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REVIEWS Designing Enzymes for Use in Organic Solvents Jonathan S. Dordick Department of Chemical and Biochemical Engineering and Center for Biocatalysis and Bioprocessing, University of Iowa, Iowa City, Iowa 52242

Enzymes are routinely used in organic solvents where numerous reactions of interest to synthetic and polymer chemists can be performed with high selectivity. Recently, it has become apparent that the catalytic properties of an enzyme can be tailored to a specific catalytic requirement by the use of solvent and protein engineering. The former involves altering the polarity, hydrophobicity, water content, etc., of the organic milieu, while the later applies site-directed mutagenesis to alter the physicochemical properties of the biocatalyst. The dominant effects of organic solvents on enzyme structure and function, and the potential of solvent and protein engineering to design enzymes to function optimally in organic media, are the major foci of this review.

Contents Introduction Dominant Effects of Solvents on Enzymes Kinetic and Thermodynamic Considerations Control of Selectivity by Organic Solvents Enzyme-Bound Water Structural Rigidity Approaches To Improve Enzyme Function in Organic Solvents Solvent Engineering Protein Engineering Conclusions

259 259 260 262 262 263 264 264 265 266

Introduction Enzymes occupy a unique position in synthetic chemistry because of their exquisite selectivities and fast catalytic rates under ambient reaction conditions. Nevertheless, synthetic chemists have been reluctant to employ enzymes as reagents in organic synthesis, although there is a need for high selectivity in synthetic chemistry, especially in the synthesis of pharmaceuticals, chiral intermediates, specialty polymers, and biochemicals. The most significant reason for this reluctance has been the strict adherence to the conventional notion that enzymes function only in aqueous solutions. Recently, however, it has become clear that enzymes can function in organic as opposed to just aqueous media (Dordick, 1991; Klibanov, 1990;Laane et al., 1987). The astonishing realization that enzymes can retain, and in some cases improve, their high degree of reaction specificity in a nearly anhydrous milieu has dramatically improved the propsect of employing enzymes in synthetic chemistry. From a biotechnological standpoint, there are numerous advantages to employing enzymes in organicmedia (Dordick, 1991;Klibanov, 1986). A brief summary is listed in Table I.

While such advantages have been realized, there remain differing views as to the efficacy of nonaqueous enzymology. For example, the seemingly simple question-What happens when an enzyme is placed in an organic solvent?-is met with vastly different answers. To the applied enzymologist or chemical engineer, enzymes are viewed as vigorously active catalysts in organic solvents. I t is not uncommon for catalytic rate enhancements of several billion-fold to be observed in organic media (Zaks and Klibanov, 1988a). Numerous reactions of interest that are nearly impossible to perform in aqueous solutions become feasible in nonaqueous media. To the biochemist, however, a typical answer may be that nothing happens. The biochemist is apt to view this question in terms of the catalytic efficiency of an enzyme (k,dK,) in organic as compared to in aqueous media. While it is well-known that enzymes do function in organic solvents, in general their catalytic efficiencies are 2-6 orders of magnitude lower than in aqueous solutions. Herein lies the paradox of nonaqueous enzymology-that commercially relevant syntheses can be performed, yet with enzymes that are only poorly active when compared to their "native" activities in water. The goal of this review is to provide a treatise on the dominant factors that govern enzyme structure and function in nonaqueous media and to offer suggestions to improve enzyme function in such environments. The studies described in this review come from both our laboratory and others. We will deal primarilywith enzyme activity and its improvement in organic solvents and not improvement of enzymic stability. This latter topic, while also vital for the commercial application of enzymes in nonaqueous media, has been reviewed in depth by Arnold (1988, 1990).

Dominant Effects of Solvents on Enzymes The consequences of placing an enzyme in nonaqueous media can be dramatic. Organic solvents can alter the secondary, tertiary, and, for multisubunit enzymes, quaternary structures of enzymes (Singer, 1962),can penetrate into the active site of an enzyme and disrupt the fine

8756-7938/92/3008-0259$03.00/0 0 1992 American Chemical Society and American Institute of Chemical Engineers

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100% buffer 70% dioxane 0 80%dioxane 959hdioxane 0

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Jonathan Dordick is an Associate Professor in the Department of Chemical and Biochemical Engineering a t the University of Iowa where he is also the Associate Director of the Center for Biocatalysis and Bioprocessing. His research interests include enzymatic catalysis in unconventional media; biological synthesis of novel polymers, including phenolic resins, carbohydrate-containing polyesters, polyacrylates, polyurethanes, etc., and chiral macromolecules; enzymes in organic synthesis; and the development of biorecognition strategies for bioseparations. He received a B.A. degree in Biochemistry and Chemistry from Brandeis University and a Ph.D. degree in Biochemical Engineering from MIT. After a year in England with Tate and Lyle Group Research and Development, he joined the faculty a t the University of Iowa in 1987.

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2

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butyl acetate ( 1 %)

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0 -I -2 -0.5

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1.0

1.5

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Figure 1. Peroxidase catalysis in nonaqueous media (both waterTable I. Potential Advantages of Employing Enzymes in Monophasic Organic Solvents (Dordick, 1991; Klibanov, 1986) 1 2

3 4

5 6 7 8 9 10

increased solubility of nonpolar substrates shifting thermodynamic equilibria to favor synthesis over hydrolysis suppression of water-dependent side-reactions alteration in substrate and enantioselectivity immobilization is often unnecessary because enzymes are insoluble in organic solvents enzymes can be recovered by simple filtration ease of product recovery from low boiling, high vapor pressure solvents enhanced thermostability elimination of microbial contamination potential for enzymes to be used directly within a new or existing chemical process

chemical and structural balance that promotes highly selective and efficient catalysis (Herskovits, 1965), and can change the thermodynamic activities of enzyme, substrate, and product from that in purely aqueous solutions (Ryu and Dordick, 1992). The native secondary and tertiary structures of enzymes are maintained by the interaction of several noncovalent forces, including hydrogen bonding, and ionic, hydrophobic,and van der Waals interactions (Tanford, 1961;Schultz and Schirmer, 1979). Disruption of these forces by a solvent less polar than water can lead to diminished substrate binding and catalytic turnover. Kinetic and Thermodynamic Considerations. The most obvious changes in solvent properties in going from water to an organic solvent are the increase in solvent hydrophobicity, decrease in solvent dielectric, and significant decrease in the water content of the medium. All three changes will affect enzymatic catalysis as both the substrate specificity and catalytic efficiencyof an enzyme initially depend on the ability of the enzyme to utilize the free energy of binding with the substrate (Kraut, 1988). This bindingenergy reflects the differencebetween binding energies of substrate-enzyme and substrate-solvent interactions (Fersht, 1985). Kinetic parameters describing enzyme function, such as binding constant with substrate,

miscible and immiscible). For the alkyl acetate solvents, the numbers in parentheses indicate the percentage (v/v) of aqueous buffer added. Substrates (in increasing degree of hydrophobicity) were p-methoxyphenol, p-cresol, p-ethylphenol, p-propylphenol, and p-tert-butylphenol. Data were from Ryu and Dordick (1989).

Kg, Michaelis constant, K m , and catalytic turnover, kat or Vmax, therefore, depend strongly upon the reaction medium. It may be expected that the replacement of water with an organic solvent would lead to profound changes in the observed kinetics of enzymatic catalysis. Peroxidase catalysis represents a vivid example of this phenomenon. In organic media, peroxidase catalysis is dependent on both solvent and substrate hydrophobicities (Ryu and Dordick, 1989). Figure 1 depicts the dependence of catalytic efficiency of peroxidase on substrate hydrophobicity, T , in aqueous buffer and several organic solvents. Peroxidase is up to 4 orders of magnitude less efficient in organic media as compared to in aqueous solution. This substrate effect becomes more pronounced as the solvent hydrophobicity increases. [The slopes of peroxidase activity vs T decrease as solvent hydrophobicity increases.] These findings are consistent with the partitioning behavior of hydrophobic phenols between the bulk reaction medium and the peroxidase’sactive site. This partitioning is likely to diminish as both substrate and solvent hydrophobicities increase, thereby necessitating a larger concentration of phenols to saturate the enzyme and resulting in a higher apparent substrate K m . Catalytic efficiency is compromised due to diminished enzyme-substrate interaction. But, is the solvent really affecting the enzyme directly to cause the drop in K m ? To answer this question, it must be noted that the observed phenomenon of partitioning can be thermodynamically described as either stabilization of the substrate ground state or destabilization of the enzyme-substrate complex (Figure 2). The distinction is significant. Ground-state stabilization of the substrate is independent of the intrinsic catalytic capabilities of the enzyme, whereas destabilization of the enzyme-substrate complex strongly suggests alteration in active-site chemistry and structure. Using

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261 ES+(transition state) +tr

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ES complex

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Figure 2. Free energy diagrams for transfer of reaction from water to organic solvent: (A) Hypothetical case where the enzymesubstrate complex (ES) is destabilized with only minor stabilization of the substrate ground state; (B)Hypothetical case where the ES complex is only slightly destabilized but the substrate ground state is significantly stabilized. For ground-state stabilization, the value of V J K , decreases (free energy difference between the ground state and transition state increases). Transfer free energies are described in the text and in Figure 3.

a transfer free energy method, it is possible to elucidate solvent effects on the free energies of the ground state and enzymeaubstrate complex, independently (Ryu and Dordick, 1992). Figure 3 depicts a simplified, yet general, kinetic and thermodynamic model for enzymatic catalysis in both water and organic solvents. The transfer free energy for substrate (from an aqueous environment to an organic solvent) can easily be calculated from thermodynamic relationships (Abraham, 1974) using the ratio of activity coefficients in a solvent relative to water (yss/ysw) where the superscripts s and w represent organic solvent and water, respectively, as shown in the following equation (note, this is the transfer free energy of the substrate from water to organic solvent and, hence, the positive sign in the expression): AG: = R T In (ysS/ysw) A similar expression for the enzyme is shown in

(1)

AGEtr = R T In ( y E 8 / y E W ) (2) Substrate binding can also be represented in free energy

InWatcr:

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Figure 3. Transfer free energy thermodynamic and kinetic model. See text for definitions of variables. terms using Figure 3 and eq 3, where AGbtr represents the free energy of transfer between water and an organic solvent for substrate binding. AG;

= RT In (K,~IK,") = AG~: - AG;

- AG;

(3)

Thus, in order to calculate the transfer free energy of the enzyme-substrate complex, eq 3 can be rearranged along

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262

2o

with enzymes, and catalytic deactivation may Iunfavorably often be the result of altered thermodynamics of enzyme

-20 -0.5

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2.5

A

Figure 4. Dependence of transfer free energy between water and 70% dioxane on substrate hydrophobicity. Symbols: ( A ) binding step, eq 3; (0) substrate ground state, eq 1;( 0 )enzymesubstrate complex, eq 4 (enzyme term is cancelled out). Note the invariance of ES complex to substrate hydrophobicity. Reprinted with permission from Ryu and Dordick (1992).

Copyright 1992 American Chemical Society. with eqs 1 and 2 to give

All values can now be calculated with the exception of the activity coefficients for the enzyme in a given solvent (aqueous or organic). However, if we use a series of substrates with systematic structural variations, effects of changes in substrate hydrophobicity on the transfer free energies of the enzyme-substrate complex can be calculated in a single given solvent without knowing the values of enzyme activity coefficients (the thermodynamic state of the enzyme is dependent on the solvent and not the substrate)-the term RT In (Y$/YE") cancels out of eq 4. Figure 4 shows an example of the dependence of substrate ground state on substrate hydrophobicity in an organic solvent (in this case 70% viv dioxane). The resulting transfer free energies of the enzyme-substrate complex were invariant to substrate hydrophobicity. Similar results were obtained in avariety of other solvents including up to 80% viv dioxane, 90% vlv methanol, and 80% viv acetonitrile (data not shown), suggesting that peroxidase maintains its native intrinsic ability to interact with substrate in these solvents. Ground-state stabilization, however, weakened the effective interaction between the enzyme and substrate. Importantly, in solvents that do not destabilize the enzyme-substrate complex, thep value of peroxidase catalysis (the Hammett constant which reflects the solvent effect on the transition state of the reaction) is unaffected by the solvent (Ryu and Dordick, 1992). Hence, these solvents do not alter the native active-site structure and chemistry of peroxidase. This is fundamentally similar to subtilisin catalysis in organic solvents wherein p values were independent of the solvent and indistinguishable from that in water (Kanerva and Klibanov, 1989). This is also similar to catalysis by a-lytic protease where 15Nmagic angle spinning NMR has shown no significant differences in active-site structure between aqueous solutions and organic solvents that support catalysis (e.g., octane or acetone) (Burke et al., 1989). However, DMSO, a solvent that cannot support catalysis, induces significant changes in active-site structure. Organic solvents, then, do not always interact

and substrate in organic solvents. In addition to stabilizationidestabilizationof the substrate due to the solvent, similar thermodynamic relationships hold for the product of an enzymatic reaction. Highly polar compounds may partition so strongly into an enzyme's active site from an organic solvent that they may become nearly irreversibly bound. The product, if also polar, may then be unable to partition from the active site into the bulk reaction medium. The result is apparent inactivation of the enzyme in organic solvents. A prime example of this is catalysis by glycosidases. Kieboom (1988) has shown that several glycosidases, while highly active in water for both hydrolysis of glycosides and transglycosylation reactions, are nearly inactive in organic media. Control of Selectivity by Organic Solvents. The effect of solvent on the ground state of the substrate may explain the alteration in catalytic specificity of other enzymes including subtilisin, chymotrypsin, and pig liver carboxylesterase in hydrophobic solvents as compared to in water (Zaks and Klibanov, 1986). For example, in aqueous solutions, the ground state of polar amino acid derivatives (e.g., esters of serine and histidine) are stabilized while that of nonpolar amino acid derivatives (e.g., esters of phenylalanine or tyrosine) are destabilized. Nonpolar amino acid esters, then, favorably partitioning into these enzymes' nonpolar active sites in aqueous buffer thereby helping to drive the reaction-enzymatic hydrolysis of a phenylalanine derivative in water is more than 4 orders of magnitude more efficient than that of a serine derivative. In octane, however, polar amino acid esters are destabilized and favorably partition into the active sites (presumably which are now more polar than the reaction medium), whereas nonpolar amino acid esters are stabilized in the hydrophobic medium. Hence, polar amino acid esters become better substrates than nonpolar esters in octane. Solvent hydrophobicity is also known to control the enantioselectivity of proteases in the transesterification of N-Ac-Ala-chloroethyl ester with n-propanol (Sakurai et al., 1988),and lipases in the esterification of 2-hydroxy acids with n-butanol (Parida and Dordick, 1991). In each case, the favored L-isomers become less reactive relative to the unfavored D-isomers as solvent hydrophobicity increases. This can be explained, in part, by a thermodynamic argument involving the mechanism of catalysis by serine protease-type enzymes. A major event in catalysis by these enzymes is the release of water from the binding site of the enzyme to the reaction medium upon substrate binding (Fersht,1985). For example, in aqueous solutions, L-isomers bind properly to the binding site of the enzyme and cause the release of this water which entropically drives the reaction. In hydrophobic organic solvents, water release into the medium is energetically unfavorable and the reactivity of the L-isomersis relatively low. In contrast, in order for D-isomersto react, they must bind differently to the enzyme than L-isomers, and this results in the release of less binding-site water. Hence, D-isomersface a less unfavorable thermodynamic barrier. Enzymic structural rigidity also affects enantioselectivity, primarily through steric considerations, and this is discussed in greater detail below. Enzyme-BoundWater. Several studies indicate that enzyme structure and function is strongly dependent on bound water (Gorman and Dordick, 1992; Zaks and Klibanov, 1988b). This is not surprising as water is involved

Blotechnd. Prog., 1992, Vol. 8, No. 4

in all noncovalent forces that hold a protein molecule together. Loss of water from an enzyme should pose dire consequences for maintenance of native enzyme structure and function. This problem becomes particularly severe in polar organic solvents that appear to strip water off an enzyme and thereby dehydrate the catalyst so that activity is destroyed. To examine the magnitude of such a phenomenon, we have directly investigated the loss of water from enzymes when placed in dry organic solvents (Gorman and Dordick, 1992). Using tritiated water bound to chymotrypsin, subtilisin, and horseradish peroxidase, the rates and extents of T2O desorption were measured as a function of the polarity and water-solubilizing character of the organic solvent. In the most polar solvents [e.g., methanol, dimethylformamide (DMF), and tetrahydrofuran (THF)], T20 desorption was nearly immediate and resulted in the highest degree of desorption (e.g., methanol desorbed from 56 to 62% of the bound T2O) while nonpolar solvents resulted in the lowest degree of desorption (e.g., hexane desorbed from 0.4 to 2% of the bound T2O). The solvent polarity, rather than hydrophobicity, appears to govern the water-stripping off of an enzymemolecule. This is easily explained as follows: water is bound to polar and charged residues of proteins through mainly electrostatic forces. Increased solvent polarity weakens these electrostatic forces and enables water to desorb off of enzymesinto the bulk solvents. Hence, waterstripping from an enzyme into a nonaqueous medium does occur and can be significant in polar solvents. Such an analysis, however, provides only a blanket statement that water is necessary for catalysis, yet it does not give us hints as to the ultimate role of water in enzyme structure and function, particularly in the enzymic active site. To investigate the role of water at the molecular level of an enzyme’s active site, we, along with Clark and co-workers at University of California at Berkeley, have studied subtilisin Carlsberg in THF with different levels of hydration (Affleck et al., 1992). Addition of only 1% v/v water to subtilisin in THF improves the catalytic efficiency of transesterification over 6-fold (Figure 5).This increase in activity is accompanied by an increase in activesite flexibility of the active site [as determined by a 10fold decrease in the rotational correlation time of a Ser221bound nitroxide spin label using saturation transfer electron paramagnetic resonance (data not shown)] as well as an increase in the local polarity of the active site (Figure 5). The greater flexibility with increasing water content is consistent with the hypothesis that protein-bound water molecules screen electrostatic interactions between polar residues of the protein and hence induce greater freedom of motion of the active site (Bone and Pethig, 1985; Bone, 1987). This, in turn, leads directly to increased catalytic activity as the interactions between enzyme, substrate, and transition state become less constrained. Structural Rigidity. The rigidity of an enzymic active site also affects substrate specificity. For example, subtilisins Carlsberg and BPN’ show high degrees of enantioselectivity in nonpolar (for the most part hydrophobic) solvents for the transesterification reaction between bulky secondary alcohols and vinyl butyrate (Fitzpatrick and Klibanov, 1991). This phenomenon may be explained by a rather simple hypothesis-namely, that enzymes in nonpolar solvents are rigid. This rigidity is a result of stronger internal electrostatic interactions and hydrogen bonding in an enzyme suspended in a nonpolar (e.g., nonsolvating) solvent. This rigidity can control not only the accessibility of bulky substrates into the active site but also the positioning of such substrates within the active site.

263

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/

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Figure 5. Effect of added water to subtilisin Carlsberg in tetrahydrofuran. (A) Catalytic efficiency of subtilisin using N-AcL-Phe ethyl ester and 1 M n-propanol as substrates in a transesterificationreaction. The drop in catalytic efficiency above 7.5rLImL is speculated to be due to an inactivation of the enzyme at higher water contents in THF, perhaps due to nonspecific denaturation. (B) Effect of water on active-site polarity of a Ser22l-boundnitroxide spin label (44 (ethoxyfluorophosphiny1)oxy)-TEMPO). The polarity of subtilisin’sactive site is indicated by the hyperfine splitting constant A0 of the active-site bound spin label. Reprinted with permission from Affleck et al. (1992). Copyright 1992 National Academy of Sciences.

Consider an active site of an enzyme with two binding pockets: one small and the other large. In a rigid state, the bulky moiety of a secondary alcohol must fit into the large binding pocket of the active site in order to undergo catalysis. Few conformational degrees of freedom exist. As the solvent becomes more polar and capable of hydrogen bonding, the strength of electrostatic and hydrogen bonding interactions decreases, and the enzyme becomes more flexible and will accommodate multiple orientations of the secondary alcohol in the active site, and thus, enantioselectivity is decreased. Consistent with this hypothesis is the finding that addition of water or other hydrogen bonding solvents (e.g., formamide or ethylene glycol) to a nonpolar solvent such as dioxane causes a dramatic relaxation in enzymic enantioselectivity (Fitzpatrick and Klibanov, 1991). The rigidity of enzymes in low-water environments is directly responsible for the activating effect of active-site ligands (inhibitors) during lyophilization, a phenomenon known as “ligand memory” (Russell and Klibanov, 1988). The ligand binds to the active site of an enzyme, makes

264

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Figure 6. Active-site structure for subtilisin BPN’. The S1and SI’sites are to the right and left, respectively, of the active-site serine. Gly166 occupies the back of the SI site, Tyr217 occupies - the back of the SI’ site. For N-Ac-L-Phe ethyl ester, the Phe occupies the Slsite and the ethyl group occupies the S I ’ site.

a structural imprint, and is washed away from the enzyme prior to addition of substrate. The molecular imprint of the active-site ligand remains and provides for a “frozen” conformationthat readily accepts the substrate’s structure leading to enhanced activity on the given substrate as well as altered substrate specificities. For example, Russell and Klibanov (1988) altered the specificity of subtilisin toward more polar amino acid esters. Similarly, Mosbach and co-workers found that by precipitating chymotrypsin with n-propanol in the presence of N-acetyl-D-tryptophan (chymotrypsinis normally highly selectiveto the L-isomer) the enzyme could catalyze the esterification of N-acetylD-tryptophan ethyl ester in cyclohexane (Stahlet al., 1990). Enzyme precipitation in the absence of the ligand or in the presence of the L-isomer did not result in esterification of the D-isomer. The aforementioned examples suggest that enzymatic catalysis in organic solvents is possible if the active-site structure is not substantially perturbed by the organic solvent and if enough water is present to promote a critical level of active-site flexibility. The activity of enzymes in organic solvents appears to be directly controlled by four factors: (1) ground-state stabilization of substrate and/or product, and ultimately enzyme, itself (thermodynamic considerations); (2) active-site flexibility/polarity (controlled via the water content of the active site); (3) waterstripping from an enzyme that will alter the fine balance of noncovalent forces that maintain the native, active

structure of enzymes; and (4) direct solvent-induced perturbation of the enzyme. Understanding how these factors control enzyme structure and function in nonaqueous media provides a framework for proposing methods to improve enzyme function in organic media.

Approaches To Improve Enzyme Function in Organic Solvents Enzyme function in organic solvents may be improved either by solvent engineering (e.g., tailoring solvent properties to achieve desired reactivity and selectivity) or by protein engineering (e.g., tailoring enzyme function to the reaction medium). Within a rational framework that encompasses the four factors that govern enzyme function in organic solvents, it is possible to alter enzyme function in a beneficial manner. Table I1 highlights proposed methods to improve enzyme function in organic solvents. Solvent Engineering. Solvent engineering is the simplest, and perhaps least appreciated, approach to improve enzyme function. It is easy to manipulate the solvent properties for a given enzymatic reaction. In contrast to aqueous solutions, there are literally hundreds of solvents and solvent mixtures that can be used for enzymatic catalysis. An ideal solvent will not induce significant structural perturbation of an enzyme or cause water to be stripped away from the biocatalyst. In addition, the solubility of compounds should not be too

Bbtechnol. Prcg.., 1992, Vol. 8, No. 4

Table 11. Proposed Methods To Improve Enzyme Function in Organic Solvents Thermodynamic Issues destabilize substrate ground state use hydrophobic solvents, when possible use polar substrates, when possible stabilize enzyme-substrate complex improve interaction between substrate and enzyme via protein engineering (e.g.,polar/charged mutations to enhance interaction with polar/charged substrates; hydrophobic mutations to enhance interactionwith hydrophobic substrates) Water-Stripping Phenomenon solvent use nonpolar solvents are less likely to strip water than polar ones protein engineering-two opposing viewpoints increase surface charges to help retain enzyme-bound water decrease surface charge to eliminate the need for enzymebound water Active-Site Chemistry increase flexibility of active site by increasing active-site polarity add water to medium-plasticizing effect of water via dielectric screening of charges and dipoles engineer polar amino acids into the active site

high such that ground-state stabilization becomes significant and causes an increase in substrate K,. Hydrophobic solvents, most especially hydrocarbons, satisfy these requirements. Furthermore, the structural rigidity of enzymes in hydrophobic solvents can improve regio- and stereoselectivity in a manner that is somewhat predictable, as previously discussed. From a thermodynamic standpoint, polar substrates are not significantly stabilized in hydrophobic media and catalytic efficiency is not compromised. Unfortunately, the low solubility of polar organic compounds in hydrophobic solvents eliminates one of the major operational advantages of nonaqueous enzymology-namely, catalysis on highly concentrated solutions. Use of polar cosolvents may help increase the solubility of the substrate without reducing the catalytic power of the enzyme; however, only small amounts of a polar solvent in a hydrophobicone are enough to drastically increase the solvent polarity and, thereby, alter the physicochemical properties of the reaction medium. Finally, maintenance of optimal hydration levels, both on the enzyme and in the solvent can also be consideredas solvent engineering. Addition of less than 1-2 % v/v water in polar organic solvents is often enough to stimulate catalytic activity without affecting the thermodynamic equilibria of hydrolase-catalyzedreactions (Affleck et al., 1992; Kise and Shirato, 1985; Nilsson and Mosbach, 1984). Protein Engineering. Unlike hydrophobic solvents, polar media are more useful for preparative organic synthesis of peptides (Ooshima et al., 19851, sugar derivatives (Therisod and Klibanov, 1986, 1987; Riva et al., 1988; Bjorkling et al., 1989; Carrea et al., 1989), and polymers (Patil et al., 1991a,b;Wallace and Morrow, 1989; Margolin et al., 1987). Solvents such as DMF, THF, dioxane,pyridine, ethyl acetate, etc., dissolve alarge number of commercially important compounds in high concentrations. Solvent engineering is often not possible. For example, sugars and amino acids are soluble in a limited number of highly polar solvents (e.g., pyridine, DMF, morpholine, etc.). There is often no great choice of solvents to use for catalysis. In such cases, a more general approach to improve enzyme function involves protein engineering. Protein engineeringhas been used successfully in aqueous biocatalysis to increase enzyme thermostability (Perry and Wetzel, 1984; Villafranca et al., 19831, alter substrate specificity (Estell et al., 1986; Craik et al., 1985),and alter pH optima (Russell and Fersht, 1987). There is no

265

fundamental reason why enzyme function in nonaqueous media cannot be altered or improved using this technique. We have studied the effect of specific mutations in the active site of subtilisin BPN’ in both aqueous and organic media with the goal of rationally designing BPN’s active site for optimal function in nonaqueous media (Xu et al., 1992). The results of our study with subtilisin Carlsberg (see above) indicated that increased polarity in the active site of the enzyme improves catalytic efficiency (Affleck et al., 1992). Instead of the addition of water, active-site polarity can be increased by protein engineering. We have examined two such mutations in the related subtilisin BPN’-namely, G166N and M222Q in which glycine and methionine are replaced by asparagine and glutamine at positions 166 and 222, respectively. Figure 6 depicts the molecular structure of subtilisin BPN’ in which the G166 resides in the hydrophobic cleft of the SIacyl binding site nucleophile binding site. and the M222 resides in the SI’, In aqueous buffer, the catalytic efficiency of the G166N mutant is similar to that of the wild-type enzyme; however, the M222Q mutant is 27-fold less efficient. In contrast, in T H F the G166N mutation is 3.8-fold more efficient than the wild-type enzyme. The M222Q does not result in a more active enzyme than the wild type; however, the catalytic efficiency of the Q mutation relative to that of the wild type in THF is substantially greater than in aqueous buffer (e.g., [k,JKm(Q)l/[k,JK,(M)] in THF is nearly 10-fold greater than in water) (Xu et al., 1992). The improved catalysis of the polar mutants relative to that of the wild-type enzyme in T H F is a combined effect of improved k,t and decreased Km. Addition of water to the organic solvents would be expected to reduce the effect of enhanced activity upon mutation to more polar activesite groups. Indeed, addition of 2% aqueous buffer to THF resulted in a 3-fold decrease in the ratio of [k,J Km(Q)I/[kc,JKm(M)I, presumably due to water diminishing (or replacing) the polar advantage of glutamine a t the 222 position. The combination of water addition and protein engineering has a powerful impact on subtilisin catalysis. For example, addition of 2 % aqueous buffer to T H F coupled with the G166N mutation results in an increase in the catalytic efficiency of 40-fold over that for the wild-type enzyme in dry solvent. In addition to active-site polarity, other factors may influence subtilisin catalysis at the molecular level. For example, stabilization of the incoming nucleophile is controlled by the chemistry of the SI’site. Specifically, it is expected that an increase in the hydrophobicity of this subsite of subtilisin should improve catalysis with hydrophobic alcohol substrates. To test this hypothesis, we studied the transesterification of vinyl butyrate with various primary alcohols (Focht et al., unpublished results). As depicted in Figure 7, the M222F mutant was progressively more active on larger (more hydrophobic) alcohols than the wild-type enzyme in THF, presumably due to the increasing stabilization of hydrophobic alcohols by the hydrophobic phenylalanine. Conversely, the M222Q polar mutation is expected to destabilize the incoming nucleophile and result in poorer catalysis than that of the wild type. In fact, this was observed (Figure 7). Finally, the issue of water-stripping must be addressed. What can be done to alleviate the detrimental effects of water-stripping? There are two approaches that can be studied. One approach is to engineer polar and charged amino acids onto the surface of a protein so that water in the vicinity of such amino acids would be less likely to partition from the protein into an organic solvent. This approach, however, may only marginally improve water

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Acknowledgment This work has been supported by grants from the National Science Foundation (CBT-8808897 and BCS8958415, Presidential Young Investigator Award), Army Research Office (No. DAAL03-91-G-0224), Mead Corp., Olin Corp., and Genencor International. I am grateful to Thomas Graycar and Dr. David Estell at Genencor International for supplying the protein engineered variants of subtilisin BPN’ and Dr. Richard Bott at Genencor International for preparing the active-site structural diagram of native BPN’.

8 ,

E

Y m

-

Literature Cited

I

E

I

0

2

Abraham, M. H. Solvent Effects on Transition States and Reaction Rates. Prog. Phys. Org. Chem. 1974, 11, 1-87.

Affleck,R.;Xu,Z.-F.;Suzawa,V.;Focht,K.;Clark,D.S.;Dordick,

U

0

a, NO. 4

0222

n

1 1

n

4

6

8

10

Figure 7. Effect of protein engineering of subtilisin B P N s SI’ site on the nucleophile specificity using straight-chain alcohols as nucleophiles and vinyl butyrate as the ester substrate in tetrahydrofuran. The reactions were performed in THF with concentrations of alcohols ranging from 2 to 200 mM and 200 mM vinyl butyrate and 1 mg/mL enzyme. The enzymes were lyophilized from a phosphate buffer solution (20 mM, pH 7.8). The enzyme suspensions were vortexed for 15 s followed by sonication for 15 s prior to the reaction and the suspensions were shaken a t 250 rpm at 30 “C. The drop in the catalytic ratio for octanol may be due to steric constraints with the bulky phenylalanine residue a t position 222.

retention in highly polar solvents. Alternatively, one may engineer the enzyme so as to replace surface charges and polar amino acids with nonpolar ones. In this manner, the enzyme may not require a large amount of water to remain catalytically active. This latter approach has been suggested to improve protein stability in organic solvents (Arnold, 1988).

Conclusions Elucidation of the factors that govern enzymatic catalysis in organic solvents should be viewed as a starting point for the rational design and optimization of enzyme function in nonaqueous media. These factors include the influence of solvent on reaction thermodynamics,enzymebound water, active-site flexibility and polarity, and direct inactivation of the enzyme by the solvent. The optimization of enzymatic catalysis in nonaqueous media is only now being addressed. I t is apparent that both solvent and protein engineering can be used effectively to tailor enzyme function in a given solvent and for a specific reaction. Future research in this field must invariably focus on the following three aspects: (1) Continued elucidation of the role of the reaction medium (including the role of water) in enzymatic catalysis. (2) Rational generation of site-specific active-site mutants that lead to improved activity and desired selectivity. This will probably involvemultiple mutants that act synergistically. (3) Development of a unified activity/stability design approach in order to develop highly active and stable enzymes for use in a wide variety of nonaqueous environments. Through such a research program, enzymes will continue to enjoy a unique position in synthetic chemistry.

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