Enhancement of Enzyme Activity in Supercritical Carbon Dioxidevia Changes in Acid-Base Conditions

Neil Harper* and Susana Barreiros

REQUIMTE/CQFB, Departamento de Quımica, FCT, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal

Enzyme performance is often impaired in supercritical carbon dioxide. We were ableto enhance enzyme activity in this medium via changes in acid-base conditions byusing ion-exchange materials (solid H+/Na+ buffer pairs and a zeolite), which wereselected on the basis of the response of an organosoluble acid-base indicator. Theconcentration of ion-exchange materials had an important effect on the catalyticactivity of subtilisin Carlsberg cross-linked enzyme crystals (CLECs), and this wasrelated to the protonation and hydration states of the enzyme. The buffer Na2CO3/NaHCO3 gave the highest enhancement in enzyme activity (by a factor of 54), probablyas a result of its high basicity and capacity to counteract the deleterious effect ofcarbonic acid to a greater extent than the other materials tested.

Introduction

Supercritical fluids (sc-fluids) are an environmentallyfriendly alternative to organic solvents as media forbiocatalysis because traces of solvent can be completelyremoved after depressurization (1). Although carbondioxide is the most widely used sc-fluid, enzyme activityis often reduced in this medium (2-5). This has beenattributed to the formation of carbonic acid in thepresence of water, and carbamates by reaction withamine groups on the enzyme. When using cross-linkedenzyme crystals (CLECs) and immobilized enzyme wherebuffer salts are present in relatively low concentrations,protection against acid-base effects is much reduced. Thepresence of counterions must be taken into account inthe protonation/deprotonation of groups on the enzymein low-dielectric media such as sc-CO2 (6). For example,deprotonation of a carboxyl group requires that a cationsuch as Na+ be available to provide electroneutrality (i.e.,enzyme-COOH + Na+ S enzyme-COO-Na+ + H+).Therefore, materials that can exchange H+ and Na+ suchas solid buffer pairs (7, 8) or zeolite NaA (9) and set afixed exchange potential of those two cations can be usedto control the protonation state of acidic residues on theenzyme in situ. Appropriate choice of buffer or zeolite canlead to dramatic rate enhancements. The exchangepotential of H+ and Na+ can be measured with anorganosoluble chromoionophoric indicator (7). This indi-cator has been successfully used to detect changes inacid-base conditions in organic media due to the forma-tion of acidic or basic species and to identify potentialacid-base buffers for tuning the protonation state ofenzymes in those media. In the present study we reportthe use of ion-exchange species present as solids in sc-CO2 to alter enzyme activity via changes in acid-baseconditions. We attempt to correlate enzyme activity withthe exchange potential of H+ and Na+ controlled by suchspecies using subtilisin Carlsberg, whose catalytic activ-ity is sensitive to changes in its protonation state.

Materials and MethodsN-Acetyl-L-phenylalanine ethyl ester (Ac-Phe-OEt),

N-acetyl-L-phenylalanine (Ac-Phe), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO), N-tris(hy-droxymethyl)methyl-3-aminopropanesulfonic acid (TAPS),piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), 3-(N-morpholino) propanesulfonic acid (MOPS), and theirsodium salts (CAPSO.Na, TAPS.Na, PIPES.Na, MOP-S.Na) were from Sigma. ChiroCLEC-BL (subtilisin) wasfrom Altus Biologics Inc. (Cambridge, MA); 1-propanol(PrOH), toluene, sodium carbonate, sodium bicarbonate,potassium carbonate, and orthophosphoric acid were fromMerck; Hydranal Coulomat A and C Karl Fischer re-agents were from Riedel de Haen; zeolite NaA (molecularsieves 4 Å, powder) was from Aldrich; and acetonitrilewas from Panreac. The molecular sieves were precondi-tioned at 300 °C overnight prior to use. PrOH was storedover molecular sieves 3 Å (Merck), and all other solventswere used as supplied. CO2, ethane, and nitrogen weresupplied by Air Liquide and guaranteed to have puritiesof over 99.95 mol %. The CO2/ethane mixtures wereprepared by weighing appropriate amounts of the twosubstances and mixing under pressure (4). The indicator(protonated) was synthesized according to the methodgiven by Harper et al. (7).

Indicator equilibrations in sc-fluids were done in avariable volume cell with two compartments separatedby filters: one where the solids and reagents are addedand another with two sapphire windows and a path of37 mm in length. Mixing in the two compartments wasachieved by means of magnetic stirring bars. Heating wasprovided by electrical resistances inserted into the bodyof the cell. Information on the operation of our variablevolume cells is provided in ref 11. The experimentsperformed are summarized in Table 1. The solids (bufferpair or zeolite) were added to the dry cell, followed by an8 mM solution of the protonated indicator in PrOH andmore PrOH to give the concentrations in the table. Thecell was then filled with the sc-fluid at 100 bar and 40°C, and both compartments of the cell were agitated. Thecell was transferred to the UV-visible spectrophotometerand spectra were measured periodically (blanked against

* To whom correspondence should be addressed. Ph: 351 212949681. Fax: 351 21 2948385. Email: nharper_pt@yahoo.co.uk.

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10.1021/bp025602w CCC: $22.00 © 2002 American Chemical Society and American Institute of Chemical EngineersPublished on Web 10/11/2002

air) until equilibrium was attained, i.e., the deprotonatedfraction remained constant (at least 2 h; only a fewminutes in CO2 in the absence of ion-exchange solids).The spectra obtained were similar to those in ref 7. Theconcentrations of the two forms of the indicator werecalculated from the absorbances at the λmax for the twopeaks, taking into account the spectral overlap (7). Sinceit was not possible to obtain 100% of deprotonatedindicator in the sc-fluids used, for both forms of theindicator the absorption coefficients obtained in toluene/1M PrOH were used (7). The absorbance of the solventwas subtracted as the background. The λmax for theprotonated indicator were 360 nm in ethane and in CO2/ethane mixtures and 356 nm in sc-CO2. The λmax for thedeprotonated indicator were 444 nm in ethane and 434nm in CO2/ethane mixtures. In the CO2/ethane mixture,the error for the deprotonated fraction obtained in thepresence of a total concentration of 46.3 g L-1 of Na2CO3/NaHCO3 buffer was 1.9%; for 9.3 g L-1, it was 36%. Intoluene/1 M PrOH, the solids were added to a solutionof the protonated indicator in glass vials at 25 °C(equilibration ca. 2 h).

We studied the transesterification of Ac-Phe-OEt(5 mM) with PrOH (200 mM) in sc-CO2 at 100 bar and40 °C. In all experiments, 11.6 µL of subtilisin CLECsuspension (0.64 mg of enzyme) was washed four timeswith anhydrous PrOH (10) and the solvent was removedby centrifugation. The enzyme was then suspended in171 µL of PrOH and added to the cell (0.046 g L-1). Afterintroduction of CO2, the system was allowed to equili-brate for at least 1 h, after which the reaction wasinitiated by the addition of Ac-Phe-OEt dissolved inPrOH. Reactions were carried out in our standard cellsfor enzymatic reaction (11) and were followed by HPLC(RP-18 HiChrom column, mobile phase 50% water, 50%acetonitrile at pH 3 by addition of orthophosphoric acid,UV detection at 220 nm). The reaction rate data (calcu-lated from the production of Ac-Phe-OPr) are the averageof at least two measurements, except for experimentswith zeolite NaA. The water concentration was measuredat equilibrium in experiments with the indicator and atthe beginning and end of reactions by Karl Fischertitration (error ca. 9%). Water activity (aw) was calculated(in all sc-fluids) by dividing the measured water concen-

tration by the water concentration at saturation (9). UV-visible spectra were measured using a diode arrayHewlett-Packard HP8452A spectrophotometer.

Results and Discussion

The acid-base indicator dissolved in sc-CO2 was usedto give direct evidence for the acidic nature of themedium. When the indicator was added initially in thedeprotonated form, it was found to become completelyprotonated upon addition of sc-CO2 (Table 1). To over-come the acidifying effect of CO2, we used basic solidadditives, which were selected on the basis of theresponse of the indicator in an organic solvent. As shownin the table, at equilibrium and in the presence of theNa2CO3/NaHCO3 buffer and zeolite NaA, high fractionsof deprotonated indicator were obtained in toluene, whichindicated a highly basic medium. However, neither ofthese solids was able to deprotonate the indicator. Asseen in the table, some of these experiments were carriedout at undetectable levels of water, but even in suchconditions the acidity of sc-CO2 was too high to depro-tonate the indicator.

Ethane, on the other hand, has no acid-base effects.Experiments carried out in this solvent showed that, inthe presence of solid buffers of varying aqueous pKa, awide range of indicator response could be obtained (Table1). We thus decided to use a CO2/ethane mixture to carryout the indicator equilibration under conditions otherwiseidentical to the equilibrations in sc-CO2. We found thatthe mole fraction of CO2 had to be reduced to 0.02 in orderto differentiate the indicator response at different levelsof the very basic Na2CO3/NaHCO3 buffer (Table 1).Provided that the capacity of the solid buffer were notexceeded, its amount should not affect the deprotonatedfraction at equilibrium. Therefore, our results indicatethat even at essentially zero aw and for a mole fractionof CO2 of only 2%, the capacity of the Na2CO3/NaHCO3pair was exceeded (12). Nonetheless, we have shown thatthis buffer was able to partially counteract the effect ofcarbonic acid.

To study the impact of the acid-base conditions of themedium on enzyme activity, we carried out transesteri-fication reactions in sc-CO2 in the presence of varying

Table 1. Summary of Indicator Response and aw in the Presence of Ion-Exchanging Materials in Different Media

medium additiveb% deprotonated

indicator at equilibriumc aw

CO2/200 mM PrOH none 0TAPS/TAPS.Na, 0-66 g L-1 0.05zeolite NaA, 2.3 g L-1 0 0.02zeolite NaA, 18.6 g L-1 0 0Na2CO3/NaHCO3, 9.3 g L-1 0 0.02Na2CO3/NaHCO3, 66 g L-1 0

ethane/412 mM PrOHa CAPSO/CAPSO.Na 75PIPES/PIPES.Na 7

(0.02 CO2 + 0.98 ethane)/200 mM PrOH Na2CO3/NaHCO3, 9.3 g L-1 6 0Na2CO3/NaHCO3, 46.3 g L-1 16 0

toluene/1 M PrOH Na2CO3/NaHCO3 92zeolite NaA 90TAPS/TAPS.Na 85MOPS/MOPS.Na + zeolite NaA 25MOPS/MOPS.Na 26

a PrOH concentrations above 300 mM were required for the indicator to be sufficiently soluble in order to give accurate absorbancemeasurements. b Equal amounts of each form of the solid buffer pair were used. In ethane and in toluene, the total buffer concentrationswere 12 and 50 g L-1, respectively. The zeolite concentration was 25 g L-1 in toluene. Buffer aqueous pKa (at 25 °C): 10.3 (Na2CO3/NaHCO3), 9.6 (CAPSO/CAPSO.Na), 8.4 (TAPS/TAPS.Na), 7.2 (MOPS/MOPS.Na), 6.8 (PIPES/PIPES.Na). c The indicator was added tothe cell to give 16.5 µM in ethane and in CO2 in the absence of ion-exchange solids, 40 µM in CO2 at all other conditions as well as inCO2/ethane mixtures, 50 µM in toluene. These values exceed the solubility of the indicator in the media, except in the case of toluene. InCO2 in the absence of ion-exchange solids, the indicator was introduced in the cell completely deprotonated (by adding crystals of anhydrousNa2CO3 to a 1.33 mM indicator solution in PrOH).

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amounts of Na2CO3/NaHCO3. As seen in Figure 1, theamount of solid buffer had a marked effect on enzymeactivity (13). Subtilisin activity first increased up to 54-fold relative to the blank (no added buffer) and thendecreased. It is known that subtilisin is more activeunder basic conditions where the catalytic triad in theactive site is deprotonated. Therefore, at optimum en-zyme activity the enzyme molecules will be in thisdeprotonated state. When the basicity is increased fur-ther, the medium may become so basic that ionogenicgroups that require to be protonated for enzyme activitydeprotonate as well. This may explain why higher Na2-CO3/NaHCO3 concentrations led to a drop in enzymeactivity. Again, if the salt pair were acting as a buffer,the amount of solid used should have no effect on theacid-base conditions. As the table shows, the amount ofavailable water decreased very slightly with increasingbuffer (14). This might also contribute to the reductionin enzyme activity at higher buffer concentrations (15).Further, it is known that the formation of carbamateson the enzyme is favored by low aw and high basicity (3).

In a recent paper (9), we rationalized the enhancementof the activity of subtilisin Carlsberg in terms of theability of zeolite NaA to exchange H+ on the enzyme forNa+. When zeolite NaA was added to our system, an 11-fold enhancement in enzyme activity was observed rela-tive to the blank (Figure 1). At higher zeolite concentra-tions, enzyme activity was reduced to the same order asthe blank. As with Na2CO3/NaHCO3, aw decreased slightlywith increasing amounts of zeolite, so excessive dryingof the enzyme may have contributed to a decline incatalytic activity. However, even at constant aw, Fonteset al. (9) observed that increasing amounts of zeolite couldhave a negative impact on enzyme activity. Zeolite NaAand Na2CO3/NaHCO3 have similar basicities, as shownby the indicator response in toluene (Table 1). The lowerreaction rates measured in the presence of the zeolitemay be due to its reduced buffering capacity comparedto that of solid-state H+/Na+ buffers such as Na2CO3/NaHCO3 (9). To present further evidence for this phe-nomenon, we equilibrated the indicator with the bufferMOPS/MOPS.Na and with equal amounts of MOPS/MOPS.Na and zeolite NaA in toluene. The MOPS buffergave a significantly different indicator response relativeto the zeolite. However, when MOPS buffer and thezeolite were used together, the response of the indicatorwas the same as with MOPS buffer alone, demonstratingthat the acid-base conditions are determined by the solidbuffer. By analogy, Na2CO3/NaHCO3 should be more

effective in buffering acid-base conditions than thezeolite NaA.

To further examine the effects of ion-exchange materi-als in sc-CO2, we used the TAPS/TAPS.Na buffer, whichis less basic than Na2CO3/NaHCO3, as shown by theindicator response in toluene. TAPS buffer gave rateenhancements up to 13-fold relative to blank (Figure 1).This suggests that TAPS buffer is able to counteract someof the acidity of the medium, but less effectively than themore basic Na2CO3/NaHCO3. However, in contrast towhat is observed with both Na2CO3/NaHCO3 and zeolite,we see that increasing amounts of TAPS buffer cause asteady increase in enzyme activity. This may be ex-plained by the fact that this buffer absorbed little waterunder the hydration conditions at which reactions werecarried out. This is reflected in the higher aw valuesachieved that remained fairly constant independently ofthe amount of buffer (Table 1). On the other hand, ahigher aw favors the formation of carbonic acid, whichmay have contributed to the lower reaction rates in thepresence of TAPS buffer relative to the Na2CO3/NaHCO3,at lower buffer concentrations.

In conclusion, both the solid buffer pairs tested andzeolite NaA were able to enhance the catalytic activityof subtilisin Carlsberg CLECs. It was found that Na2-CO3/NaHCO3 was a particularly good candidate foreffecting changes in acid-base conditions in sc-CO2. Itssuitability is probably related to its high basicity andcapacity to counteract the deleterious effect of carbonicacid to a practically useful extent. Although a wide rangeof enzyme activity response was obtained with the ion-exchange materials used, the indicator was unable todiscriminate the changes in acid-basic conditions imposedby those materials in sc-CO2. An indicator more acidicin sc-CO2 would be very useful. Also, elucidation of acid-base effects in CO2 would be facilitated by using fixedaw. This would require the use of salt hydrates in situthat do not have any acid-base effects, unlike those mostcommonly used (Fontes et al., unpublished results).

Acknowledgment

This work has been supported by Fundacao para aCiencia e Tecnologia (FCT, Portugal) through the con-tract POCTI/35429/QUI/2000 and the TMR network“Super-Clean Chemistry 2” (research contract no. ERBFM-RXCT970104). We would like to thank Dr. Mark Dolmanfor the gift of the indicator.

References and Notes(1) Mesiano, A. J.; Beckman, E. J.; Russell, A. J. Supercritical

biocatalysis. Chem. Rev. 1999, 99, 623-633.(2) Marty, A.; Chulalaksananukul, W.; Willemot, R. M.; Con-

doret, J. S. Kinetics of lipase-catalyzed esterification insupercritical CO2. Biotechnol. Bioeng. 1992, 39, 273-280.

(3) Kamat, S. V.; Beckman, E. J.; Russell, A. J. Enzyme activityin supercritical fluids. Crit. Rev. Biotechnol. 1995, 15, 41-71.

(4) Borges de Carvalho, I.; Correa de Sampaio, T.; Barreiros,S. Solvent effects on the catalytic activity of subtilisinsuspended in compressed gases. Biotechnol. Bioeng. 1996, 49,399-404.

(5) Fontes, N.; Almeida, M. C.; Garcia, S.; Peres, C.; Partridge,J.; Halling, P. J.; Barreiros, S. Supercritical fluids are superiormedia for catalysis by cross-linked enzyme microcrystals ofsubtilisin Carlsberg. Biotechnol. Prog. 2001, 17, 355-358.

(6) Halling, P. J. Biocatalysis in low-water media: understand-ing effects of reaction conditions. Curr. Opin. Chem. Biol.2000, 4, 74-80.

(7) Harper, N.; Dolman, M.; Moore, B. D.; Halling, P. J. Acid-base control for biocatalysis in organic media: New solid-state

Figure 1. Initial rate of transesterification as a function ofconcentration of ion-exchange material in sc-CO2, at 40 °C and100 bar: (]) Na2CO3/NaHCO3 buffer; (0) zeolite NaA; (4) TAPSbuffer. aw was always below 0.08.

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proton/cation buffers and an indicator. Chem. Eur. J. 2000,6, 1923-1929.

(8) Partridge, J.; Halling, P. J.; Moore, B. D. Solid-state proton/sodium buffers: “Chemical pH stats” for biocatalysts inorganic solvents. J. Chem. Soc., Perkin Trans. 2 2000, 465-471.

(9) Fontes, N.; Partridge, J.; Halling, P. J.; Barreiros, S. Zeolitemolecular sieves have dramatic acid-base effects on enzymesin nonaqueous media. Biotechnol. Bioeng. 2002, 77, 296-305.

(10) Partridge, J.; Hutcheon, G. A.; Moore, B. D.; Halling, P. J.Exploiting hydration hysteresis for high activity of cross-linked subtilisin crystals in acetonitrile. J. Am. Chem. Soc.1996, 118, 12873-12877.

(11) Fontes, N.; Almeida, M. C.; Barreiros, S. Biotransforma-tions in supercritical fluids. In Enzymes in NonaqueousSolvents, Methods in Biotechnology Series; Vulfson, E. N.,Halling, P. J., Holland, H. L., Eds.; The Humana Press: NewJersey, 2001; Vol. 15, pp 565-573.

(12) Although none of the buffers act as such in sc-CO2, we keepthe term buffer for convenience.

(13) Because of the low aw at which transesterification wascarried out, it was not possible to detect Ac-Phe as a resultof hydrolysis of the substrate ester. Thus, the buffers usedonly had to counteract the effect of carbonic acid.

(14) Both Na2CO3 and NaHCO3 have an affinity for water.(15) Affleck, R.; Xu, Z. F.; Suzawa, V.; Focht, K.; Clark, D. S.;

Dordick, J. S. Enzymatic catalysis and dynamics in low-waterenvironments. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 1100-1104.

Accepted for publication August 30, 2002.

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