Alcoholysis catalyzed by Candida antarctica lipase B in a gas/solid system : effects of water on kinetic parameters

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1 Alcoholysis ctlyzed by Cndid ntrctic lipse B in gs/solid system : effects of wter on inetic prmeters Mrinne Grber, Mrie-Pierre Bousquet-Dubouch, Sylvin Lmre, Mrie-Dominique Legoy To cite this version: Mrinne Grber, Mrie-Pierre Bousquet-Dubouch, Sylvin Lmre, Mrie-Dominique Legoy Alcoholysis ctlyzed by Cndid ntrctic lipse B in gs/solid system : effects of wter on inetic prmeters BBA - Proteins nd Proteomics, Elsevier, 3, 648, pp4-3 <hl-3968> HAL Id: hl Submitted on 3 Oct 8 HAL is multi-disciplinry open ccess rchive for the deposit nd dissemintion of scientific reserch documents, whether they re published or not The documents my come from teching nd reserch institutions in Frnce or brod, or from public or privte reserch centers L rchive ouverte pluridisciplinire HAL, est destinée u dépôt et à l diffusion de documents scientifiques de niveu recherche, publiés ou non, émnnt des étblissements d enseignement et de recherche frnçis ou étrngers, des lbortoires publics ou privés

2 Alcoholysis ctlyzed by Cndid ntrctic lipse B in gs/solid system : effects of wter on inetic prmeters Mrinne Grber, Mrie-Pierre Bousquet-Dubouch, Sylvin Lmre nd Mrie- Dominique Legoy Université de l Rochelle, Lbortoire de Génie Protéique et Cellulire, Pôle Sciences et Technologies, Bâtiment Mrie Curie, Avenue Michel Crépeu, 74 L Rochelle, Cedex, Frnce Foot note : Corresponding uthor : Dr Mrinne Grber, fx: , Emil: mgrber@univ-lrfr EY WORDS Lipse B from Cndid ntrctic inetics trnsesterifiction wter solid/gs bioctlysis - hydrtion

3 ABSTRACT The influence of wter on the inetics of lcoholysis of methyl propionte nd n-propnol ctlyzed by immobilized lipse B from Cndid ntrctic ws studied in continuous solid/gs rector In this rector, the solid phse is composed of pced enzymtic smple which is percolted by gseous nitrogen, simultneously crrying gseous substrtes to the enzyme while removing rection products In this system, interctions between the enzyme nd non-recting molecules re voided, since no solvent is present, nd it is thus more esy to ssess the role of wter To this end, lcohol inhibition constnt, substrtes dissocition constnts s well s cyltion rte constnt nd rtio of cyltion to decyltion rte constnts hve been determined s function of wter ctivity ( W ) Dt obtined highlight tht n- propnol inhibition constnt nd dissocition constnt of methyl propionte re lot ffected by W vritions wheres wter hs no significnt effect on the ctlytic cyltion step nor on the rtio of cyltion to decyltion rte constnts These results suggest the wter independent chrcter of the trnsition step INTRODUCTION The ctivity of bioctlysts in non-conventionl medi, especilly in orgnic solvents, is usully gretly ffected by the level of wter present in the medium Numerous ttempts hve been mde to investigte this dependence As reported by mny uthors, wter vilbility in non-conventionl medi cnnot be quntified using its concentrtion becuse the cpcity of orgnic phse to dissolve wter vries considerbly with the polrity of solvent The influence of wter is best nlyzed in terms of its thermodynmic ctivity ( w ) [,] nd ccurte comprisons of enzyme ctivity cn only be mde if wter ctivity is controlled [3,4,5] For mjority of enzymes, the rection rte is low or zero fter exhustive drying, but increses s slightly more wter is present This rise is considered to reflect smll quntity of wter essentil for enzyme ctivity, the hydrtion llowing to form the ctlytic

4 conformtion [6] It is cler tht different bioctlysts require different levels of wter to exhibit substntil ctivity In prticulr, lipses from different sources differ considerbly in their ctivity- w profiles [7,8] Some lipses show n ctivity optimum t very low wter ctivity (Rhizopus rrhizus, Rhizopus niveus, Cndid ntrctic), some hve incresing ctivity with incresing W (Pseudomons sp), nd others hve intermedite profiles with broder optim (Mucor miehei nd Cndid rugos) As mentioned by mny uthors, the only wy to ssess the influence of wter on enzyme inetic behvior is to perform full inetic description bsed on properly determined ct nd M vlues t different wter ctivities [9] Indeed it leds to the true mximl enzyme ctivity, nd not substrte concentrtion-dependent ctivity Severl uthors studied the influence of wter on the inetic prmeters of lipses in orgnic medi, in the cse of esterifiction or lcoholysis rections, obeying Ping Pong Bi Bi mechnism [,,] It ws shown tht wter rises M vlues for lcohol by cting s competitive inhibitor of the nucleophile lcohol [,] As fr s the effect of wter on the M of cyl substrte is concerned, it ws shown tht, in the cse of R rrhizus lipse-ctlyzed esterifiction, the M for the cid presents no significnt vrition with w [] The explntion ws tht this substrte binds to the enzyme first nd does not compete with wter in tht sense Nevertheless in n other study, incresing w ws shown to led to n increse of the M vlue for the cid in the cse of esterifiction rection [] The mechnism involved in this cse ws supposed to be prtil obstruction of the ctive site by wter molecules binding close to it Wter ws lso shown to cuse hydrophilic hindrnce ginst hydrophobic substrtes thereby inhibiting ccess to the enzyme [3] Concerning the evolution of ct with w, it ppers tht ct generlly increses with w [,,] This corresponds to the ctivtion of enzyme by hydrtion, involving generl role of wter in ming the enzyme structure more flexible nd mobile, incresing the turnover rte However high wter levels hve lso been shown to reduce the rte of lipse- 3

5 ctlyzed esterifiction or trnsesterifiction [,] This decline cnnot be ttributed to wter competition with the lcohol becuse ct is obtined t sturting substrte concentrtion, but might be due to physicl ggregtion of enzyme, reducing interfcil re nd limiting mss trnsfer [8,] To ssess this hypothesis, system in which ggregtion plys minor role, ie polyethylene glycol-modified lipse dispersed in toluene, ws used It ws shown tht ct increses continuously with w, the vrition of ct s function of w resembling wter dsorption isotherm for protein in orgnic solvent [] All these studies were performed in orgnic medi, which mes the sole role of wter impossible to ssess Indeed it ws shown tht t constnt w, ct vries with the orgnic solvent used, decresing with incresing solvent hydrophobicity in the cse of PEG-lipse ctlyzed trnsesterifiction [] In severl studies, it is shown tht M vlues obtined t constnt w in different orgnic solvents vry with the solvent used, but s they re expressed in molr concentrtion insted of thermodynmic ctivity, it is impossible to distinguish between the vrition scribed to different prtitioning of the substrte nd wter between the enzyme nd the orgnic phse or to the influence of the solvent lone [] In the study of Wehtje nd Adlercreutz [], M obtined in different solvent were subjected to tretment with the UNIFAC group contribution method [4], to be expressed in thermodynmic ctivity, but this ttempt filed to give dequte lignment of the M in the different solvents This would indicte tht solvent hs proper effect on this prmeter Contrry to the clssicl solid/orgnic liquid system, the solid/gs process offers severl dvntges to perform fundmentl studies bout the influence of wter on bioctlysis First in this system, the study is limited to the enzyme nd the substrtes/products/wter since no species re predominnt, contrry to synthesis in orgnic solvent As consequence, the role of wter cn be better ssessed Second it permits to perfectly control nd djust thermodynmic ctivities of rection species [5] 4

6 In previous pper, the lcoholysis of methyl propionte by -propnol using immobilized lipse B from Cndid ntrctic (CALB) s bioctlyst ws studied in solid/gs system The inetics obtined for lcoholysis were shown to fit Ping Pong Bi Bi mechnism with ded-end inhibition by the lcohol [6] The effect of wter thermodynmic ctivity ws lso investigted Wter ws found to ct s competitive inhibitor of methyl propionte, with higher inhibition constnt thn n-propnol This determintion ws obtined t fixed propnol thermodynmic ctivity, which does llow to ssess dequtely how wter content ffects the ctlytic behvior of the enzyme in detils In this rticle, full inetic studies of the lcoholysis of methyl propionte by -propnol using immobilized CALB s bioctlyst t wter ctivities rnging from to,3 re reported EXPERIMENTAL Enzyme nd chemicls Chirzyme L (Type B lipse from Cndid ntrctic lyophilized) ws gift from Roche Industrie (Penzberg, Germny) All esters of propionic cid (methyl-, ethyl-, propyl- nd butyl propionte) were purchsed from Flu (S t Quentin-Fllvier, Switzerlnd) Propionic cid ws from Sigm (St Louis, MO) All substrtes were of the highest purity (99% minimum) nd checed by gs chromtogrphy before use Chemicls were dried over moleculr sieves nd filtered just before use Pure wter ws obtined vi Milli Q system (Millipore, Frnce) Adsorption of lipse onto solid support Enzyme dsorption ws performed onto Chromosorb P AW DMCS), mesh 6-8 (Acid Wshed DiMethylChloroSilne) (Prolbo, Frnce) s described in previous wor [6] 5

7 Experimentl setup for solid-gs ctlysis The biorector used in this study hs lredy been described in previous publiction [5] The pced bed biorector itself ws composed of 9 cm long glss tube (65 mm OD, 35 mm ID) in which 5 mg of the enzymtic preprtion were pced between two glss wool lyers Substrte feeding ws done by pssing dried nitrogen, s crrier gs, through the substrte flss Substrtes were continuously flowed through the biorector, recting with the lipse Thermodynmic ctivities of these compounds in the rector re defined s the rtio of their prtil pressure in gs entering the biorector to their sturtion pressure t the woring temperture The desired thermodynmic ctivities of substrtes were obtined by djusting the volumetric flows of the crrier gs in the different lines t pproprite vlues, ccording to the clcultions explined previously [5] Clcultions were performed considering tht the gs ws close to n idel one Then fugcity ws not ten into ccount The gs leving the biorector ws injected into gs chromtogrph for nlysis Acquisition nd control of prmeters (volumetric flows, molr flows, substrte nd wter thermodynmic ctivities, prtil pressures, tempertures) were monitored on-line using n IBM personl computer E is the corrected thermodynmic ctivity of functionl enzyme It is defined s the product of coefficient vrying from to nd ET, the thermodynmic ctivity of totl enzyme As the enzyme is in the solid stte, ET is by definition equl to The coefficient llows to correct ET for modifiction of functionl enzyme quntity A typicl experiment ws run t 7 C, with 5 mg of dsorbed enzyme nd the totl flow pssing through the rector ws set t mmolmin - giving volumetric flow of 85 mlmin - In these conditions, less thn 5% of the substrtes were converted llowing thus to ssume tht initil rtes (expressed in µmoles of product formed per minute nd per mg of enzymtic preprtion) were mesured nd tht inhibition by the products did not occur 6

8 Chromtogrphic ssys The vpor phse leving the biorector ws smpled using 5 ml loop on six-wy vlve (Vlco) mintined t 5 C Smples were utomticlly injected in the split injector of gs chromtogrph (Hewlett Pcrd model 589 A) equipped with Flme Ioniztion Detector (FID) for detection of ll products The column used ws CP sil 9-CB fused silic cpillry column (5 m x 3 mm id x µm film thicness, Chrompc, Frnce) The split rtio ws 5: The injector nd the detector were ept t C nd 5 C respectively Crrier gs ws nitrogen nd the flow rte in the column ws mlmin - Hydrogen nd ir were supplied to the FID t 39 nd 375 mlmin - respectively For the quntifiction of methnol, ethnol, propnol, methyl propionte, ethyl propionte, propyl propionte nd propionic cid, the column temperture ws held t 6 C for min, then progrmmed to increse t 3 Cmin - to 8 C For the quntifiction of butnol nd butyl propionte, the column temperture ws held t 6 C for min, then progrmmed to increse t 3 Cmin - to 7 C nd t 5 Cmin - from 7 C to 3 C Quntittive dt were obtined fter integrtion on HP 3396A integrtor An externl stndrd method ws used for the clcultions 3 RESULTS 3 Stbility of the enzyme in the solid/gs rector As mentioned in precedent study [6], it ws checed tht ctivity losses never exceed 5% during experiment, the durtion of which ws inferior to hours 7

9 3 Determintion of propnol inhibition constnt nd enzyme-substrte dissocition constnts t different wter thermodynmic ctivities As demonstrted in previous wor [6], the rection of lcoholysis between methyl propionte nd propnol ctlyzed by CALB cn be modelized by Ping Pong Bi Bi mechnism with ded-end inhibition by the lcohol In this rection sequence, the lipse rects with methyl propionte to yield the lipse-methyl propionte complex or with propnol to yield ded-end enzyme-propnol complex Then the lipse-methyl propionte complex is trnsformed into n cyl-enzyme intermedite nd the first product (methnol) is relesed The second substrte, propnol, next intercts with the cyl-enzyme to form ternry complex (Acyl-E-P), which finlly yields propyl propionte nd free lipse (scheme ) The inetic rte expression for the Ping Pong Bi Bi mechnism of the lipse, considering tht the experiments were performed under conditions in which the influence of the products cn be neglected, cn be written s shown in Eqution () At given ctivity of propnol, eqution cn be treted s n nlog of the Michelis- Menten eqution, with the pprent V Mpp nd Mpp being function of P (Equtions 3 nd 4) Experimentl dt (initil rtes versus MP t severl P vlues) were nlyzed nd gve V Mpp nd Mpp vlues for ech P P nd MP were then determined using equtions 3 nd 4 s explined erlier [6] Further detils for the determintion of dissocition constnts ( I, MP nd P ) re extensively detiled in previous pper [6] Wehtje nd Adlercreutz [] nd Bovr et l [] hve lredy pplied this method for the clcultion of V M nd M of cyl nd lcohol substrtes t different W, for esterifiction nd trnsesterifiction rections in orgnic medi respectively Results re summrized in Tble I The vritions of I nd MP with W follow the sme trends, ie slight decrese for W comprised between nd nd deep increse for W superior to As fr s P is concerned, no significnt devition hs been observed 8

10 33 Inhibition studies using different chin lengths for liner primry lcohols The effect of the lcohol polrity on its inhibitory chrcter ( I ) t W ner ws lso investigted using the sme method s the one previously described Typiclly, s deduced from eqution, the dissocition constnt of the lcohol-enzyme complex, I, cn be deduced from the plot of Mpp /V Mpp (obtined from the plot of reciprocl initil rtes versus reciprocl MP t different P ) versus P (Figure ) I vlues obtined for ethnol, propnol nd butnol, rnging from 3 to 46, showed tht I ws incresing with chin length of the lcohol moiety As wter is not vilble, dsorption of lcohol molecules on the enzyme is liely to occur thus probbly fvoring the inhibitory effect for the most polr molecules 34 Determintion of ctlytic constnts t different wter thermodynmic ctivities A simplified inetic scheme for the cyl-enzyme mechnism in the presence of n dded nucleophile hs been proposed by Grci-Alles nd Gotor [7] nd is displyed on scheme From this simplified mechnism, inetic equtions were determined using the stedy stte pproch for the formtion of the cyl-enzyme intermedite (Acyl-E) These equtions enble ctlytic constnts t different wter ctivities to be determined since they te into ccount the hydrolysis of methyl propionte even if the ltter tes plce in very prticulr conditions ie for very low thermodynmic ctivities of propnol ( P < 5) For inetic experiments t W superior to ( to 3), the W ws set t constnt vlue nd the initil rtes of formtion of propyl propionte (Vi PP ) nd propionic cid (Vi AP ) were mesured t different MP vlues ( to 5) From reciprocl initil rtes versus reciprocl MP plots, four pprent mcroscopic rte constnts (V Mpp ) PP, (V Mpp ) AP, ( Mpp /V Mpp ) PP nd ( Mpp /V Mpp ) AP were extrcted for ech P The vlues of V M re not determined by the y- xis intercept t / MP =, but rther by the /V vlue for / MP =, since the mximum substrte concentrtion is [6] These experiments were repeted t severl P vlues 9

11 (from 6 to for the quntifiction of the propionic cid formtion rte nd up to 5 for the quntifiction of the propyl propionte formtion rte) Then, for ech W, /(V Mpp ) PP nd (/ V Mpp ) AP vlues were plotted ginst / P nd P respectively (Figure nd 3 respectively) These representtions gve stright lines from which the slopes nd the y-intercepts could be extrcted using equtions 7 nd 9 giving A(w), B(w), C nd D(w) vlues t ech W These experiments being run t fixed W vlues, severl prmeter vlues ( E, / 4 nd 4 / 3 ) could be clculted for ech W (Tble ) For W equl to, the sole product is propyl propionte t every P vlue nd the /V Mpp expression using the simplified mechnism is reduced to eqution () So the y-intercept nd the slope of the /V Mpp versus / P plot give directly ( E ) - nd ( 4 E ) - E increses slightly for n W increse from to, then decreses t W = to similr vlue s the one observed without wter On the contrry, E is divided by fctor between W vlues of nd 3 For P vlues higher thn 5, no hydrolysis of methyl propionte could be observed (no propionic cid formed) whtever W Therefore, eqution () enbled S /( E ) to be determined, giving finlly S (Tble ) S vlues re not significntly different s the ones of MP One cn ssume tht both constnts probbly vry by chnge of - which increses with W (since MP = S + / nd S = - / ) nd tht nd re rther stble with W Therefore, the vritions of E previously mentioned cn be resonbly ssigned to vritions of functionl enzyme quntity with W rther thn to vritions of 4 DISCUSSION It ws estblished tht I (propnol) nd MP decresed slightly when W increses from to This cn be relted to the generl wter-induced enzyme ctivtion, observed when bioctlysis is performed in nhydrous medium Wter would fvor interction between enzyme ctive site nd ester or nucleophile inhibitor by cting s moleculr lubricnt,

12 incresing mino cid mobility [8] or incresing ctive site polrity [9] To demonstrte the ltter effect, Wngir et l [9] studied the combined effects of protein nd solvent engineering using subtilisin from Bcillus myloliquefciens s model serine protese The effect of wter in polr (cetone) nd polr (hexne) solvent on ctlytic efficiencies of wild-type enzyme nd two mutnts ws investigted for trnsesterifiction rection One of the mutnts nmed polr mutnt presented muttion in the bc of the S cyl-binding pocet of the enzyme (Gly 66 Asn) It ws shown tht in hexne the incresed polrity of the polr mutnt improved the interction of the enzyme with polr substrte, which is expected to te plce minly in the S subsite of subtilisin In the presence of smll concentrtions of wter dded, however, this dvntge over the wild-type enzyme ws mde negligible, presumbly by polr wter molecules penetrting into the ctive site of the enzyme When W is incresed from, to,3, the opposite effect is obtined, ie both constnts increse significntly Wter would thus ffect enzyme-methyl propionte nd enzymepropnol interctions This is in ccordnce with our previous results [6], which stted tht wter cted predominntly s competitive inhibitor of methyl propionte, fter optiml hydrtion level for ctlysis ws reched ( w >,) Wter would bind close to the ctive site to form ded-end complex, s the lcohol inhibitor does too The effect of wter on I (propnol) would ccount for mutul exclusivity of the two competitive inhibitors close to the ctive site Indeed it ws shown tht propnol is ble to compete with wter for the dsorption t hydrtion sites of proteins, in prticulr t ionizble groups t the surfce of the protein [] In principle, the precise dependence of binding constnt of propnol to enzyme on w gives n estimte of the number of wter molecules displced, though mss ction reltionship Vlivety et l mde such n estimte in cse of esterifiction of dodecnol nd decnoic cid in hexne ctlyzed by lipses t different W, where wter nd cid were in competition for binding to the ctive site [] They estimted the number of wter molecules displced by cid substrte, by using substrte dissocition

13 constnt vlues t two different W, insted of binding constnts of cid to enzyme t different W In tble, it ppers tht I (propnol) chnges 6 fold with fold chnge in w (when W increses from, to,), This could be ccounted for if 3 wter molecules were displced per lcohol molecule When W increses from, to,3, I (propnol) is multiplied by fctor 8, which corresponds this time to the displcement of 6 wter molecules per lcohol molecule Therefore n dditionl effect of wter on I (propnol) could be ssumed: the possible formtion of solvent brrier of wter molecules t ey position ner the enzyme Indeed, if the use of thermodynmic ctivities (insted of concentrtions) is method llowing correction for substrte vilbility t different hydrtion conditions, it does not permit to ccount for the differences between enzyme-wter nd enzyme-substrte interctions [] Moreover it hs to be pointed out tht t n W close to zero, I (propnol) is lmost ten times higher in the orgnic medium thn in the gs phse (Tble I) One cn suppose tht -methyl -butnol, which is modertely polr tertiry lcohol, is liely to interct with the protein nd to shield enzyme from other species, s wter does, thereby limiting the ccess of propnol to the ctive site [] Besides, the vlue obtined for I (propnol) in the orgnic medium without wter is similr to the one obtined in the gs phse t W equl to,3 (Tble) -Methyl -butnol might even ct s competitive inhibitor of methyl propionte, s propnol nd wter do, which would ccount for the higher vlues of I nd MP in the orgnic phse compred to the ones in the gs phse Vn Tol et l [] lso suggested competitive inhibition of the solvent by non-specific interctions of the solvent nd the ctive site With regrd to the increse of MP when W increses from, to,3, the sme explntion cn be put forwrd for the role of wter on the binding of the ester first substrte on the enzyme, i e the formtion of n hydrophilic brrier in the vicinity of the ctive site, hindering the ccess to it

14 Grci-Alles nd Gotor [7] studied the inetics of immobilized CALB-ctlyzed trnsesterifiction in non-queous orgnic medi They mesured how the solvent ffects the first substrte (ester) binding constnt nd the nucleophile inhibition constnt independently It ws first estblished tht the support did not interfere with solvent effects on the enzyme nd tht the ctlytic mechnism seemed to be the sme regrdless of the solvent used, ie Ping Pong Bi Bi with cyl-enzyme intermedite It ws shown tht the binding nd inhibition constnts were significntly incresed when the polrity of the solvent incresed; these vritions could not be explined using thermodynmic rguments (solvtion of the ester or the inhibitor) The solvent itself could thus led to decrese of the ffinity between enzyme nd ester or nucleophile by n other effect thn solvtion of these molecules, lie direct hindrnce effect of the ctive site for exmple It ws shown tht this effect increses with the polrity of the solvent Moreover in n other study, it ws found tht the binding of ester nd lcohol to CALB ws lower in polr cetonitrile thn in polr heptne nd tht these differences could not be explined by the difference in solvtion of the substrtes by the solvents [3] The possible explntion proposed ws tht cetonitrile intercts more strongly thn heptne with the ctive site nd thereby increses the M vlue of the substrtes The effect of the lcohol polrity on its inhibitory chrcter ws investigted nd it ppers tht the more hydrophobic the lcohol is, the higher the I (lcohol) vlue is Grci-Alles nd Gotor [4] lso relted n incresed inhibitory effect of short chin lcohols for lcoholysis of ethylcette ctlyzed by CALB in nhydrous cyclohexne In n other study, n explntion for higher inhibitory effect of short chin lcohols is tht they increse the system polrity thereby shifting the wter distribution towrds the fluid phse (supercriticl CO) which results in the removl of essentil wter molecules from the immobilized enzyme [5] Another possible explntion is tht polr molecules such s wter or short chin lcohols my bind close to the ctive site nd form n hydrophilic brrier impeding the 3

15 binding of the cyl substrte The hydroxyl of the lcohol or of wter my bind to the region round the ctlytic serine (Ser-5) which hs been reported to be polr in nture [6] This ltter hypothesis is supported by our results concerning the effect of wter on I of the lcohol since possible explntion for mutul exclusivity of the two competitive inhibitors is tht they compete for the sme site on the enzyme suggesting they intercts with the enzyme with the sme type of interction, ie polr interctions The effect of W on P (dissocition constnt of the cyl-enzyme-propnol complex) ws found to be negligible, which is opposite to results obtined by severl uthors in orgnic medium Indeed, severl studies report tht the M for the second substrte lcohol increses with W, in cse of esterifiction ctlyzed by different lipses [,] In the present cse, the fct tht P is not influenced by W seems to men tht the second step of the rection (decyltion) is not influenced by the microenvironment contrry to the first step (cyltion step) which is both influenced by the wter ctivity (increse of I nd MP with W for W superior to ) nd the medium (gs or orgnic phse) Indeed, I nd MP re more thn ten times lower in the gs phse thn in the orgnic phse t W ner, contrry to P which is the sme in both medi One cn suppose from this lst result tht the first step of the rection (cyltion or nucleophile inhibition) tes plce with substrtes or more generlly species in the gs stte wheres the cyl-enzyme intermedite would hppen in pseudoliquid stte, fter condenstion of substrtes molecules through binding with the enzyme Further experiments could be run in the gs phse to determine dissocition constnts in the presence of incresing thermodynmic ctivities of -methyl -butnol or of n inert solvent Concerning the influence of w on the ctlytic cyltion rte ( ), it ws shown tht smll quntity of wter dded ( w =,) leds to smll increse of E This is consistent with n increse of ctive-site flexibility nd s result, flexibility of the enzymic trnsition stte fforded by wter In the previously mentioned study [9], it ws shown tht smll quntity of wter dded (5% in nhydrous THF) resulted in n increse in the rte constnt of 4

16 cyltion for subtilisin-ctlyzed trnsesterifiction This increse ws more pronounced for the wild-type enzyme thn for the polr mutnt It ws suggested tht, in ddition to its role of plsticizer, wter cn increse the proton conductivity within n enzyme Serine proteses utilize generl cid-bse ctlysis nd proton trnsfer to the ctlytic histidine hs been suggested to be rte-limiting during cyltion [7] In the present study, wter would thus increse this proton-trnsfer rte nd increse the vlue When w is incresed from, to,3, the opposite effect is obtined, ie E decreses As explined in the results section, this ltter effect could be ssigned to decrese of functionl enzyme quntity, rther thn to decrese of This could be proved by ctive site titrtion t different w, but vilble methods for this titrtion need to be performed in orgnic solvents nd it ws shown tht solvents hve n influence on this prmeter [9, 4] The influence of the polrity of orgnic solvent on individul rte constnts in the cse of trnsesterifiction of ethyl cette nd n-butnol ctlyzed by immobilized CALB ws studied by Grci-Alles nd Gotor [7] It ws found tht chnges on cyltion rte nd decyltion rte were much less importnt thn the vritions of substrte binding with the solvent, indicting tht the orgnic solvent lmost did not interfere with enzyme-trnsition stte interctions Furthermore the rtio of these two rtes ws found to be independent on the polrity of the solvent used, which ws interpreted s n indiction of the trnsition-stte desolvtion In the present study, this rtio ( / 4 ) is lmost constnt for W equl to,, nd, (Tble ), s if cyl-enzyme intermedite ws shielded from its microenvironment To conclude, the results obtined in the present study highlight tht wter my ply multiple roles in the enzyme s ctive site, either directly or indirectly This wor provides more evidence tht enzyme hydrtion is complex phenomenon As demonstrted in this wor, wter ffects minly the first step of the rection, ie the cyltion step, by incresing the dissocition constnts between both enzyme nd cyl substrte nd enzyme nd lcohol It ws suggested tht wter, s polr lcohols, binds to the 5

17 enzyme through polr interctions nd thereby blocs the ccess to the ctive site However, wter hs no significnt effect on the ctlytic constnt Once the cyl-enzyme intermedite hs been formed, wter does not seem to hve n influence nymore on the binding of the second substrte nor on the ctlytic constnt of decyltion Similr results hve been reported in the literture concerning the independence of decyltion on W nd solvent polrity Results suggest tht ddition of wter in the solid/gs rector mimics n increse of solvent polrity in orgnic medi Further experiments re in progress to evlute the effect of solvent of different polrities on inetic prmeters in the solid/gs rector 6

18 REFERENCES []- Hlling, PJ (987) Wter ctivity in biphsic rection systems In Bioctlysis in orgnic medi, Lne C, Trmper J, Lilly MD (Eds), Elsevier Science Publishers, Amsterdm, 5-3 []- Cssels, JM nd Hlling, PJ (988) Effect of thermodynmic wter ctivity on thermolysin-ctlysed peptide synthesis in orgnic two-phse systems Enzyme Microb Technol,, [3]- Goldberg, M, Prvresh, F, Thoms, D nd Legoy, MD (988) Enzymtic ester synthesis with continuous mesurement of wter ctivity Biochim Biophys Act, 957, [4]- Goldberg, M, Thoms, D nd Legoy, MD (99) Wter ctivity s ey prmeter in synthesis rections : the lipse exmple Enzyme Microb Technol,, [5]- Ljunger, G, Adlercreutz, P nd Mttisson, B (994) Enzymtic synthesis of octyl-β-dglucoside in octnol t controlled wter ctivity Enzyme Microb Technol, 6, [6]- Rupley, JA, Grtton, E nd Creri G (983) Wter nd globulr proteins Trends Biochem Sci, 8, 8- [7]- Vlivety, RH, Hlling, PJ nd Mcre, AR (99) Lipses from different sources vry widely in dependence of ctlytic ctivity on wter ctivity Biochim Biophys Act,, [8]- Vlivety, RH, Hlling, PJ nd Mcre, AR (99) Rection rte with suspended lipse ctlyst shows similr dependence on wter ctivity in different orgnic solvents Biochim Biophys Act, 8, 8- [9]- Vn Erp, SHM, mensy, EO nd hmelnitsy, YL (99) The effect of wter content nd nture of orgnic solvent on enzyme ctivityin low-wter medi Eur J Biochem,,

19 []- Vlivety, RH, Hlling, PJ nd Mcre, AR (993) Wter s competitive inhibitor of lipse-ctlysed esterifiction in orgnic medi Biotechnol Lett, 5, []- Bovr, R, Crre, G, Ottolin, G nd Riv, S (993) Effects of wter ctivity on V mx nd M of lipse ctlyzed trnsesterifiction in orgnic medi Biotechnol Lett, 5, []- Wehtje, E nd Adlercreutz, P (997) Wter ctivity nd substrte concentrtion effects on lipse ctivity Biotechnol Bioeng, 55, [3]- Mrty, A, Chullsnnuul, W, Willemot RM nd Condoret JS (99) inetics of lipse-ctlyzed esterifiction in supercriticl CO Biotechnol Bioeng, 39, 73-8 [4]- Hnsen, H, Rsmussen, P, Fredenslund, A, Schiller, M nd Gmehling, J (99) Ind Eng Chem Res 3: [5]- Lmre, S nd Legoy, MD (995) Woring t controlled wter ctivity in continuous process: The gs/solid system s solution Biotechnol Bioeng, 45, [6]- Bousquet-Dubouch M P, Grber M, Sous N, Lmre S nd Legoy M D () Alcoholysis ctlysed by Cndid ntrctic lipse B in gs/solid system obeys Ping Pong Bi Bi mechnism with competitive inhibition by the lcohol substrte nd wter Biochim Biophys Act, 55, 9-99 [7]- Grci-Alles LF nd Gotor V (998) Lipse-ctlyzed trnesterifiction in orgnic medi : solvent effects on equilibrium nd individul rte constnts Biotechnol Bioeng 59: [8] Schmite JL, Wescott CR, libnov AM (996) The mechnistic dissection of the plunge in enzymtic ctivity upon trnsition from wter to nhydrous solvents J Am Chem Soc 8: [9]- Wngir PP, Grycr TP, Estell DA, Clr DS nd Dordic JS (993) Protein nd solvent engineering of subtilisin BPN' in nerly nhydrous orgnic medi 5:

20 []- Mc Minn, JH, Sow, MJ, Chrnic, SB nd Pulitis, ME (993) The hydrtion of proteins in nerly nhydrous orgnic solvent suspensions Biopolymers 33, 3-4 []- Grber M, Bousquet-Dubouch M P, Sous N, Lmre S nd Legoy M D (3) Wter plys different role on ctivtion thermodynmic prmeters of lcoholysis rection ctlyzed by Lipse in gseous nd orgnic medi Biochim Biophys Act, in press []- Vn Tol, JBA, ryveld, DE, Jongejn, JA nd Duine, JA (995) The ctlytic performnce of pig pncres lipse in enntioselective trnsesterifiction in orgnic solvents Bioctl Biotrns : 9-36 [3]- Mrtinelle, M nd Hult, (995) inetics of cyl trnsfer rections in orgnic medi ctlysed by Cndid ntrctic lipse B Biochim Biophys Act 5: 9-97 [4]- Grci-Alles LF nd Gotor V (998b) Alcohol inhibition nd specificity studies of lipse B from Cndid ntrctic in orgnic solvents Biotech Bioeng 59: 63-7 [5]- Goddrd, R, Bosley, J nd Al-Duri, B () Esterifiction of oleic cid nd ethnol in plug flow (pced bed) rector under supercriticl conditions Investigtion of inetics J of Supercriticl Fluids 8: -3 [6]- Uppenberg J, Öhrner N, Norin M, Hult, leywegt GJ, Ptr S, Wgen V, Anthonsen T nd Jones TA (995) Crystllogrphic nd moleculr modelling studies of lipse B from Cndid ntrctic revel stereospecificity pocet for secondry lcohols Biochemistry 34 : [7]- Creri G, Gerci M, Ginsnti A nd Rupley JA (985) Protonic conductivity of hydrted lysozyme powder t meghertz frequencies Proc Ntl Acd Sci USA 8:

21 FIGURE LEGEND Figure Determintion of the dissocition constnt of the enzyme-lcohol complex ( I ) in the solid/gs biorector for 3 liphtic lcohols using eqution () Figure Reciprocl V Mpp versus reciprocl P t different W vlues V Mpp were obtined from the plot of reciprocl initil rtes of formtion of propyl propionte versus reciprocl MP ( to 5) t different P (6 to 5) nd for ech W ( to 3) Synthesis ws crried out in the solid/gs biorector t 7 C, t molr flow of mmolmin - in the presence 5 to 5 mg of dsorbed CALB onto Chromosorb AW DMCS Figure 3 Reciprocl V Mpp versus P t different W vlues V Mpp were obtined from the plot of reciprocl initil rtes of formtion of propionic cid versus reciprocl MP ( to 5) t different P (6 to 5) nd for ech W ( to 3) Synthesis ws crried out in the solid/gs biorector t 7 C, t molr flow of mmolmin - in the presence 5 to 5 mg of dsorbed CALB onto Chromosorb AW DMCS

22 SCHEME EP P I P E + MP E-MP Acyl-E Acyl-E-P E + PP - M - V Vi = Mpp Mpp MP + MP () with V Mpp Mpp + P () = MP VM I nd MP = +, V ( P VMpp P ) VM P M V Mpp = VMpp = + P + P (3) P = ' +' ', Mpp = ( + ) P P I MP (4) P + P E = enzyme, MP = methyl propionte, P = propnol, M = methnol, PP = propyl propionte P nd MP re thermodynmic ctivities of propnol nd methyl propionte respectively nd I, MP nd P re the dissocition constnts of the propnol-enzyme, enzyme-methyl propionte nd cyl-enzyme-propnol complexes respectively

23 SCHEME AP PP Mpp MP AP PP Mpp Mpp AP PP i V V V,,, + = (Eqution 5) with If w > P P W E W E W AP Mpp P W E S E S W W I P AP Mpp Mpp w B w A V V ) ( ) ( == + + = = P P E W E PP Mpp P E W S E S W W I P PP Mpp Mpp w D C V V ) ( = + = = If w = 4 E P E PP Mpp I P E S PP Mpp Mpp V V + = + = (Eqution 6) (Eqution 7) (Eqution 8) (Eqution 9) E + MP E-MP Acyl-E E + AP E + PP S 3 W 4 P I EP P W EW W M (Eqution ) (Eqution )

24 Tble I : Effect of W on dissocition constnts of propnol-enzyme, enzyme-methyl propionte nd cyl-enzyme-propnol complexes respectively ( I, MP nd P ) nd on V M W I MP P 9 47 Gs phse Orgnic phse * 6 65 * results from Bousquet-Dubouch et l [6] Tble II : Effect of W on ctlytic constnts W E (min - ) / 4 4 / 3 S 49 3 nd nd

25 FIGURE 3 5 Mpp /V Mpp lcohol ethnol propnol butnol 4

26 FIGURE w = w =, /VMpp 5 /VMpp / P / P w =, w =,3 /VMpp 5 /VMpp / P / P 5

27 FIGURE /V Mpp P w=, w=, w=,3 6