Biocatalysis in Organic Solvents K. R. Natarajan Annamaiai University. Annamalainagar-606 002, Tamil Nadu, India Evolution has taken place in an environment of which water is one of the major components. I t is not surprising, therefore, that the biochemical reactions in living cells take place in an aqueous medium. As a "biomolecule" i t plays a central role in many enzyme-catalyzed processes and is ahsolutely, directly or indirectly, in all noncovalent interactions-hydrogen-bonding, hydrophohic, and van der Wads interactions-that maintain the native, catalytically active enzyme conformation (1-3). For this reason, virtually all studies in enzymology (extraction, purification, characterization, reaction, and industrial applications) thus far have been carried out in aqueous medium. However, recent developments in biotechnology demand wider applications of hiocatalysts. From this point of view there are numerous advan: tages of carrying out enzymatic reactions in organic solvents or mixed solvent systems (water-organic solvent mixtures) instead of water alone. Zaks and Klibanov (4) list several advantages of biocatalysis in nonaqueous solvents as opposed to water. These include
.
high solubility of moat organic compounds in nonaqueous solvents greater stability of enzymes in organic solvents ability to carry out new reactions that are impossible in water because of kinetic or thermodynamic restrictions relative ease of product recovery from organic solvents compared to water insolubility of enzynes in organic solvents, which permits their easy recovery and reuse and thus eliminates the need for immobilization undesirable side reactions such as hydrolysis of acid anhydrides caused by water can be prevented
Many enzymes were shown to he catalytically active in a wide variety of different organic solvents (4-9). Their catalytic efficiency in organic media is comparable to that displayed in water. Water-immiscible organic solvents have been employed for immobilization of enzymes hy microencapsulation method (10) in which an enzyme is entrapped or enclosed in a semipermeable membrane without loss of enzyme activity. But removal of water no doubt drastically distorts the native (biologically active) conformation and inactivate the enzyme. Although this is undoubtedly correct, the real question should be not whether water is indeed required hut how much water is needed. I t is hard to imagine that the enzyme molecule will need the entire 55.5 mol of water per liter that surround i t in an aqueous solution. It is more plausible that only a few monolayers of water around the enzyme molecule are truly necessary. I t is probable that enzyme may he able to carry out its action with constitutional water located within the protein molecule (about 0.0003 g HpO/g protein) and interfacial water located on the surface of the protein and in small crevices (11). As long as this water is available around the enzyme molecule, the rest (i.e., the hulk) of the water can probably be replaced by an organic solvent without adversely affecting the enzyme structure and function. Since the absolute amount of water contained in that monolayers is very small, this situation is equivalent to an enzyme functioning in a nearly anhydrous organic medium. The work of Klihanov and others (4-9) has confirmed this rationale.
Crlterla for Solvent Selection Selection of a suitable solvent is imoortant because manv solvents can inactivate enzymes. I t is usually desirable to finda solvent in which the enzvme isnot onlv thermodvnamically stable hut also catalyt~callyactive. H O W active and stable are the enzymes in organic solvents? Often these questions cannot he directly answered because comparable data for aqueous catalysis is not available. However, most studies with organic solvents are carried out with mixed solvent systems that contain at least 50% water. In such cases, activity and/or stability has been correlated with a number of solvent properties, of which the partition coefficient in the solven&ter svstem is imoor&nt. unless i t is desired to utilieea two-phasesystem (12;. othercriteria that should be taken into consideration in the selection of oreanic " solvents for enzymic catalysis include solubility of reactants, stabilitv of enzwnes. toxicitv. and flammahilitv (13).In addition, the enzyme should becofactor-independent, as common cofactors are insoluble in organic solvents and water should he a nonreactant in the enzymatic process. The nature of the solvent is crucial for maintaining the layer of essential water around the enzyme molecule. The most useful solvents are very hydrophohic, such as hydrocarbons. They will not pull water away from the enzyme. The less hydrophobic the solvent, the higher its affinity for water and hence the more likely i t is to strip the essential water from the enzyme molecules. But less hydrophohic solvents will work as long as they are resaturated with aqueous solution so that their thirst for water is quenched. Even in the case of hvdro~hilichut still water-immiscible solvents such as ethyl acetate, i t is essential to saturate the enzymes with water. Most enzymes are inactive in hydrophilic, water-miscihle organic solvents. However, there are some enzymes in which the water is hound so tightly that hydrophilie solvents . . cannot usurp the water. such enzymes are active in hoth hydrophohic and hydrophilic solvents. Presumably, some degree of hydration of the enzyme is necessary for the maintenance of catalytic activity in organic solvents. Although our understanding of the effects of organic solvents on enzyme activity and stability is growing, it is not yet at the stage where i t can he used predictively tochoose asolvent for a particular enzyme-catalyzed reaction (14). An intriguing question concerning the action of enzymes in organic solvents is that of pH. All enzymatic reactions in water are strongly pH dependent. But organic solvents have no pH. How does the enzyme know what the pH is then? To quote Klibanov (5),an enzyme "remembers" the pH of the last aqueous solution t o which i t has been exposed, i.e., the enzyme's ionogenic groups acquire the corresponding ionization state, which then remains hoth in the solid state and in organic solvents. Therefore, i t is better, before placing an en&me in an organic solvent, to dissolve it in water of optimal pH for the enzymatic activity. This should be followed by freeze-drying or soi1,ent precipitation. Similarly, it is better to saturate a water-immiscible organic solvent with an DH.instead of olain water. aaueous huffer oftheaoorooriate .. . p;ior to adding the enzyme poGdei. In other'words, thd enzymes must be presented to the organic solvent in a catalytically competent ionic state. Volume 68 Number 1 January 1991
13
Effects of Organlc Solvents on Enzymes Organic solvents produce various physicochemical effeds on enzyme molecule (15,16). The effects can he unexpected and potentially very useful depending upon the kinds of organic solvents and the enzymes. One must have knowledge about the effects of organic solvents on properties and functions of enzymes if they are to he used in reaction systems. Enzymes may he stahilized or destabilized owing to the chanee not onlv in the tertiaw structure hut also in the secondary structure such as alpha helix and beta structure. While the usual effect of hieh concentration of nonaaueous solvents is the destahilizatyon of enzymes, low concentrations have the opposite effect, i.e., thev stabilize them. This phenomenon haibften heen applied fbr the stabilization of enzymes during purification and storage. In many cases, low concentrations ojorganic rolvents do not affect the catalytic activities of enzymes. In some cases, the catalytic activities of enzymes are even enhanced by organicsolvents. The hasis for this enhancement of enzyme activity andlor stability may be the restoration of conditions more closely resembling those in the cellular environment, in which these are presumablv ootimal. Studies with such solvent svatems have are shown ihai all the protein structure-stahilizin~solvents ~referentiallvexcluded from contact with the protein surFace. A detaiied analysis of the preferential interactions hetween proteins and solvent components in water-structurestabilizing solvent systems indicates that the structural stabilization is due to a microphase separation of the solvent a t the protein surface, with water molecules accumulating a t that surface. This layer of water molecules ordered on the protein surface may account for the activity and stability of enzymes in nonaqueous solvents (17). There exists the possihilitv that enzvme snecificitvmav he altered bv-the oresence " . of an organic solvent, presumahly by modifying the active site conformation. Thus. hvdrolvtic enzvmes. which reauire water as one of the suhstr"ates in hydrolyticreactions;can catalvze reactions that are imnossihle in water. For example. . . in water, lipase catalyzes hut a single reaction-hydrolysis: hut in oreanic solvents. it can catalvze a t least six other reactionsu including t;ansesterific&ion, esterification, aminolvsis, thiotransesterification, oximolvsis, and acvl exchangi (18). Other examples include thk enzyme alphathrompin, which normally exhibits esterase and amidase activity but had the amidase activity reduced while the esterase activity was increased in the presence of dimethyl sulfoxide (20%vlv), and chymotrypsin, which in water prefers to act upon aromatic amino acids as substrates, hut for which in hydrocarbon solvents serine was the better substrate (19). The enzymes also show greatly enhanced thermal stahility. Apparently all chemical and structuralchanges that lead to irreversible thermal inactivation have one thine in common-they require water. I t follows that, if enzymes are placed in an essentially water-free environment, they will become more thermostable. This has been confirmed by Zaks and Klibanov (20). who showed that pancreatic l i ~ a s e is extremely thermostable and can withstand heating a i l 0 0 OC. At this temoerature the enzvme is 10 times more active than a t room temperature for several hours. Thus organic solvents have different effects on enzyme activity, stability, and specificity because of, in the words of Klibanov (21,22), "their differing ability to strip essential water from the catalyst".
-
-
At the present time organic synthesis has reached the level a t which..eiven adeauate resources. nearlv"anv.stahle oreanic compound can he prepared. However, two major problems do exist making many syntheses complicated, difficult, and uneconomic. These are regio- and stereospecificity. These properties are the most obvious attributes of enzymes. But
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14
Journal of Chemical Education
most of the compounds and intermediates encountered in organic syntheses are hydrophobic in nature and do not dissolve in water. Enzymes' regio- and stereospecificity coupled with their ability to work in organic solvent may be exploited in the synthesis and resolution of a wide variety of optically active compounds. For drugs and agrochemicals there is an increasine trend toward the use of ooticallv . pure stereoisomers, which are more target-specific and show fewer side effects than racemic mixtures of isomers. Klibanov and his group (23-25) have developed a novel enzymatic approach t o the production of optically active alcohols and esters from racemic mixtures. I t involves the use of lipasecatalyzed transesterifications in biphasic aaueous-organic solvent systems. As a result, a number of optically active alcohols and esters can he produced with high yield and enantiomeric purity. For example, using a yeast lipase-catalyzed esterification in organic media, they resolved racemic (R,SJ-2-bromo- and cRS-2-chloro propionic acids. This lipase catalyzes a stereoselective esterification of the R-2halopropionic acid with primary alcohols like n-butanol, n hexanol, or n-octanol in organic solvents such as hexane or chloroform. Only R isomer is esterified, whereas S isomer is not. If a racemic mixture of the acid is used, an equimolar mixture of the R-ester and unreacted S-acid are produced.
.
-
+
(R,S)=2-halocarboxylic acid
Butanol
I
I
Butyl (R)=2-halacarboxylate
I
(S)=Z-halocarhoxylic acid
These two chiral acids are versatile intermediates for the production of phenoxy propionic acids, an important class of herbicides (23). These products may be separated and recovered by simple extraction, providing both isomers in greater than 90% optical purity. The yeast lipase is exceedingly stable under operating conditions, allowing repeated use of the enzyme and thus making the economics of the process attractive. Another a~olicationof stereoselectivitv of nonaaueous hicatalysis i s & the resolution of DL-menihol in n-hLptane (26). L-menthol. a compound havina a peppermint flavor. is used in the food and pharrnaceutici i"d;sirieu. Development in the svnthesis of chiral intermediates for drugs and agrochemicals, in some cases, is well advanced: a t Chemie-Linz in Austria, a process for the resolution of racemic alpha-halopropionic acids by lipases is operating on pilot-plant scale (27), and a t Andeno-DSM in Holland, the resolution of racemic alkanoic and oxyranyl methyl esters (glycidyl esters) is performed on a commercial scale (28). These 6rocesses arefeasible onlv in oraanic solvents. HiocatalysW are increasingly used in pharmaceutical industries for selective biotransformation of steroids for the production of steroidal drugs (29-31). The hiotransformation of steroids is limited by the poor soluhility of steroids in water. A major improvement not only in percentage conversion but also in selectivity can be obtained by carrying out these reactions in organic solvents. Immobilized enz.vmea and cells in the presence of organic solvents (table) have been used to catalyze regio- and stereoselective reactions in the steroid structure. Both biphasic and monophasic systemsareused (31-33). For example, an imponant stepin the production of cortisone and other antiinflammatory agents is introduction of a hydroxyl group at carbon atom 11 in the steroid structure. T o obtain cortisone by chemical synthesis (from desoxycholic acid, requires 31 step*, of which nine steps are involved in moving an oxygen function from position 12 to I1 (with a low overall yield). This oxygen function at carbon atom 11 is essential for biological activity of c o d . sone. But in hiotransformation using immobilized catalvsts selective hydroxylation a t the nonactivated carbon atom 11 occurs in a single step (Fig. 1).
.
?.
Sterold BlotranslormatlonIn Water-Water-lmmlscibte Organlc Solvents Biohansformatlon
Biocatalyst Bktydroxy sterold dehydrogenase (free or immobilized) 20-8-Hydroxy steroid dehydrogenase (free or immobilized) Nocardia sp. (free or immobilized) Nocardia rhcdochmus (free or Immobilized) Arthrobacter simplex (immobilized)
Organlc solvent
Oxidation of Bhydroxy steroids
Ethyl w butyl acetate
Reduction of 20-keto steroids
Ethyl w bulyl acetate
Oxidation ot cholesterol
Carbon tetrachloride or tolwne Benzene, chloroform. mheptane, cyclohexane Methanol
Various steroid hansformatlons A'dehydrogenation of hydrocortisone to predni~olone
Figwe 1. Bioconvenion of compound S by Immobilized C-iaria dimethyl sulfoxide.
lwrata in
Another example of a nonaqueous bicatalytic process of industrial interest is in the processing of fats and oils, since the substrates, triglycerides, are insoluble in water. Interesterification (resynthesis of ester bonds) is a process that is used in the edible oil and fat industry to alter the composition and therefore the physical properties of triacyl glycerol mixtures (Fig. 2). I t is used for production not only of margarine and shortenings but also speciality fats required for food industries. Microbial lipases initially catalyze the hydrolvsis of fats with the auantities of water in the oil follo~&"dby reesterification 2 the resultant acids and alcohols. This reaction can be used to retailor low-arade oils to ~ r o duce cocoa butter substitutes, which are in great demand in food industriel. The uniaue ~hvsicaland textural qualities of cocoa butter stem from its very unusual triglyceiol compounds. In the predominant molecular species found in high-quality cocoa butter, oleic acid residue occurs at the Sn-2 position flanked by the saturated fatty acids, stearate and palmitate. By using 1,3-specific and fatty acid-specific lipases, the stearate content of low-grade oils such as palm oil or olive oil in which oleate exclusively occupies the Sn-2 position, can be increased to produce speciality fats including cocoa butter substitutes that cannot be obtained by conventional interesterification methods using sodium or sodium alkoxide as catalyst (34,35). Lipase-catalyzed interesterifications in organic solvents have been commercialized (36.37). ' dth$r applications of nonaqueous biocatalysis include Novo's method for conversion of semisynthetic porcine insulin to human insulin (38),synthesis o i aspartame, an artificial sweetener (39), synthesis of chiral intermediates for optically active P-adrenergic blocking agents for hypertension and anaina pectoris (40),synthesis of grandisol, a sex pheromone : d l ) , broduction of flavor esters (421, enzymatic determination of cholesterol (43). and synthesis of peptides containing D-amino acids, which is impossible in water because of the enzyme's strict stereoselectivity (44).
Figure 2. lntere~terlficatlonreaction with chemical or nonspecific ilpase (a-trlacyl glycerol mixture) and wilh 1.3-specific lipse (b-trlacyl glycerol mixture, and c-triacyl glycerol plus free fatty acid). B-oleic acid. Concluslon
and Future Prospects
From the foregoing discussion, i t is obvious that enzymes can work in organic solvents, and the reactions discussed illustrate well the power of enzyme catalysis in organic solvents. The use of enzymes in organic milieux represents unexpected opportunities that confound our preconception of enzyme catalysis that have been developed from studies carried out in ~ svstems and has chanaed . u r e-l vaaueous . - the way we think about the potential applications of eneymes. We are now witnessina various and enthusiastic activities of R and D in nonaqueous biocatalysis. This represents a significant advance in enzvme biotechnolow. Accordinn to Klibanov (6), biocatalysis in organic solv&ts is a field of immense potential and application that have been ruled out because of enzymes' shortcomings should be reevaluated. As the knowledge of nonaqueous biocatalysis is advanced and combined with recombinant DNA technology that enables production of virtually any enzyme in unlimited quantities, nrotein eneineerine in redesiened forms with imnroved site-dyrected muggenesis, and immobifization properties iy technolow. we are o~timisticthat we will see several novel enzyme-catalyzed industrial processes in the near future. Volume
68
Number 1 January 1991
15
Literature Clted 1. 2. 3. 4. 5. 6. 7. 8. 9, 10. 11. 12. 13. 14. 15. 16. 17. 16. 19. 20. 21. 22. 23. 24.
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Tanford, C. J. Am. Cham. Sor. 1962.84,424&4247. Frerht, A. R. Trends Biocham. Sri. 1987.12,301-305. Pace,C. N. C ~ i tR . e~.Bbeh~m 1975.3.143. . Zaks. A ; Klibsnov, A. M. Science 1985,228.1280-1284. Zaks,A.:Klibanov.A. M.Ploc.Nal. Acad Sci.. USA 1985.82,31923196. Jones,J. B.;Schwsrtz. H. M.Con. J Chrm. 1982,61,1030-1033. Dastoli, F. R.;Price, S. Arch. Bioehem. Biophys. 1967,118, 163-165. Kazandiian, R. 2.; K1ihanou.A. M. J.Am. Chem. Soc. 1986.107,544%5450. Natarsian, K. R.: Rarnschandrarnurfhy, B., unpublished results Chibata, I., Ed. ImmoUliaodEnzymsa; Halsted: New York, 1978: Chapter 2. Fennerna. 0. In Food Protrina: Whitaker, 3. R.: Tannenbaum. S. R.. Eds. Connecticut. 1977. Lanne. C.; Boeren,S.;Vos,K.:Veeger,C.Bioterhnol.Bioeng. 1987,30,81-87. Lilly, M. D. J. Chem. Technol. Biol~rhnol.1982.32.162-169. Halline, P. J. Trends Biof~chnol.1989, 7,5&52. Sin8er.S. 3.Adu.Piotein C h ~ m1952,I7,1-68. . Leaanie. S. Phvairo-Chemical Aamcia of Protein Denoturofion; Wilw New i978. Timashsff, S. N. InBiophyairs of Wafer; Felix Frank, Ed.; Wilay: New York. 1982; p 70. Zaks.A.;Klibanov, A.M. J A m Chem.Soc. 1986,108,2767-2768. Pal,P.K.;Gertler, M. M. ThrombosisRes. 1983,29, 175-178. &k%, A ; Klibsnov.A. M. Scisnea 1684,224, 1249-1251. Zaks,A.;Klibanov, A. M. J.Bioi. Chem. 1988,263,3194-3201. Zaks,A.:Klibsnov,A.M. J.Biol.Chem. 1988,263,8(117-8021. Klibanov, A. M.U.S. Patent No.4.601.987, 1986. Camhou,R.; K1ibanou.A.M. J.Am. Cham. Soc. 1984,106,2687-2692,
Journal of Chemical Education
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W~:
