenreensne seocuca resus,rec or a cemca comoun or mxure o comounsorme omeer amouns.omers essena o reean srucura unse.., a monomer. m zaon an conssn oneo oer moecuesoorm aomer.omoomonomers a moecuea cane on onee o monomer un. coomers aomeroremres aomeras mae u o es o monomers arenene sameomer canas oo wenwoor moreeren on one monomers use. sasca coomers aomeseo aomoomer were are arranen a seuenceaoows some s rn wcwo or more monomers euaoeroa onn a resue oaas mcoa rue.e moeracon o a monomeres a ranomomer.eseomers areener nomer a anonne can,eomer meo. a snesze vaeree racaomerzaon suraces,s w uesere aeorenm,erererm “ase maera” reerso a susraerovn one or more eraeo or oer us canerownrome surace, oro wcomerruses can we vare ouncwosnea axe.s conemaeaeomerruses cane axeo a oroene. ze suraces,ncun sca, resns,omereas, aarose, an sormesr uosme aeorenm,erezaeromn s “urussra”e ora “von am rearcraus-”o remeersrzoa aeoermmenrac sroue cana eomerzae susraesease maera, or cane enraeo or oe , weren ase maera,ere su rwse axeoe s us sanaaneorm oease maera. caarcanen ae rarencaeneurcme “reraacnve r meaocnoomne.r” ree reerascove a m coomnomouern canaes an ca maaeer oa cae o mormnomers asescre aove aneren,or exameu nomeo e same ceacrae , or eene.ease maera an reacve monomer mae o oromeromoun,or exame, aoeenease maera ma uze eeene monomers an manuss acon urrereacra e.c.,nn re aea ac mceon aeo. osr o a ceonnavenneron co amrerursnerrove. “”snene omerzaonrecursor mxure oenvenon aeas one reaen, e.., a as a unorerormne meos oeresen.nve maeromoe,srue, or so aacaenserescrne an meosorse unse.on.n ona,es ma conan rove wn conanersaroecemrome exerna o ern a oe reaens mae conaners vronmen, suc asn seae escoo o suwro re asuc reanes an. roxamae numerca ranes anarameers senoreroa scoe o reore asrecs ons,e numerca vaues seorne secc exames are necessar resuen arsomosse se.annar numeveracaon vaouune,onweeverr, renseecrenvee csonnan m ceeran errors urermore, wen numerca ranes o varn scoe are s asuremens. an comnaon oese vauesncusve oe receeoreren,s conemaea an meso ussenceureen,ee reeerrmen “cceom cormsnon”enssn oern see va s,ou mes uea m nna oae ueser.oucs, comosons essena o” wen useoeneroucs, comoson excun oers. “onssn oer comonens or ses o an essena snc s an meos, sa mean excun oe rece comonens wou no ance.us, a comoson conssn essena carrers. “onss excuerace conamnans anarmaceuca acceae ses. n o” sa mean excun moreanrace eemens o oer comonens or ar e avanaes seor aove, anose mae aarenromeoreonescron, eea erncenrom aea snceo.e oncee cenrvaenno cn,anessn meanee maa ae mnaeer as coovnea cnoensrnuceono wreoonu aesmcrnon se or sownne accomannrawns saenerree asusrave an non rnesee. reerenceorenece esx cene nonnceoresen acaon arencororaener enreeren oreon wescre soeenae avnanssaeens seereowr. aove, anose mae aarenrome consrucon woun, aerea erncenrom aaen se an snce ceran canes mae maene aove conaneneoreonescron or socwonen oe aeccnovmenanon,nrsanenea a maers asusrave an non amn sen wns saenerree an secsc aseoauorees u oneresonovenase one. eroenowenscr caem,s a anre ane snaeemeon cosv oer ae o scoeenercnvenon wc, as a maer oanuae, m e sao e oenvenonaseenescre, aereeween.owae

[0002] ae.eaures o muans useoes e eec o emnan aromac resues.

[0003] º º waereee.raumeceorr oes r aenmaaznenr aaeecro aresnor eacer snn cree mroaeceuscer exeernmeen ceonoson se.coens.e . c meac . x -asco rae . erc ac ur2u wre - w caaaes..ereo rnearre rasenoss oe reaerecnen rsaanoes. oe raos are r neoacrmoanszeo oro eeac se cnoonceesmnser oraorouns omearec crou.seesr

[0004] exareesse. wenes a o-ermn maueaxnas- wscne weare cnarraocuecrescn ooe exr-esso.n veecsoer.enes were -onomeenrecoen eer meacrae rucure --nraceneme meacrae ae.rucures o seec aromac monomersor coomerruses. onomer rucure ae . .

Claims

a.s c aro ca mom-e aos c mm: oenromruesr comrsn an aromac monomern comnaon w a non- neereo., werene co-omerruss aaeo saze an enzmeoe . moee co-oamaceraruess a acaccormno camurer comrsn an aona monomer or .e co-omerrus en o an enzmeoe coomerrus. c accorno camurer comrsn an enzme aaceoe .o roeo u com s-eor asmru e,es cr. arruosnc a acncorno cam werene enzmes seecerome osaase, rase, cocrome ,enzaeease, aane aceconesrerassen,, cucmosoersosmne,rasero,mennc, sunsn,orseraseroxase, ,ransamnase, nroreucase, unsecceroxe ancasea,s aen, emmneera rsee,ase,roen .e co-omerrus accorno cam werene ucase. rou conssn oana ruosaa enzmes seecerome omucor meease ,acsues ,ana anarccaase , suerracceroasease, susase ,seuomonas aseromnomona ermomcesanunosusase , an .roeu co s-eoecmeerrormuse accs ro s or. un conos c-am. werene enzmesrom an enzme a sn o oxoreucases,ranserases,roases, .roese m cso,-s eoom romer uearses cor, nussa s as ncecso, or an enraons en co eacmases. co weernen ee aromac monomers seece en meacrae,enz meacrae,eener meacrae , reenecoen eer meacra ecoen eer meacrae, meacrae,enaeenecoee,eraeenecoen eer en eer meacrae,-a n eer meacrae,oeeneco reneme meacrae,- meacrae,-a meacrae,- en eer acrae,en acrnaraec,eneenmze acr meaea,creae,e snerenec,o eeenne eceora accrraaee,,reenaeeneenecocoenen ee er aecrr acare,aee,raeoeeneenecocoen eer acrae,-a acrae,-a acrae,-reneme a en eer nraceneme acrae, eenecoen eer acra crae,- enz acrame,eenecoen eer ac me,en acrame, eer acrame,eraeenecoen e rame,reenecoen en eer acrame,oeenecer acrame,enaeeneco acrame,-a acrame,-renoeen eer acrame,-a nraceneme acrame, eenecomeen acrame an- meacrame,enz meacrame,e eer meacrame,en meacrame,reenecoen eer meneeaccoen eer en eer meacrame,enaeenec rame,eraeeneco oeenecoen eer meacramoeen eer meacrame, a meacrame,-reneme meacr,-aa meacrame,- nraceneme meac me an-.e co-omerrus arame. see ccorno cam werene non-aromac monomers ocseorromerou conssn o suoeane meacrae , oeecneonceo me meacraae ,cosoxe meacrae ,. seeec coe-orommererurosu ac ccoonrcr ssnae no o cam- w.erene non-aromac monomers mmeeaaccrrooaomnorormeammonum core,- suor xe rmeammonum core,-suoro meacrae,- amceacoro acorxaee,-sumoero- a-csruaomroe,-asmumoonroum meroaxcre,a mmeea,cr-c o,e acrecne ac,c aocr acarmaee,, moeaecraemnee,cooe acreanmee,coo meeaecrae, meacrame,-eamnoe meacrae,-eamnoeneco eamnoe acrame,-eamnoe meacrame, acrae,- soroacrame,--moronoe meacrae,--mor n- acrae,--moronoe acrame, an--moronoonoe .e co-omerrus accorno cam were e meacrame. zweronc monomer. ne non-aromac monomers a. seeec coe-orommererurosu ac ccoonrssno cam werene zweronc monomers suoeane meacrame,nos oo srucooeannee ac arcraae,e, suosoeane acrame, acrame,osorcone meacrame, orcone acrosne, an acro serne, acro ornne,.omerrus com acrrson zwuameraoen. eenecoen ee caromacsuoeane meacrae.eomerrus accorr meacrae coomers. a no cam werenerus comrses aou o.ou . aoeuomerrus accorno cam werenerus comrses aou o.eo.merrus. accorno cam werenerus comrses aou..eoomemrerrus comrsn zweroncaromac coomers. coen eruser a a mccorno cam werene aromac monomers eene.eomerruscecoracnraoe cam w.erenerus comrses aou o. aoeuo.merrus. accorno cam werenerus comrses aou . sueoor omr seorrus accorno camurer comrsnurer comrsn a. aoeuome arromru s asur c aacc mce oonror omn aa er,oc a cm oae umn o we o aero oeunmeer arrruuss. comrses aou.o aou aromac monome omac monomer, aou o.roemoemreoruru cosns acsconr onr, o eor c a eao nmu ec w ar oeormea ennc m ee aorn eoo rmmaerc. monomers seece en meacrae,enz meacrae,ee meacrae , reenecoen eer m necoen eer meacrae, meacrae,en eacrae,eraeenecoen eer en eer meaaceraeen,e-acoen me eacerr maee,a-craae,o meeacreneae,c-orceonemeen e meera accrraaee, anen-n acrracaenee,meenz m aceraacrae, srene, eene eer acrae,reenecoen eer acra e,eenecoen acrae,enaeenecoen eer e,eraeenecoen eer acrae,-a acrae,-a a acrae,oeenecoen eer nraceneme acrae, ee crae,-reneme acrae,- enz acrame,e necoen eer acrame,en acrame, eer acrame, enecoen eer acrame,reenecoen en eer eraeenecoen eer acrame,enaeeneco acrame, a-crame,oeenecoen eer acrame,-a nracenemea ac acrame,-reneme acrame an- meacrame,rame, eenecoen eer meacrame,en meacrame,reenze meacrame,eenecoen eer en eer meacnecoen eer meacrame,eraeeneco oeenecoramee,enaeenecoen eer meacrame, a meacramen,- eer meacrame,-a meacrame,- nraceneme meacramrenee.me meacrame an-. surace-or aaoeu co.o mmoearr orus aro smuaocr comose o an aou. . surace-rae coomer . moar o aromac orus suors comose o anreaeran.e surace-rae coomer a acou moar o aromac . monomer or moe corno camurer comrsn an aona.e surace-rae coaoacmera aecsco aracnmoen ca om an en wzemreenoee a cooonmae mrorus. moes seeceromerou conssn oc meac nomer or roxsuccnme meacrae -,-roxsuccnrmaee a,- acrae,-socanaoe acrae,-socanao crae,c acrame,-socanaoe me e meacrae,-socanaoe nroracec ac acrame, nroracec ac meacrae, mea acrae, nroracec ac acrame, nroracec ac mnocracaemc aec,m acnroamaceec, amcno meacaecrc aace, mmenoacracaemc aec. acrae,. mee saucrracae-erae c,o woermeenr acecornso creasemn a cuornceer comrsnc.e surace-rae coomer acco nraon o aou. meacrae , werenernso cramurer comrsnc aou esen a concenraon o aou.o. enze smuera ccoe. v-areae coomer accorno one o cams-urer comrsn an.e surace-n aaceoe . c rae coomer accorno cam werene enzmes , rarsonn,c c anmorrases, cn,ocromromne, su,senn,zaoresease, aaneosaase, ac eraseroxase, or .e roem sucro eacren o-es uraera coese. ns csono omera anccora rnuoosa camase werene enzmes seece ,omucor meease ,acus s,ana anarccaase seuomonas suerracceroaseaseu,sase , ase , anaseromnomon ermomcesanunosus.e surace-rae coomer accornaos c s. -. enzmerou seecer am werene enzmesrom an roases,ases,someoramses,e anroua csoens.ssn o oxoreucases,ranserases,. coeo surmaecre-sraraee cos ao smcear s aucrcoarceno cam werene suraceo wce. coeo surmaecre-sraraee cos ao mmcerors acceorr. e.no cam werene suraceo wce. coveaeonmerrus accorno one o cams-urer comrsn an enzme.eo aaceoeomerrus. .eommeerrrruuss a accccoorrnn c caamm w weerreennee e ennzzmmeess seaescee. conssn oana ruosaase ,ana ana romerou zomucor meease ,acus susaserccaas,e , suerracceroasease,ermomc seuomonas aseromnomonas s. -. esanunosusase , an. seeeceomroemrruesr aocucor connss canm o werene enzmesrom an enzmerousomerases, ases anransocases. oxoreucases,ranserases, roases,ases,

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