lipase-catalyzed transesterification in organic media: solvent effects on equilibrium and individual...

Lipase-Catalyzed Transesterification inOrganic Media: Solvent Effects onEquilibrium and IndividualRate Constants

Luis F. Garcı́a-Alles, Vicente Gotor

Departamento de Química Orgánica e Inorgánica, Facultad de Química,Universidad de Oviedo, 33071 Oviedo, Spain; telephone: +34 8 510 34 51;Fax: +34 8 510 34 48; e-mail: [email protected]

Received 5 September 1997; accepted 6 February 1998

Abstract: The kinetics of the immobilized lipase B fromCandida antarctica have been studied in organic sol-vents. This enzyme has been shown to be slightly af-fected by the water content of the organic media, and itdoes not seem to be subject to mass transfer limitations.On the other hand, some evidence indicates that thecatalytic mechanism of reactions catalyzed by this lipaseproceeds through the acyl-enzyme intermediate. More-over, despite the fact that the immobilization supportdramatically enhances the catalytic power of the enzyme,it does not interfere with the intrinsic solvent effect. Con-sequently, this enzyme preparation becomes optimumfor studying the role played by the organic solvent incatalysis. To this end, we have measured the acylationand deacylation individual rate constants, and the bind-ing equilibrium constant for the ester, in several organicenvironments. Data obtained show that the major effectof the organic solvent is on substrate binding, and thatthe catalytic steps are almost unaffected by the solvent,indicating the desolvation of the transition state. How-ever, the strong decrease in binding for hydrophilic sol-vents such as THF and dioxane, compared to the rest ofsolvents, cannot be easily explained by means of ther-modynamic arguments (desolvation of the ester sub-strate). For this reason, data have been considered as anindication of the existence of an unknown step in thecatalytic pathway occurring prior to formation of theacyl-enzyme intermediate. © 1998 John Wiley & Sons, Inc.Biotechnol Bioeng 59: 684–694, 1998.Keywords: immobilized enzymes; organic solvents;mechanism; kinetic studies; microscopic rate constants;rate-limiting step

INTRODUCTION

Using enzymes in nonaqueous solvents is a generally ac-cepted strategy in organic synthesis (Boland et al., 1991;Dordick, 1989; Klibanov, 1990; Waldmann and Sebastian,1994; Wong and Whitesides, 1994). They display two fea-tures which are appealing to organic chemists: catalyticpower and selectivity. However, both of them are extraor-

dinarily dependent on the choice of reaction conditions(Parida and Dordick, 1991; Sakurai et al., 1988; Tawaki andKlibanov, 1992; Zaks and Klibanov, 1988a). Often, the un-predictability of enzyme behavior and the lack of clear in-formation for selecting the most suitable conditions limittheir exploitation. It is therefore necessary to develop pre-dictive models, which correlate enzyme efficiency to sol-vent properties, substrate and active-site polarities and hy-dration, which are known to govern enzyme function(Wangikar et al., 1993; Xu et al., 1994).

There has been strong experimentation in this field duringthe last few years. Despite the fact that studying enzymes isnot an easy task because so many variables influencing theircatalytic activity have to be controlled, some explanationsare emerging. For instance, it has been possible to explain,at least partially, the dramatic activity decrease observedwhen an enzyme is used in anhydrous environments com-pared to that observed in water (Schmitke et al., 1996). It isalso possible to understand, by means of thermodynamicmodels, the large specificity changes observed for an en-zyme, depending on the solvent used (Ke et al., 1996;Wescott and Klibanov, 1993; Wescott et al., 1996).

As far as organic solvent effects on enzyme catalyticpower are concerned, they have not been completely ex-plained yet, even when some conclusions have been drawnand are widely accepted such as: (1) The local and globalstructure of enzymes is not significantly altered when goingfrom aqueous solution to the most organic solvents (Adamset al., 1990; Burke et al., 1989; Kanerva and Klibanov,1989). However, enzyme flexibility and amino acid mobil-ity are very much reduced (Guinn et al., 1991; Zaks andKlibanov, 1985), providing the most probable reason for thehigher stability of enzymes in those media (Zaks and Klib-anov, 1988a). (2) Catalytic activity usually increases withthe amount of water retained by the enzyme (Zaks andKlibanov, 1988b). This is the reason why the most popularpredictive tool for estimating enzyme activity has been thepartition coefficient of the solvent, logP, which is an indi-cator of solvent hydrophobicity (Laane et al., 1987). (3)Therefore, it is often observed that the presence of water in

Correspondence to:Vicente GotorContract grant sponsors: CICYTContract grant numbers: BIO-95-0687

© 1998 John Wiley & Sons, Inc. CCC 0006-3592/98/060684-11

the organic environment enhances catalytic efficiency (Zaksand Klibanov, 1988). (4) The organic solvent is known todrastically affect the ground state of the substrate, and con-sequently the free energy of substrate binding to the enzyme(Ryu and Dordick, 1992; Schmitke et al., 1996).

In most of the published work to date, investigations havecentered around the effect of solvent on Vmax/Km(app)for theester substrate (acylation specificity constant). This com-plex term simplifies under appropriate conditions to k2[E0]/KS. However, even if this is true, much care must be takenwhen interpreting this kind of data, because it is not knownwhether the changes observed with solvent variation are dueto effects on substrate binding (KS), on the catalytic acyla-tion rate (k2), or on the concentration of functional enzyme([E0]). Thus, it is necessary to measure how the solventaffects each of these parameters independently. This is thepurpose of the present work, to study the mechanism ofaction of the immobilized lipase B fromCandida antarctica(CAL) by measuring the equilibrium and individual rateconstants in different organic solvents. Despite the highlevel of experimentation, the data have been relatively easyto analyze.

MATERIALS AND METHODS

Enzyme Preparation

The immobilized and lyophilized lipase B fromCandidaantarcticawere kindly gifted by Novo Nordisk. The sameinitial batch of immobilized enzyme was used throughoutthe realization of the work (Novozym 435 with specificactivity of 7300 pLU/g). CAL was stored under nitrogen at5°C, and its activity was found to be constant for almost oneyear. The water content of the immobilized particles wasdetermined to be approximately 1% (w/w) (by comparisonof its weight before and after heating at 105°C for 3 h). Theprotein content of a sample of beads with a mean diameterof 0.50–0.63 mm was found to be around 4.7% (w/w)(Bradford assay).

Chemicals and Solvents

All solvents used were of high purity, being stored undernitrogen atmosphere and dehydrated over molecular sieves(4Å) for at least 24 h prior to use. Consequently, hydrolysisis expected to be significantly reduced, compared to trans-esterification, at the nucleophile concentrations employedthroughout this work. Chemicals used were of commercialquality (most of them purchased from Aldrich Chemie).

Initial Rate Determinations

A 5 mL solution containing the ester (0.01 to 1.2M) and thealcohol (0.01M to 0.11M) in a given solvent was prepared inan Erlenmeyer flask. The solution was closed to air with arubber septum and introduced in an orbital shaker (260

rpm, which in our case had been found to be enough toovercome external mass transfer limitations), and allowedto stand for at least 30 min at 29–31°C. Finally, the immo-bilized enzyme (3–5 mg) was added to the solution, and atregular intervals of time (usually 1 min) a 20-mL samplewas extracted from the flask (five samples per reaction) andinjected into a Hewlett-Packard 5890 Series II gas chro-matograph (equipped with an HP1 crosslinked methyl sili-cone gum capillary column). Isocratic conditions were used;the oven temperature adjusted to detect the products 2–5min after injection. They were detected with the aid of aflame ionization detector (350°C), their signal peaks inte-grated, and their concentrations determined by using inter-nal standards (ethylbenzene for butyl and propyl acetate andacetophenone for decyl and dodecyl acetate, Rangheard etal., 1992). Standard curves were produced enabling the con-centration of the ester product to be followed over time.Reactions never exceeded 10% of total conversion.

Determination of Apparent Vmax, Km

and/or Vmax/Km

Initial rates were determined for 8 AcOEt concentrations(0.01–1.20M), at the same alcohol concentration (Table I).Data obtained were fitted to a rectangular hyperbola using anonlinear regression analysis. This analysis provided valuesfor the apparent Vmax and Km acylation constants. Valuesfor the apparent acylation specificity constant (Vmax/Km)were extracted using the Lineweaver-Burk analysis.

Support Effects

Initial rates were determined in different organic solventsusing a 0.08M concentration of 1-butanol and a 0.10M con-centration of AcOEt, employing both the immobilized (3–5mg) and lyophilized samples (5–10 mg).

Diffusion

CAL was sieved, and several samples were collected, theirdiameter ranging between the following values (mm):<0.10, 0.10–0.25, 0.32–0.50, 0.50–0.63, 0.63–0.71, 0.71–0.80, >0.80. Initial rates were measured intBuOMe (0.08M,1-butanol and 1.1M AcOEt) using each one of these enzymesamples. Representation of the initial rates against the in-verse of the mean particle diameter rendered an excellentlinear correlation.

Hydration

A 0.5M AcOEt and 2M 1-butanol solution in the dry organicsolvent was prepared (solution A). A second solution (B)saturated with water was also prepared (in the case of THFand 1,4-dioxane the latter was a 0.66M solution of water).The final reaction mixture was obtained by mixing variablequantities of solution B (from 0 to 4.5 mL) with 0.5 mL ofsolution A, and adjusting the final volume to 5 mL by

GARCIA-ALLES AND GOTOR: KINETIC STUDIES OF ENZYMES IN ORGANIC SOLVENTS 685

addition of dry organic solvent. The initial transesterifica-tion rates were determined and plotted against the watercontent present in the organic environment (Fig. 3).

It is important to point out that during these experimentshydrolysis is also expected to occur to some extent, espe-cially at high water contents, but it could not be measuredbecause of chromatographic reasons. The presence of hy-drolysis might mask an enzyme activation due to hydration.However, we believe that if significant enzyme activationwas indeed occurring due to hydration, an increase in trans-esterification rates should be observed at least at low watercontents, where the water concentration is still much lowerthan the concentration of 1-butanol.

Competent Active-Site Concentration

Two solutions containing 50 mg ofp-nitrophenyl-N-butylcarbamate in 5 mL of hexane ortBuOMe were prepared.Twenty mg of CAL were added to each solution, and theywere stirred at 260 rpm and 30°C over 15 h. At this time, theenzyme was filtered, washed with solvent (5 × 1 mL) anddried at vacuum. The activity of both samples was assayedby comparing the initial transesterification rate intBuOMe(using 0.08M 1-butanol and 0.05M ethyl acetate concentra-tions) with that of the untreated enzyme. This experimentrevealed that the enzyme had been almost completely in-hibited in both solvents.

Formation of EAN Complexes

Solution A containing AcOEt and 1-butanol, and solution Bcontaining 1-propanol were prepared in an organic solvent.Eight reaction mixtures were prepared by addition of vari-

able quantities of solution B to 0.5 mL of solution A, andadjusting final volume to 5 mL. Final concentrations were0.08M of 1-butanol, 0.10M of AcOEt, and 0.02–0.25M of1-propanol. The initial rate of formation of both productswas measured for each reaction mixture.

Determination of KS and the IndividualRate Constants

Method 1: Figure 1 represents the expected mechanism forour system, where alcohol competitive inhibition is in-cluded (Garcı´a-Alles and Gotor, 1998). Assuming that re-actions will take place under initial rate conditions (catalyticsteps are irreversible due to the absence of products, andsubstrate concentrations will change by less than 10%), thissystem will follow a Michaelis-Menten behavior, with theapparent Vmax and Km constants given by Equations (1) and(2).

Consequently, we can determine the Vmax and Km valuesat several nucleophile concentrations and correct Km valuestaking into account the alcohol competitive inhibition (KN),using Equation (2) to obtain the Km* term. Next, these datacan be fitted to rectangular hyperbolas (Fig. 4), and thevalues for the k2[E0] and KS constants can be extracted fromtheir maximum values if saturation is observed for both theVmax and Km* values with the nucleophile concentration(the [E0] term is included because the enzyme has not beentitrated in each solvent).

Thus, Km and Vmax values were determined at six 1-butanol concentrations, in the range of 0.01 to 0.11M, inTHF and tBuOMe. Km* values were calculated usingKN(THF) 4 0.291M and KN(tBuOMe)4 0.025M. Satura-tion was not observed intBuOMe (Fig. 4B), and conse-

Table I. Specificity constant values for the acylation step in the CAL-catalyzed reaction between AcOEt and 1-butanol (0.08M) in different organicsolvents. The ratio of initial rates of the reaction catalyzed by the immobilized and lyophilized lipases are also shown.a

Solvent LogPbVmax/Km

× 102 (min−1)ck2[E0]/KS ×102 (min−1)d

Vo immob. ×104 (M min−1)e

Vo lyoph. × 104

(M min−1)e Vo immob./Vo lyoph.f

DMF −1.0 <0.0001 — — — —Dioxane −1.1 0.48 0.70 3.9 0.06 62Acetone −0.23 0.06 — — — —THF 0.49 0.20 0.25 1.9 0.05 34tBuOMe 1.5 1.62 6.8 11.8 0.24 49PhCOMe 1.8 0.27 — — — —iPr2O 1.9 1.19 5.5 9.4 0.14 67Toluene 2.5 0.31 9.2 3.1 0.05 58Bu2O 2.9 0.65 8.1 5.8 0.10 57Cyclohexane 3.2 0.10 8.9 1.0 0.05 20Hexane 3.5 0.19 15.4 2.0 0.06 32tMPentg 4.5 0.07 — — — —

aKinetic values are given for a 1 mg/mL CALconcentration.bValues taken from Laane et al. (1987).cValues obtained using the immobilized lipase. Ethyl acetate concentrations were varied in the range 0.01–1.1M. Specificity values determined using the

Lineweaver-Burk analysis.dSpecificity values for the immobilized lipase corrected taking into account KN values of Table III.eValues measured using 0.08M 1-butanol and 0.1M AcOEt concentrations.fThe different percentage of protein present in each enzyme preparation has not been taken into account.g2,2,4-trimethylpentane.

686 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 59, NO. 6, SEPTEMBER 20, 1998

quently the data had to be adjusted to a straight line, ren-dering the values for k3[E0] and k2[E0]/KS.

Method 2: The second procedure employed is known asthe added nucleophile method (Bender et al., 1964; Berezinet al., 1971). Chatterjee and Russell (1992) and Wangikar etal. (1993) have already applied this method to measure in-dividual rate constants for subtilisin-catalyzed transesterifi-cations in organic solvents. A second nucleophile (N2) com-peting for the same EA intermediate is introduced in thereaction (Fig. 2), and saturation kinetics are measured atdifferent concentrations of the second nucleophile (usingalways the same concentration of the first nucleophile). Inour case, the values for the apparent macroscopic rate con-stants were determined as usual, employing a 0.08M con-centration of the first nucleophile, in the presence of 0 to0.08M concentrations of the added nucleophile. The rates offormation of both esters were measured and data weretreated as described. Because of the presence of nucleophilecompetitive inhibition, Km/Vmax values were corrected em-ploying Eq. (4) with the KN values depicted in Table III(1-propanol and 1-butanol inhibition constants values wereconsidered to be equal, KN1 ≈ KN2).

Method 2a: A (Vmax)−1 and (Vmax/Km*)−1 linear increase

for the formation of the first product (P1) was observed withincreasing concentrations of the second nucleophile (Fig. 5).The values for the kinetic constants were obtained fittingthese data to Equations (5) and (6): The ratio of slopes gaveKS, which was used in combination with k2[E0]/KS, y-intercept of Equation (5), to extract k2[E0]. k3[E0] was es-timated using the last value and the y-intercept of Equation(6).

Method 2b: In some cases it was also possible to measureformation of the second product (P2) (Fig. 6), and to fit datato Equations (7) and (8). The y-intercept of Equation (8)afforded k2[E0]. This value was combined with the k2[E0]/KS value to obtain KS. However, k3[E0] was estimated usingthe k2[E0] value and the y-intercept value of Equation (6).

RESULTS AND DISCUSSION

We chose to study the simplest reaction—the lipase-catalyzed transesterification of ethyl acetate with 1-butanol.The enzyme selected was the immobilized lipase B fromCandida antarctica(CAL). Firstly, the apparent specificityconstant (Vmax/Km)AcOEt was determined in a wide range oforganic solvents (Table I). The results attained were differ-ent from what would be expected on the basis of the solventhydrophobicity (logP). Though for very hydrophilic sol-vents, such as DMF or acetone, results were in agreementwith general knowledge (null or low catalytic power), dis-crepancies appeared for the most hydrophobic solvents,where the enzyme should have displayed the highest cata-lytic power. However, we knew from previous studies thatalcohol competitive inhibition was indeed occurring, inhi-bition being stronger in such hydrophobic solvents (Garcı´a-Alles and Gotor, 1998). Thus, for some of these solvents theVmax/Km term could be corrected to estimate real k2[E0]/KS

values (also collected in Table I). In this case, the results aremore in accordance with the logP rule, showing that im-portant errors can be committed when interpreting Vmax/Km

data.

Preliminary Studies

Because our aim was to analyze the organic solvent influ-ence on each step of the catalytic mechanism, it was im-portant to control other factors that are known to influenceenzyme efficiency as well.

Immobilization Support

Immobilization can facilitate studies of enzymes in organicsolvents and increase their catalytic power (Suzawa et al.,1995). Nevertheless, it is known that the chemical nature ofthe immobilization support can change the partitioning ofwater and/or substrates from the solvent to the enzyme(Brockman et al., 1973; Clark, 1994). Therefore, it wasnecessary to check whether the support could interfere bymasking real solvent effects. With this purpose, initial rateswere measured in several organic solvents, using both thelyophilized (non-immobilized) and the immobilized en-zyme. A low AcOEt concentration was selected to securethat initial rates were a reflection of Vmax/Km. As far as thesolvent variation effect was concerned, results were inde-pendent of the kind of enzyme preparation employed, as wecan see in Table I (the ratio of initial rates, Vo

immobilized/Vo

lyophilized, is almost constant for all the solvents tested).Similar information has been provided by van Tol et al.(1995) and Suzawa et al. (1995) for other enzymes. We canalso observe that the immobilized enzyme is around 2–3orders of magnitude more active than the lyophilized coun-terpart. Several reasons may account for this activity differ-ence, among them being aggregation and denaturation ofthe enzyme upon lyophilization (Dabulis and Klibanov,1993; Mishra et al., 1996). This last reason has been used toexplain the aqueous-like activity ofa-chymotryspsin dis-solved in organic solvents (Paradkar and Dordick, 1994),and the enzyme activation by molecular imprinting (Russelland Klibanov, 1988).

Diffusion

In heterogeneous processes there appear two classes of dif-fusional barriers: external and internal diffusion (Russelland Beckman, 1991). External diffusion can be alleviatedusing vigorous shaking. Indeed, in our case, shaking speedsabove 150 rpm were enough to eliminate such limitation(260 rpm were used throughout this work). As far as internaldiffusion is concerned, the problem is more difficult to ad-dress. This factor is dependent on the pore and particle sizes(when using lyophilized enzymes, both of them can changewith the solvent). Thus, the effect of the enzyme particlesize on activity was measured intBuOMe. The immobilizedenzyme was sieved to eight different bead sizes, with di-


ameters ranging from 0.10 to 0.80 mm. The activity of thesesamples was determined and an excellent correlation withthe reciprocal particle diameter was obtained (data notshown). Activity was higher as the particle size was re-duced, indicating that the only acting enzyme was spreadonto the particle surface. This could be due to the existenceof internal mass transfer limitations precluding the access ofthe substrate to the enzyme located in the internal porestructure of the support. Nonetheless, this reason appearedto be improbable, because the mean pore size of immobi-lized CAL (140–170 nm) was larger than that reported forthe lipase efficiency to become independent of pore diam-eter (pores >100 nm are enough in the case of immobilizedRhizomucor mieheilipase; Bosley and Clayton, 1994). Itwas also possible that there was no enzyme located in thecentral region of the particles.

To find an answer this question, two samples differing insize were selected (with bead diameters of 0.10–0.25 mmand 0.71–0.80 mm, the first sample was 2.5 times moreactive than the second sample), and two experiments wereperformed. In the first experiment, both samples werecrushed and the catalytic activity of the powder obtainedwas measured. The 0.10–0.25 mm powdered sample re-tained the same activity as the sample of beads, and it wasstill 2.0 times more active than the 0.71–0.80 mm powderedenzyme. This result indicated the heterogeneous distributionof the enzyme on the immobilized particles, and the lack ofinternal diffusion, at least in the case of the small particles.In the second experiment, the protein content of these pow-dered samples was determined using the Bradford assay.Once again, a clear difference in the protein content for bothsamples was observed, despite the fact that the differencewas not as high as expected (a protein content relationshipof 1.7 was obtained). Anyhow, these studies demonstratedthat the enzyme content was different depending on theparticle size, and for that reason during the remaining part ofthis work we used immobilized particles with a mean porediameter of 0.50–0.63 mm. Thus, error due to dispersion inenzyme concentration ([E0]) were also reduced.

Hydration Effects

Water can have a dramatic influence on catalysis, dependingupon its effect on the solvent used (Gorman and Dordick,1992; Xu et al., 1994; Zaks and Klibanov, 1988b). Severalmechanisms have been proposed for water-induced enzymeactivation. It may act as a molecular lubricant, increasingamino acid mobility (Schmitke et al., 1996), and reducingunfavorable protein-protein interactions, responsible forstructural distortion and/or aggregation, or else it may in-crease active site polarity (Guinn et al., 1991; Xu et al.,1994). Both effects have been studied recently by means ofelectron spin resonance (ESR), comparing water activationof immobilized and suspended chymotrypsin (Suzawa et al.,1995).

Bearing this in mind, the CAL catalytic power was stud-ied at different water contents in the following systems:

1,4-Dioxane, THF,iPr2O, and Hexane (Fig. 3). The resultsindicated that the water influence on transesterificationrates, far from being dramatic, was almost non-existent.Enzyme activity was only enhanced in hexane (by less than50%). For the rest of the solvents, the rate of product for-mation was reduced as the water content was increased. Asimilar result had been previously described for other im-mobilized lipase (van Tol et al., 1995). However, in our casehydrolysis could not be detected, and consequently muchcare must be taken when interpreting this information (seeMaterials and Methods).

Competent Active-Site Concentration

The organic solvent can modify the catalytic efficiency ofthe enzyme (kcat/Km) or the concentration of competent en-zyme ([E0]). To test for the second possibility, one mustdetermine the percentage of enzyme that is acting in eachsolvent. However, a recent work has shown that the usualmethods for titration of active sites can be erratic (Wangikaret al., 1996). Moreover, in the case of lipases there are nodescribed methods to perform such titration in organic me-dia. For this reason, we have assumed that the active sitepercentage is rather similar for all the solvents. Even whenthis can be a very rough approximation, final results suggestthat this effect is not significant. Besides, when thep-nitrophenyl-N-butyl carbamate (inhibitor of serine hydro-lases, Hosie et al., 1987; Scofield et al., 1977) was used withCAL, the enzyme was almost completely inhibited (around90%) after a 15 h treatment, both in hexane andtBuOMe,indicating that the amount of functional enzyme was similarin both solvents. However, because we have not titrated theactive-site concentration, nor studied other solvents, the re-sults shown in this study are analyzed taking into accountthe [E0] may change with solvent hydrophobicity.

At this point, it is worth mentioning that alternative ex-periments have shown that immobilized CAL becomes in-activated when exposed to 1-butanol. Catalytic activitystrongly decreases within the first minute after mixing theenzyme and the alcohol. This is only evidenced at very lowalcohol concentrations (much lower than those employedthroughout this study), probably because the inactivationprocess is slower under these reaction conditions. At theseconcentrations, a strong burst of product formation takesplace during the first minute of reaction, before reaching astationary state where the enzyme is not further inactivated(activity is maintained for hours). This burst can be elimi-nated in two ways: (1) by using an enzyme which has beenalready exposed to a 1-butanol solution, washed with or-ganic solvent, and dried under vacuum; or (2) by adding theenzyme to the reaction mixture some minutes before start-ing the reaction by addition of the ester substrate. In anycase, data also indicate that the final inactivation degree isindependent of the alcohol concentration, and that the latteronly modifies the rate at which inactivation occurs. For thatreason, and because the same alcohol concentration has


Figure 1. Kinetic scheme illustrating CAL-catalyzed transesterificationin initial rate conditions. S represents the acyl-donor substrate, N the nu-cleophile, and P the product formed during the reaction. E is the freeenzyme, ES and EA are the non-covalent enzyme-substrate complex andthe acyl-enzyme intermediate, respectively. EN is the nucleophile inhibitedcomplex. KS is the dissociation constant of the ES complex, whereas KN isthe dissociation constant of the inhibited complex. k2 and k3 representindividual rate constants.

Figure 2. Kinetic scheme for the acyl-enzyme mechanism in the pres-ence of an added nucleophile. N1 and N2 represent the first and the addednucleophile, respectively, whereas P1 and P2 are the ester products. EN1

and EN2 represent the nucleophile inhibited complexes, and their disso-ciation constants are KN1 and KN2, respectively. k4 represents an individualrate constant. See Figure 1 for other information regarding the notation used.

Figure 3. Transesterification initial rate of the reaction catalyzed by theimmobilized CAL (1 mg/mL) between AcOEt (0.05M) and 1-butanol(0.2M) as a function of the water content of the organic solvent. The X-axisrepresents the water content of the solvent: For cyclohexane andiPr2O, itgives the percentage relative to the water-saturated monophasic solvent. Inthe case of THF and dioxane, it indicates the percentage of a water 0.6Msolution.

Figure 4. Kinetics of immobilized CAL-catalyzed transesterification ofAcOEt in the presence of varying concentrations of 1-butanol in THF(graph A) andtBuOMe (graph B). Km* values have already been corrected,taking into account 1-butanol inhibition. (s) Vmax, (h) Km.


been used throughout the experiments described in thiswork, the kinetic data presented will not be affected by thisparameter, except in the sense that it is feasible that theinactivation degree varies from solvent to solvent (Titrationof the functional enzyme in each organic solvent in presenceand absence of the alcohol might give some explanation).This inactivation effect might be related with the KC termintroduced in Figure 7 (see below).

Catalytic Mechanism

In aqueous solution, serine-hydrolases follow the classicalacyl-enzyme mechanism (Fersht, 1985). Several studiessuggest that the same mechanism of action (Ping-Pong) isfollowed in organic solvents (Adams et al., 1990; Chatterjeeand Russell, 1992; Kanerva and Klibanov, 1989). We havealso obtained some experimental evidence (Chatterjee andRussell, 1993), which confirms that CAL-catalyzed reac-tions proceed through the acyl-enzyme intermediate:

1. When ethyl acetate (Ac-OEt) and isopropenyl acetate(Ac-OiPr), that presumably produce a common acyl-enzyme intermediate (EA), were used as acyl-donor sub-strates, the partition between two competing acceptors(0.05M 1-butanol and 0.05M 1-propanol) gave a similarratio of products (Table II). The butanolysis/propanolysisratio obtained in THF was 0.91 for AcOEt and 0.92 forAcOiPr, whereas it was around 0.73 for ethyl butyrate,suggesting that only with the last substrate a differentacyl-enzyme intermediate was formed.

2. The reaction rate must be the same for substrates onlydiffering in leaving group, when deacylation is rate-limiting. Two substrates clearly differing in leavinggroup ability, ethyl and isopropenyl acetate, were used inpresence of 0.04M of 1-butanol in THF. The results ob-tained are shown in Table II. They indicate that despitethe fact that the values obtained for the apparent Vmax/Km and Km constants differ by around one order of mag-nitude, the Vmax values (which represents k3, when

Figure 5. Kinetics for the formation of the first product (P1) in transesterification reactions catalyzed by immobilized CAL between AcOEt and 1-butanol(0.08M), in the presence of an added nucleophile (1-propanol, 0–0.08M). The following solvents were used: (A) THF, using isopropenyl acetate insteadof ethyl acetate as acyl donor; (B) THF; (C) Dioxane; (D)iPr2O; (E) Bu2O; (F) Cyclohexane, using 1-dodecanol and 1-decanol, instead of 1-butanol and1-propanol, respectively. (s) 1/Vmax, (h) (Km/Vmax*).


k3[Nu]!k2) are practically the same for both kind ofsubstrates.

Formation of the Acyl-Enzyme-NucleophileComplex (EAN)

It has already been mentioned that in our case, as well aswith other lipases, the nucleophile is known to be a com-petitive inhibitor. On the other hand, it has been reported forother enzyme-catalyzed processes that more than one nu-cleophile molecule may bind to the EA intermediate, form-ing different EA(N)n complexes (Gololobov et al., 1993).This information prompted us to study whether the possiblemode of action of the nucleophile on the second part of thereaction mechanism (after the EA intermediate has beenformed) would be solvent dependent. With this purpose, thepartition of the EA intermediate between two competingacceptors, N1 and N2, was studied in different organic sol-vents. Transesterification experiments were performed withthe N1 concentration kept constant (0.08M 1-butanol) whilevarying the N2 concentration (0.02–0.25M 1-propanol). Thedependence of the ratio of product formation (VoP2/V

oP1)on the N2 concentration was determined. For all the solventstested (toluene, Bu2O, iPr2O, tBuOMe, and THF) a lineardependence was obtained, and the partition constant (r 4[N2] × VoP1/V

oP2) was found to be invariable in the rangeof N2 concentrations used, and very similar for all the sol-vents tested (data not shown). These results indicated thatthe same mechanism of action for the nucleophile was validregardless of the solvent used, and that formation of EA(N)n

(with n ù 2) could be dismissed, because this kind of

mechanism would hardly render a linear VoP2/VoP1 rela-

tionship with the N2 concentration.However, there are still two mechanistic models which

can be proposed to account for the linear dependence ob-served: with or without formation of the EAN complex. Forsimplicity, we will use a model that does not include for-mation of the EAN complex (as represented in Figs. 1 and2); it will not affect the general conclusions of the presentwork.

Determination of Individual Rate Constants inDifferent Organic Solvents

Once we knew that (1) the support did not interfere withsolvent effects on the enzyme, (2) diffusion was not goingto be rate-limiting, (3) the water effect was not very impor-tant, and that (4) the catalytic mechanism seemed to be thesame regardless of the solvent used, we confronted the de-termination of solvent effects on the individual rate con-stants. Unfortunately, the application of pre-steady-state ki-netics to obtain this kind of information is severely limitedin heterogeneous systems (Fersht, 1985). For this reason,one is restricted to steady-state techniques to do researchinto enzymes suspended in organic solvents.

The equilibrium and microscopic rate constants, KS, k2,and k3 for transesterification reactions were measured usingtwo approaches (methods 1 and 2 in Materials and Meth-ods). Data obtained using method 1 have been representedin Figure 4, whereas Figures 5 and 6 show plots of dataobtained using methods 2a and 2b, respectively. KS, k2[E0],and k3[E0] values measured at 30°C in different organic

Figure 6. Kinetics for the formation of the second product of the reaction (P2), when AcOEt was used as the acyl-donor substrate in the presence of1-butanol (0.08M) and an added nucleophile (1-propanol, 0–0.08M)). (s) 1/Vmax, (h) (Km/Vmax*). Solvents were: (A) THF; (B)iPr2O; (C) Bu2O; (D)Cyclohexane (using 1-dodecanol and 1-decanol).


solvents have been collected in Table III. We can see thatthe different methods afforded similar values in THF, indi-cating the validity of the kinetic model employed. On theother hand, when isopropenyl acetate was used in THF, onlythe values for the k3[E0] and for the k2[E0]/KS terms couldbe obtained. This was due to the fact that for this substratethe k2 value is at least one order of magnitude higher thank3[N]. Therefore, deacylation will be rate determining, andformation of butyl acetate at AcOEt saturation (Vmax) willbe independent of the 1-propanol concentration (Fig. 5A).

In cyclohexane, data were obtained using 1-dodecanoland 1-decanol, instead of 1-butanol and 1-propanol, becausewith the last substrates saturation is not observed (and con-sequently Km and Vmax values cannot be determined). Thiseffect is probably the result of the relaxation of inhibitionpromoted by short-chain alcohols, when trying to reachsaturation (addition of the ester substrate improves solvationof the inhibitor). This effect is less pronounced using long-chain alcohols, because the effect of changing the esterconcentration does not greatly influence their solvation (asit has been demonstrated by means of UNIFAC calcula-tions).

Data presented in Table III show that the major effect dueto variation of the anhydrous environment is on substratebinding (Chatterjee and Russell, 1992; Ryu and Dordick,1992). Changes on k2[E0] and k3[E0] are much less impor-tant. Furthermore, the k2/k3 values are very similar for all ofthem, indicating that the active site is mostly deshieldedfrom the organic solvent. Similar conclusions have alsobeen extracted from Hammett constant and deuterium iso-tope exchange analyses with other serine-hydrolase en-zymes; in those studies, data was interpreted as an indica-tion of the transition-state desolvation (Adams et al., 1990;Kanerva and Klibanov, 1989).

On the other hand, our results are also in agreement withother published data, which demonstrate that changes inrate-limiting steps can be tailored by means of solvent en-gineering (Chatterjee and Russell, 1992; Wangikar et al.,1993), and that these rate-limiting switches are due to an

effect on the acylation rate, rather than on the deacylationstep. In this sense, we have already reported that for CAL-catalyzed transesterification processes, acylation is more af-fected by solvent than deacylation, suffering an importantdecrease in hydrophilic solvents like THF or dioxane (Gar-cı́a-Alles and Gotor, 1998). Moreover, inhibition constantvalues (KN) were also very high in these solvents. Thesedeviations could not be explained using thermodynamic ar-guments (solvation of the ester or the inhibitor), and weproposed that some other effect might be exclusively affect-ing acylation (and KN values). If we represent this hypoth-esis in a general way, by means of a kinetic scheme depictedin Figure 7, it can be shown that only the reading of the KS

and KN equilibrium constant values would be modified bythe presence of the new step, represented by KC, Equations(9) and (10). In this case, both of them would result equallyoverestimated.

Data presented in Table III are in agreement with thishypothesis: KS values are clearly responsible for the bigdrop in specificity values for the acylation step in THF anddioxane. Moreover, if we compare the KS/KN relationshipfor solvents which solvate the ester and the alcohol verysimilarly (in THF, dioxane andtBuOMe, the UNIFAC-calculated activity coefficients for AcOEt and 1-butanol are:gAcOEt 4 0.91, 1.3, 1.0, andgButOH 4 2.1, 2.4, 2.5, respec-tively), values obtained are very similar (5.5, 4.2, and 4.1),indicating that the source for the overestimation of the KN

and KS values can be the same. In any case, these kinds ofinterpretations have to be taken with much care; no otherevidence for this unknown step have been presented.

CONCLUSIONS

In summary, it has been shown that once other effects suchas hydration or diffusion have been relaxed using an immo-bilized enzyme preparation, it is easy to perform kineticstudies in organic media. In this manner, the kinetic mecha-nism of action of the lipase B fromCandida antarcticahasbeen evidenced to proceed through the acyl-enzyme inter-mediate. Furthermore, the validity of the kinetic model hasbeen ratified when the equilibrium and microscopic rateconstants were measured. These studies demonstrate that

Table II. Apparent kinetic constants for the acylation step ofCandidaantarctica lipase-catalyzed reactions.a

Acyldonor

Vmax/Km ×102 (min−1)

Vmax × 102

(M min−1)Km

(M)Vo

ButOHb/

VoPrOH

AcOEt 0.26 ± 22% 0.12 ± 7% 0.52 0.91 ± 5%AcOiPrc 1.95 ± 27% 0.11 ± 7% 0.05 0.92 ± 4%ButOEt — — — 0.73 ± 9%

aKinetic values are given for a 1 mg/mLconcentration of immobilizedCAL. They were obtained using a 0.04M concentration of 1-butanol inTHF at 30°C.

bRatio of initial rates when 1-butanol and 1-propanol were used at thesame concentration.

cIsopropenylacetate.

Figure 7. Ping-Pong kinetic model for lipase-catalyzed reactions, includ-ing competitive inhibition by the alcohol and a hypothetical equilibriumstep represented by KC. See Figure 1 for other information regarding thenotation used.


the major solvent effect on catalysis is on binding of thesubstrate to the enzyme. Catalytic rate constants, k2 and k3are slightly influenced, indicating that the organic solventalmost does not interfere with enzyme-transition state inter-actions. However, a different kinetic model, which includesan initial equilibrium step, has been suggested to explain thebig increase observed in both the KS and KN values in themost hydrophilic solvents.

We want to thank Novo Nordisk A/S, Denmark, for the enzymeand the information supplied. L.F.G.A. also thanks the II P.R.I.(Asturias) for a predoctoral scholarship.

References

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Table III. Individual rate constants for transesterification reactions catalyzed by the immobilizedCandida antarcticalipase in anhydrous organic solventsat 30°C.*

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THFc,d 2a 0.29 1.6e 3e 2.7 (7%) 1.3e 0.8 (20%)THF 2a 0.29 1.6 (29%) 0.4 (35%) 6.4 (39%) 0.07 1.1 (18%)

2b 1.6 (48%) 0.7 (41%) 4.2 (45%)f 0.17 1.4 (24%)THF 1 0.29 1.4 (21%) 0.5 (6%) 4.1 (40%) 0.12 —Dioxaned 2a 0.17 0.73 (17%) 0.5 (20%) 5.8 (21%) 0.09 2.1 (13%)tBuOMe 1 0.025 0.10g 0.8g 6.3 (30%) 0.13g —iPr2O 2a 0.022 0.13 (38%) 0.8 (49%) 11 (54%) 0.07 1.1 (31%)

2b 0.09 (58%) 0.7 (38%) 13.5(43%)f 0.05 1.0 (32%)Bu2O 2a 0.007 0.04 (80%) 0.3 (90%) —h — 1.9 (44%)

2b 0.06 (42%) 0.7 (7%) 7.0 (18%)f 0.10 1.3 (43%)Toluenei 2a 0.003 0.13 2.1 11 0.19 1.9Cyclohexanej 2a 0.007 0.14 (120%) 1.2 (130%) 3.4 (140%) 0.35 0.9 (23%)

2b 0.01 (130%) 0.3 (26%) 16 (33%)f 0.02 4.0 (110%)

*Kinetic values are given for a 1 mg/mLconcentration of immobilized CAL. AcOEt was used as the acyl donor substrate.aProcedure employed: 14 method 1; 2a4 method 2, but measuring formation of P1, and using Equations (5) and (6), 2b4 method 2, analyzing

formation of P2 by means of Equations (7) and (8).bValues taken from Garcı´a-Alles and Gotor (1998).cIsopropenyl acetate (AcOiPr) was used as the acyl donor.dFormation of propyl acetate (P2) could not be detected.eData obtained assuming that values for (KS)AcOiPr and (KS)AcOEt in this solvent are similar.fThese values were determined using a combination of Equations (8) and (6).gSaturation for Km* and Vmax was not observed, and consequently these data have been obtained assuming that in this solvent and iniPr2O, the k2[E0]

value should be similar.hNegative value.iOnly two points were used to extract these data.jThe nucleophiles used were 1-dodecanol and 1-decanol. Therefore, k3 or k4 values cannot be compared with those obtained in the rest of solvents. In

this case, the inhibition values employed were KN1 4 6.8 mM and KN2 4 9.7 mM.


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