Influence of Water-Miscible Aprotic Solvents on r-ChymotrypsinStability

Pedro Lozano, Teresa de Diego, and Jose L. Iborra*

Departamento de Bioquımica y Biologıa Molecular B e Inmunologıa, Facultad de Quımica,Universidad de Murcia, P.O. Box 4021, E-30001 MURCIA, Espana

The influence of five different water-miscible aprotic solvents (dimethyl sulfoxide,dimethylformamide, acetonitrile, acetone, and tetrahydrofuran) on the stability ofimmobilized R-chymotrypsin adsorbed onto Celite, has been studied. In all cases,R-chymotrypsin exhibited non-first-order deactivation kinetics, which were adequatelyanalyzed by a two-step series-type deactivation model. The main effects of solventswere observed in the first-step of the kinetic mechanism. The most hydrophilic solvent(DMSO) enhanced greatly the enzyme stability, while the increase in solventhydrophobicity determines a loss in their protective effect. For the most assayedhydrophobic solvent (THF) a denaturative effect was always showed. These facts werealso observed from the analysis of the evolution of the half-live of the enzyme, as afunction of the solvent concentrations and the water distribution between both themacro- and microenvironment of the enzyme.

IntroductionAn active area of research in biotechnology is bioca-

talysis in nonconventional systems, involving all thereaction media where the water activity (Aw) parameteris reduced (i.e., organic solvents, supercritical fluids, etc.)(Klibanov, 1986; Khmelnitsky et al., 1988; Dordick, 1992;Halling, 1994). This is not surprising given the potentialadvantages of conducting biocatalytic reactions in aque-ous-organic solvent mixtures or in pure organic solvents,such as the increase in solubility of poorly water-solubleorganic substrates and/or the ability to shift thermody-namically hydrolytic reactions to the synthetic way(Arnold, 1990; Combes and Lozano, 1992; Iborra et al.,1992; Manjon et al., 1992). In the case of proteases, theseenzymes have been growing attention due to theirpotential ability to synthesize dipeptide products withhigh potential pharmacological and industrial uses fromamino acid derivatives (Kise et al., 1990; Lozano et al.,1995). However, in many cases, the use of organicsolvents in biocatalytic reactions is seriously limitedbecause many organic solvents can be shown as dena-turative agents of biocatalysts (Tanford, 1968; Mozhaevet al., 1989). In other cases, organic solvents (i.e.,polyhydric cosolvents) act as protective agents of enzymestoward thermal denaturation (Gekko and Timasheff,1981; Graber and Combes, 1989; Lozano et al., 1993,1994).Several parameters have been chosen to correlate the

nature of the organic solvent and its influence on enzymestability. Solvent polarity, measured by the Hildebrandparameter or by the log P parameter, appears in theliterature as the most important criterium to describethe ability of an organic solvent to distort the essentialwater shell around biocatalysts and so to produce theconsequent modification in its activity or stability (Laaneet al., 1987; Mozhaev et al., 1989; Gupta, 1992; Manjonet al., 1992). In other cases, the thermodynamic wateractivity (Aw) of the media has been proposed as the bestparameter to analyze the influence of organic cosolventson the enzyme behavior because it describes the influence

of these binding-water additives on the concentration of“free” water molecules (Graber and Combes, 1989; Comb-es and Lozano, 1992; Manjon et al., 1992; Lozano et al.,1993, 1994; Halling, 1994). Only for the most hydropho-bic and water-inmiscible solvents (log P > 2) have severalcorrelations between the enzyme behavior and the log Pand/or Aw been shown (Laane et al., 1987; Mozhaev etal., 1989), while for the most hydrophilic cases (polyols),their stabilizing effect on enzymes has been related tothe Aw parameter (Graber and Combes, 1989; Lozano etal., 1993, 1994). However, in the case of the aprotic andwater-miscible solvents, no systematic study has beencarried out to explain their influence on enzyme stabilityas a function of their chemical characteristics.The aim of this paper is to analyze the deactivation

process, induced by five aprotic and water-miscibleorganic solvents, of the R-chymotrypsin immobilized onCelite. This derivative has been previously used tosynthesize the dipeptide kyotorphin in these nonconven-tional media with excellent results (Lozano et al., 1995).In this way, the results were fitted to a two-step non-first-order enzyme deactivation model. A relationshipbetween the deactivation data and several mediumparameters such as the solvent concentration, Aw, watercontent, and log P was established.

Experimental Section

Materials. R-Chymotrypsin (EC 3.4.21.1) type II frombovine pancreas was purchased from Sigma Chemical Co.N-Acetyl-L-tyrosine ethyl ester (ATEE, Sigma ChemicalCo.) was used as the standard substrate. Dimethylsulfoxide (DMSO), N,N′-dimethylformamide (DMF), ac-etonitrile (ACN), acetone (AC), and tetrahydrofuran(THF) were Merck, analytical grade. Celite 545 (0.01-0.04 mm particle size) was obtained from Merck. Allremaining reagents were analytical grade and were usedwithout additional purification.Immobilization Method. R-Chymotrypsin (40 mg)

was dissolved in 4 mL of 0.1 M phosphate buffer (pH 7.8),and mixed with 1 g of Celite. The mixture was shakenfor 30 min at room temperature and then lyophilized. Allthe placed protein was adsorbed into the support, mea-* To whom correspondence should be addressed.

488 Biotechnol. Prog. 1996, 12, 488−493

S8756-7938(96)00011-2 CCC: $12.00 © 1996 American Chemical Society and American Institute of Chemical Engineers

sured by the Lowry’s method; the immobilized derivativeshowed an esterase activity toward N-acetyl-L-tyrosineethyl ester of 10.45 U/mg of support and a water contentof 0.18 mg of H2O/g of support.Assay of Enzyme Activity. The esterase activity of

R-chymotrypsin was determined by the pH-stat methoddescribed in detail by Wilcox (1970). A videotritratorVIT-90 equipped with an autoburette (ABU 91) and asample station SAM 90 (Radiometer, Copenhagen) wereused. The protocol was as follows: a 3 mL sample of 50mM ATEE in 30% w/v aqueous ethanol solution contain-ing 20 mM CaCl2 was placed into a thermostated (40 °C)reaction vessel. The reaction was started by addition of50 µL of either a 0.1% (w/v) R-chymotrypsin aqueoussolution or 1% w/v aqueous suspension of the immobilizedderivative previously homogenized. The pH was main-tained constant at 7.0 by continuous addition of 50 mMNaOH as titrant. One unit of activity was defined as theamount of enzyme that hydrolyzes 1 µmol of ATEE/minunder standard conditions of assay (pH 7.0, 40 °C).Study of Stability in the Presence of Aprotic

Organic Solvents. Into a screw-capped test tube con-taining 50 mg of the immobilized R-chymotrypsin deriva-tive were added 5 mL of aqueous solutions of each solvent(DMSO, DMF, AcN, AC, or THF) at the different assayedconcentration, and the mixture was incubated at 30 °C.At regular intervals of time, homogeneous aliquots of 50µL were extracted from the incubation mixture and theresidual activity was measured as described above. Theresults obtained were fitted to modeled theoretical curvesusing a nonlinear regression program of iterative con-vergence by the Marquardt-Levenberg algorithm methodincluded in the Sigmaplot 5.1 (1992) software.Measurement of Water Activity. Water activity

was determined using a humidity and temperaturedigital indicator HUMIDAT-IC II (Novasina, Zurich,Switzerland), with a humidity sensor model BS-3(4)/PP(Novasina). The humidity sensor was checked andperiodically recalibrated at three points, with controlsaturated salt solutions (LiCl, Aw ) 0.113; Mg(NO3)2, Aw) 0.544; BaCl2, Aw ) 0.905) for the overall measuringrange.Determination of Water Content. The water con-

tent of immobilized enzyme preparation in each reactionmedia was determined as follows: 10 mg of R-chymot-rypsin-Celite complex were suspended into 2 mL of eachassayed water-organic solvent mixture, and homogenizedduring 30 min. at room temperature. Then, the im-mobilized derivative was separated by centrifugation, andtheir water content was measured by the optimized Karl-Fischer method using a moisture titrator MKS-210(Kyoto Electronics, Japan).

Results and Discussion

Kinetic Analysis of the Enzyme DeactivationProcess. Figure 1 shows the deactivation profiles,depicted by the experimental points, of both the solubleand the immobilized R-chymotrypsin derivative in aque-ous solution at 30 °C. The analysis of these data by aone-step first-order deactivation model did not representadequately the enzyme deactivation behavior, their bi-phasic character being noticeable (Mozhaev et al., 1989,1992; Owusu and Berthalon, 1993; Lozano et al., 1994).A first-order enzyme deactivation kinetic requires thata homogeneous inactivation should occur independent ofthe microenvironmental conditions. However, consider-ing the complexity of the enzyme molecule, the fractionalrate of decrease in enzyme activity will not always beconstant, suggesting that complex internal events take

place in its conformational transitions to the deactivatedstate. Therefore, it is reasonable to expect that morethan one type of bonds must be broken before a loss ofactivity occurs, which should be followed by differentconformational stages during the deactivation process(Henley and Sadana, 1985). However, Wada et al. (1983)concluded for 17 denaturing proteins that an only oneintermediate state of transition should be consideredbecause a second stage of transition could be an artifactdue to lack of sensitivity of the experimental measure-ments (i.e., calorimetry). Additionally, in the case ofR-chymotrypsin, several authors (Mozhaev et al., 1989;Owusu and Berthalon, 1993) concluded a two-step deac-tivation process studying the structural changes in theenzyme during thermal deactivation by fluorescencespectroscopy. In this way, it has been previously pro-posed (Lozano et al., 1994) that the general mechanismof deactivation of R-chymotrypsin follows a two-stepseries model developed by Henley and Sadana (1985) asfollows:

where k1 and k2 are first-order deactivation rate constants(min-1); E, E1, and Ed are conformational enzyme stateshaving different specific activities; and R1 and R2 are theratios of specific activities E1/E and Ed/E, respectively.Different authors (Mozhaev et al., 1992; Owusu andBerthalon, 1993) concluded that the R-chymotrypsinthermodeactivation is an irreversible process, where thetime course for enzyme unfolding as the same as foractivity loss and E and E1 are two protein fractionsdiffering in stability. In our case, it was experimentallyproved, at long deactivation times, that the final state ofR-chymotrypsin (Ed) is an irreversible fully deactivatedstate, where the R2 parameter of the model should beequal to 0, and then the overall activity can be writtenby eq 2 (Henley and Sadana, 1985). The fit of the

experimental enzyme-deactivation data of Figure 1 bythis equation using a nonlinear regression procedurebased on the Marquardt-Levenberg method of iterativeconvergence yields the theoretical curves depicted in thesame figure, which were obtained by substitution of thecalculated convergence values of all kinetic parametersin eq 2. The good agreement between the experimental

Figure 1. Deactivation of both the soluble (2) and the im-mobilized derivative onto Celite support (b), (R-chymotrypsinin aqueous media at 30 °C). Residual activity of R-chymotrypsinwas measured at 30 °C using 50 mM ATEE at pH 7.0.

E98k1E1R1

98k2EdR2

(1)

a ) [1 + R1k1/(k2 - k1)/(k2 - k1)][exp(-k1t)] -[R1k1/(k2 - k1)][exp(-k2t)] (2)

Biotechnol. Prog., 1996, Vol. 12, No. 4 489

and theoretical data showed the suitability of the pro-posed model, which was corroborated by a correlationcoefficient higher than 0.998. The calculated values ofdeactivation parameters of both the soluble and theimmobilized R-chymotrypsin derivative are included inTable 1. In both cases, the R1 parameter was lower than1, indicating the loss of activity showed by the enzymein the transition to the intermediate state E1. Addition-ally, the deactivation rate constant of the first step wasfive-times higher than the second one, showing thebiphasic nature of the process, which is clearly depictedby the convexity (toward the origin) of these curves. Ahigher degree of convexity implies more protection to-ward denaturation or a lower rate of deactivation. Inthis way, the adsorption of R-chymotrypsin to the Celitesupport involved an eventual stabilization of the enzyme,indicated by both the decrease in the deactivation rateconstants (k1 and k2) and the increase in the stabilizationlevel (R1), which also implies an increase in the degreeof convexity of the curve. These facts were also observedby the increase in the half-life of the enzyme after theimmobilization process (more than 10 times), suggestingthat the presence of the support should be involved inboth the maintenance of the conformational structure ofthe enzyme and the limitations of the autolysis phenom-ena.On the other hand, the thermodynamic parameter

standard free energy (∆G°) of the deactivation processcan be calculated from the following equation fromexperimental data:

where ki is the first-order deactivation rate constant foreach step (h-1); kB is the Boltzmann’s constant (J‚K-1); his the Planck’s constant (J‚h); R is the gas constant(J‚mol-1‚K-1), and T is the temperature (K). As it canbe seen, in Table 1, the protective effect of Celite on theR-chymotrypsin deactivation is also supported by anincrease in the standard free energy of denaturation.Although not very large, this standard free energyincrement should be sufficient to stabilize efficiently theprotein, since the net free energy for stabilization ofproteins is in general small (Gekko and Timasheff, 1981).Effect of Aprotic Organic Solvent Concentration

on the Immobilized r-Chymotrypsin Stability. Theeffect of five different aprotic organic solvents (DMSO,DMF, AcN, AC, and THF; log P ) -1.3, -1.01, -0.33,-0.23, and 0.49, respectively) on the stability of theimmobilized R-chymotrypsin derivative has been studiedat different solvent concentrations at 30 °C. In all cases,the analysis of the experimental deactivation data by theproposed series-type model also yielded kinetic behaviorswith biphasic nature, similar to the obtained previouslyin water media. Figure 2 depicts the evolution of alldeactivation parameters (R1, k1, and k2) of the im-mobilized derivative as a function of solvent concentra-tions. The decrease in R1 parameter upon increase insolvent concentration clearly reflects an important de-naturative effect of these solvents, which was enhancedby increase in solvent hydrophobicity (THF > AC > AcN),except in the most hydrophilic cases (DMSO and DMF),

where a slight increase in this R1 parameter was observedfor low assayed concentrations, previously to a final decayof enzyme activity at high concentration. In the sameway, the kinetic rate constants (k1 and k2) decreasedlightly for the most hydrophilic solvents, and increasedgreatly for the most hydrophobic cases. Thus, at lowsolvent concentration, the most hydrophilic solvents(DMSO and DMF) act as protective agents towardenzyme deactivation, changing to a denaturative effectat high concentrations. So, in the same way of polyols,at low concentration, these aprotic hydrophilic solventsaid to maintain the solvophobic interaction into theprotein, which play the key role in supporting the activeconformation, as well as to preserve the water shellaround the protein molecule (Bull and Bresse, 1978; Backet al., 1979; Lozano et al., 1994). However, at highsolvent concentration, the negative effect of DMSO andDMF should be attributed to the direct interaction withthe protein molecule by the disruption of the hydrationshell, which could contribute to the desorption of theenzyme from the support, as well as to the break out ofhydrophobic intramolecular interactions, to finally yieldan inactive protein molecule (Mozhaev et al., 1989, 1992).On the other hand, the most hydrophobic solvents

(AcN, AC, and THF) always produced a denaturativeeffect on the immobilized enzyme, which was enhancedby the increase in their hydrophobicity, which can beattributed to the increase in the direct enzyme-solventinteractions breaking the hydrophobic core of the protein.In agreement with Khmelnitsky et al. (1988), the higherthe solvent hydrophobicity (adequately measured by thelog P parameter) the stronger denaturing effect. In ourcase, the enhancement of the deactivation process by thesolvent hydrophobicity was higher than that induced bythe increase in solvent concentration in each case.Mozhaev et al. (1989) studied the influence of several

polyhydric cosolvents on the stability of R-chymotrypsin,measuring the denaturing efficiency of solvents by athreshold concentration parameter (C50, defined as theconcentration of solvent to reduce to one-half the enzyme

Table 1. Kinetic and Thermodynamic Parameters ofr-Chymotrypsin Deactivation in Aqueous Media at 30 °C

enzymeform R1

k1(h-1)

k2(h-1)

t1/2(min)

∆G°(kJ‚mol-1)

SE 4.2 × 10-2 2.0 0.3 21.9 93.2IME 57.8 × 10-2 0.6 0.1 231.1 96.2

-∆Gi° ) RT ln[(kBT)/(kih)] (3)

Figure 2. Effect of solvent concentration on kinetic deactivationparameters of R-chymotrypsin at 30 °C. The residual activityof R-chymotrypsin was measured at 30 °C using 50 mM ATEEat pH 7.0. Figures inset depict the deactivation rate constants(k1 and k2) of the enzyme in the presence of THF in the fullscale. DMSO (9), DMF (2), AcN (O), AC (0), and THF (∆).

490 Biotechnol. Prog., 1996, Vol. 12, No. 4

activity). These authors established a linear relationshipbetween the C50 parameter and the hydrophobicity of thesolvent, measured by the log P parameter, showing thatthe more the hydrophilicity of solvent (or lower log P)the better cosolvent. Tanford (1968) established thatinactivation of proteins by organic solvents is followedby binding of organic cosolvent molecules with proteinmolecules. Thus, good cosolvents are those that allowgreat reduction of the overall water content of themedium without significant loss of enzyme activity. Onthis order, Gekko and Timasheff (1981) attributed theprotein stabilization phenomena by hydrophilic cosol-vents to a preferential exclusion of solvent molecules fromthe domain of the protein molecule, where the solvationshell around the exposed nonpolar residues remainedintact.Role of Water on the r-Chymotrypsin Stability

in Aprotic Water-Miscible Organic Solvents. Innonaqueous environment or low-water media, enzymescan function provided if the essential water layer aroundthem is not stripped off (Laane, 1987; Gorman andDordick, 1992). In this way, the essential role of wateron the enzyme stability in water-miscible organic mediashould be studied from both a macro- and a microenvi-ronmental point of view.The changes in water properties in the macroenviron-

ment of an immobilized enzyme could be adequatelymeasured by the thermodynamic water activity of themedia (Aw, defined as the ratio of the water vaporpressure over a medium to that over pure water). Figure3 shows the influence of the Aw of the assayed organicmedia on the half-life of the immobilized R-chymotrypsinderivative induced by these additives. As it can be seen,the decrease in Aw produced by the most hydrophilicsolvents (DMSO, DMF, and AcN) increased the half-lifeof the enzyme to an optimal value, while the mosthydrophobic assayed solvents (AC and THF) produced acontinuous decrease in this stability parameter. Ad-ditionally, it can be seen that the maximum half-life ofthe immobilized enzyme is enhanced by the increase insolvent hydrophilicity, which was observed at a morereduced Aw of the medium. Thus, the obtained team ofcurves allowed classification of these organic solvents asa function of their stabilizing power, which was in thesame order of their increase in hydrophobicity, as fol-lows: DMSO > DMF > AcN > AC > THF. These resultsshould be explained as a function of the decrease inconcentration of “free” water molecules in the media by

the increase in solvent hydrophilicity. The changesproduced on the macroenvironment of the enzyme bythese solvents could be considered as an immobilizationof the water medium, which influences directly themicroenvironment of the immobilized derivative (Lozanoet al., 1994).A quantitative criterion of water properties to analyze

the influence of organic solvents on the microenvironmentof the immobilized enzyme could be carried out by thechanges in water content. Additionally, a parametercalled the protective effect, and defined as the ratio ofenzyme half-life in the presence of cosolvents to the half-life of enzyme without cosolvents, was used. Figure 4shows the profiles of the protective effect of these cosol-vents as a function of the water content of the im-mobilized derivative. As it can be seen, the decrease inwater content produced by these aprotic organic solventsproduced a behavior in the enzyme stability similar tothat observed in Figure 3, showing the direct incidenceof the Aw depressing power of solvents in the macroen-vironment on the water content of the microenvironment.The higher the capacity decrease in Aw the macroenvi-ronment, the higher the decrease in water content of themicroenvironment of the immobilized derivative. In thisway, hydrophilic solvents enhanced the enzyme stabilityto an optimum value with a minor water content, whilehydrophobic solvents produced a continuous deactivationprocess. It is also necessary to mention that the protec-tive effect can attain important value: the half-life of theenzyme was increased 15 times in the presence of 4.3 MDMSO, which involved a water content in the im-mobilized derivative of 1.5 mg of H2O/g of support and aAw of the water media of 0.8 (see Figure 3).Furthermore, in the same way that the Aw depressing

power of solvents influenced the water content of theimmobilized derivative, the nature of the immobilizationsupport could imply a partition effect on water distribu-tion between the macro- and microenvironment, involvingsome artifacts in the interpretation of the results. Reslowset al. (1988) studied the influence of water partitionbetween solvents and immobilized R-chymotrypsin de-rivatives as a function of the aquaphilicity of the supportmaterials, a parameter defined as the ability to absorbwater from a water-saturated diisopropyl ether system(µL of H2O support/µL of H2O media). These authorsobserved not only the lowest aquaphilicity of Celite (0.36)with respect to other supports usually employed to adsorbenzymes, i.e., CPG (0.98), Sephadex G-25 (12.3), or Bio-Gel P4 (13.8), but also that the enzyme activity decreaseswhen the support increased in aquaphilicity. These facts

Figure 3. Effect of water activity (Aw) of the organic media onthe half-lives of immobilized R-chymotrypsin at 30 °C. Figureinset depicts the half-lives of the enzyme in presence of DMSOin the full scale: DMSO (9), DMF (2), AcN (O), AC (0), andTHF (4).

Figure 4. Protective effect of aprotic organic solvents onimmobilized R-chymotrypsin stability at 30 °C as a function ofthe water content of the immobilized derivative. Figure insetdepicts the protective effect of DMSO in the full scale. DMSO(9), DMF (2), AcN (O), and THF (4).

Biotechnol. Prog., 1996, Vol. 12, No. 4 491

minimized any contribution of the support on a positivepartition effect of water to the microenvironment of theimmobilized enzyme.In order to summarize the influence of these five

aprotic water-miscible organic solvents on the stabilityof the immobilized R-chymotrypsin, Figure 5 depicts themaximum protective effect produced by the assayedsolvents as a function of both, their hydrophobicity,measured by the log P parameter, and the water contentof the immobilized derivative. As it can be seen, theincrease in solvent hydrophobicity reduced exponentiallythe protective effect of the solvent, which changed theiraction to a denaturative role, which was clearly shownfor the most hydrophobic cases (AC and THF). However,the water content of the immobilized derivative necessaryto obtain the maximum stability level increased from 1.5g of H2O/g of support to 2.18 g of H2O/g of support(corresponding to a 100% aqueous media) with theincrease in solvent hydrophobicity. These results in-volved that the molecules of hydrophilic solvents can beapproached or introduced into the environment of theenzyme, resulting in a more rigid conformation of theenzyme structure that increased its stability. So, theincrease in solvent hydrophilicity not only allows reduc-tion of the water content of the medium to have anefficient enzyme function (i.e. to carry out a peptidesynthesis process) (Lozano et al., 1995), but also allowsenhancement of the enzyme stability.

ConclusionThe deactivation process of R-chymotrypsin adsorbed

onto Celite support followed a two-step kinetic model withexcellent agreement, where the deactivation parameterswere highly influenced by presence of the aprotic water-miscible organic solvents. The experimental resultsclearly showed that the solvent hydrophobicity is a keyparameter in the enzyme stability, which changes in therole of the solvent in the enzyme activity, from aprotective action to a denaturative effect, when the logP was increased. Furthermore, the study of the role ofwater in the enzyme stability from both macro- andmicroenvironmental water parameter (Aw and watercontent of the immobilized derivative) clearly showedthat the stabilization or denaturation process of theenzyme per se does not depend of the amount of waterthat has been dispared from the microenvironment of theenzyme, but is mainly governed by the nature of theorganic cosolvent used.As a function of the overall results, the assayed aprotic

water-miscible cosolvents could be classified in agreement

with their efficiency as nonconventional media in thesame order of their increase in hydrophobicity, measuredby the log P parameter. These results clearly shows thatlog P as a good parameter to correlate the nature of theorganic media and their influence on enzyme action, aswell as the efficiency of hydrophilic cosolvents to modifyboth the macro- and microenvironments of enzymes toimprove their stability.

Notationa remaining fractional activity (dimensionless)AC acetoneAcN acetonitrileAw water activityDMF N,N-dimethylformamideDMSO dimethyl sulfoxideE initial enzyme activity (U‚mg-1)E1 active intermediate enzyme activity (U‚mg-1)Ed final enzyme activity (U‚mg-1)h Planck’s constant (J‚h)IME immobilized enzymek1 first-order deactivation rate constant for first

step (h-1)k2 first-order deactivation rate constante for second

step (h-1)kB Boltzmann’s constant (J‚K-1)R gas constant (J‚mol-1‚K-1)SE soluble enzymet time (min)t1/2 half-lifeT temperature (K)THF tetrahydrofuranR1 ratio of specific activities of the active intermedi-

ate to the initial enzyme formR2 ratio of specific activities of the final state to the

initial enzyme form∆G° standard free energy (kJ‚mol-1)

Acknowledgment

This work was partially supported by the “Consejerıade Cultura, Educacion and Turismo. Comunidad Au-tonoma de la Region de Murcia, ” Spain (Grant No. PTC93/27).

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Accepted February 8, 1996.X

BP960011P

X Abstract published in Advance ACS Abstracts, April 1, 1996.

Biotechnol. Prog., 1996, Vol. 12, No. 4 493