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Enzymes in Reversed Micelles as Catalysts for Organic-Phase Synthesis Reactions John W. Shield, Holly D. Ferguson, Andreas S. Bommarius, and T. Alan Hatton’ Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02 739
Industrial exploitation of the specificity and high catalytic activity of enzymes is becoming an important technique for the synthesis of commercially important compounds. Traditionally, enzymes are used in an aqueous medium, but an organic solvent is more attractive when the reactants or products are lipophilic or a low-water environment is desired. This review discusses a method for utilizing enzymes as catalysts in organic solvents through the encapsulation of the enzymes within reversed micelles. Extensions of traditional enzyme kinetics appear to apply to enzymatic reactions within reversed micelles, but further modeling to account for the low-water-concentration environment is required. Only recently have enzymatic reactions within reversed micelles been examined, but early results involving a large variety of enzymes suggest that reversed micelles will become a useful system for enzymatically catalyzed synthesis reactions.
Introduction “The economy of chemical manufacture depends largely upon establishing high rates of production of desired products and minimizing the production of undesired by-products.” The importance of this maxim has not diminished in the 40 years since Olaf A. Hougen and Kenneth M. Watson used these opening words in the third volume of Chemical Processing Principles, a series of books that has had a profound influence on generations of chemical engineers ( 4 5 ) . It is certainly as valid now for the emerging technologies associated with biocatalytic processes as it was then for the more traditional petrochemical industries. Perhaps nowhere is the minimization of byproduct formation more important than in the new biotechnology growth areas of pharmaceuticals, food products, and agricultural chemicals. In these industries, the requirement for pure, biologically active compounds can be stringent, since unwanted isomers can have undesired properties. Moreover, the production of inactive isomers, sometimes in stoichiometric amounts, represents a substantial loss of feedstock materials and increases the bulk material handling requirements for the product per unit of active compound. Since separation of the desired product from structurally similar compounds is difficult and expensive, it is particularly important to minimize byproduct formation during the manufacture of these classes of compounds. Many of the compounds of interest can be obtained via conventional organic synthesis, although these techniques are often not selective for specific regio- or stereoisomers. Enzymatically catalyzed synthesis, on the other hand, is selective and can be used to produce only the specific compound of interest. Indeed, a number of commercially important compounds can be synthesized by using enzymes as catalysts, including steroids, peptides, lipids, aromatics and long-chain alcohols. Enzymes have been adopted or show potential in the manufacture of food products, fragrance compounds, herbicides, pesticides, and pharmaceuticals (18,95, 108,109). For instance, steroids can be regioselectively oxidized or reduced by enzymes, as in the oxidation of cholesterol by cholesterol oxidase (48) and the reduction of progesterone solely at the 20P-position (59). Chemical catalysis of such reactions, on the other hand, is difficult because of the multitude of possible reaction sites on the substrate molecule. Likewise, enzymatic resolution of stere3isomers by selective reaction of only one
of the enantiomers has been used commercially with success (102). Enzymes with high degrees of substrate specificity produce few byproducts from mixed substrates, resulting in both increased yields and reduced downstream separation costs (104, 106). Additionally, since an enzyme generally catalyzes the reaction of a specific moiety, it is often not necessary to protect other functional groups on the substrate, which can simplify complicated multistep organic synthesis pathways. For example, the residues of a substrate peptide can frequently be left unblocked in protease-catalyzed peptide-bond formation (17 ) . Most enzymes are water-soluble proteins normally found in an intracellular environment consisting of an aqueous medium a t physiological temperature and pH. Usually enzymes demonstrate optimal activity a t these mild conditions. Operation a t relatively low temperatures and a t nearly neutral pH permits reactions with labile reactants and products and can lower energy requirements and capital costs. Furthermore, a t these conditions, many enzymes are known to be high in catalytic activity, some to the extent that the apparent second-order rate constants for the reaction of the free enzyme with substrate is close to the diffusion-controlled association rate (30). In contrast, a large number of processes employing conventional catalysts use extremes of temperature, pressure, or pH to achieve similar reaction rates. Relative to conventional catalysts such as acids, bases, metals, and metal oxides, enzymes have seen little use for large-scale synthesis reactions (108). However, the technical difficulties inhibiting the use of enzymes are being considerably reduced with the introduction of new techniques for enzyme purification, stabilization, and assay. But there is a time lag between these advances in technology and the application by industrial chemists and process engineers to practical synthesis probiems. Familiarity with the recent technological advances in enzymology is spreading, as is evidenced by the recent rapid growth of commercial interest in enzymatically catalyzed syntheses (93). This will lead to future increases in enzyme stability and availability. As the range of commercial products based on the knowledge of biological systems increases, the use of enzymes as catalysts will certainly become an increasingly important and necessary technique for industrial synthesis. In this paper we provide an overview of an enzymecontaining medium that shows considerable promise for the commercial realization of enzymatic synthesis reac-
0196-4313/86/1025-0603$01.50/0@ 1986 American Chemical Society
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tions. Specifically, we are concerned with reactions in an organic medium where the enzyme is in solution, protected from the apolar medium by being sequestered within the polar cores of reversed micelles. We begin by discussing the more general question of why organic solvents may be advantageous for the reactions of interest and follow with a description of reversed micelle systems and a historical perspective on reversed micellar catalysis. After listing some interesting potential applications for this technology, we discuss the problem of determining reversed micellar enzyme reaction kinetics. The paper concludes with some of the engineering questions to be addressed if these applications are to be implemented on a large scale.
Enzymatic Catalysis in Organic Media While enzymes are typically employed in aqueous media, there are several important advantages of enzyme-mediated catalysis in organic solvents. For example, many interesting compounds are poorly soluble in water but quite soluble in organic solvents. In such cases an organic medium makes it possible to obtain higher reactant and/or product concentrations than with aqueous reaction systems. Organic reaction media can also reduce other problems associated with aqueous enzymatic reactions, such as product inhibition, which is directly related to the concentration of product in the environment of the enzyme. Often, in a synthesis reaction, the desired product is preferentially organic soluble. If such a reaction is carried out in an aqueous phase in contact with an immiscible organic phase, the product partitions away from the enzyme into the organic phase. This can effectively alleviate product inhibition and can drive equilibrium-controlled synthesis reactions toward completion. Water can react with substrates, products, or other species in the reaction broth, producing byproducts. In such cases, reduction or near elimination of water from the system by using organic solvents can significantly increase yields. Additionally, Klibanov reported that the thermostability of enzymes may be substantially improved when the enzyme is not fully hydrated, as is often the case in organic solvents (113). There is a variety of possible approaches to designing a process employing enzymes in a partially to mostly organic reaction medium. These systems fall into four major categories depending upon the reaction medium used: (1) one phase, aqueous/water-miscible organic solvent medium (77,88),( 2 ) two phase, aqueous/water-immiscible organic solvent medium (1, 16, 77, 881, ( 3 ) two phase, organic solvent/solid enzyme medium (52, 224), and (4) reversed micellar medium (50, 63, 70). Synthesis reactions studied in these systems are of three general types, each having potential industrial applications: synthesis of oligopeptides, many of which are or will be commercially important products in the food and pharmaceutical industries; esterification or transesterification reactions to synthesize or resolve optically active acids, alcohols and esters; and oxidation or reduction reactions. Enzymatic peptide synthesis can be carried out by employing hydrolytic enzymes such as pepsin, papain, and a-chymotrypsin, whose natural function is the breaking of peptide bonds. Because the reaction equilibrium in an aqueous medium is weighted heavily in favor of hydrolysis and not synthesis, the reaction conditions must be adjusted such that product formation is favored. Either an equilibrium or a kinetic approach can be used. The former depends upon the use of a medium containing organic solvent to lower the concentration of water, driving the synthesis reaction toward completion. The kinetic ap-
proach involves synthesis of the peptide, not from the free amino acid, but from the amino acid ester. This reaction, catalyzed by acyl-enzyme-forming enzymes such as CYchymotrypsin, is significantly faster and can result in higher yields (47). The bulk of the work in the area of peptide synthesis has been carried out using a one-phase batch process with a water-miscible organic solvent and employing a hydrolytic enzyme (57,83, 85, 89). The medium is usually selected such that the substrate amino acids or peptides are soluble but the product precipitates. An aqueous/waterimmiscible organic solvent medium has been investigated for peptide synthesis by Jakubke (46, 56) and Martinek (49, 100). Ethyl acetate is often the solvent chosen when i t is desired to maintain product solubility. Enzymatically catalyzed esterifications and transesterifications in organic-solvent-containingreaction media have typically employed either a-chymotrypsin or lipase. The system of choice has been either a biphasic aqueous/water-immiscible organic solvent system or a solid enzyme/water-immiscible organic solvent system (78. 212, 114). Oxidation/reduction reactions require cofactors and electron donors or acceptors. This has led to the use of not only isolated enzymes but also whole cells to catalyze the desired reaction. The major focus of recent research efforts has been the modification of steroids for pharmacological applications using a two-liquid-phase medium. Carrea and Cremonesi (15) have concentrated on the use of isolated enzymes, cofactors, and electron carriers, while other researchers have investigated cells as the reaction catalysts ( 1 2 , 34). Other types of reactions that have been studied include cell-catalyzed epoxidations (9, 10, 19,98),L-menthol resolution (87),and L-tryptophan enzymatic synthesis (5). In 1985, Klibanov reported on a two-enzyme system (horseradish peroxidase/cholesterol oxidase) for the quantification of cholesterol (48). The enzymes were used in solid form, dispersed in a water-saturated, water-immiscible organic solvent such as toluene or ethyl acetate. Another example of organic-phase synthesis involved subtilisin employed in a two-liquid-phase medium for the preparation of D-arylglycines for use as side chains on semisynthetic penicillins (97). The above results demonstrate the potential of enzyme-catalyzed organic-phase reactions. However, enzymes employed in organic solvent containing reaction media are often denatured or can experience undesired changes in specificity. These difficulties led to the consideration of reversed micellar organic-phase systems for use in enzyme-catalyzed synthesis. In reversed micelles, the enzyme is isolated from the solvent by a surfactant layer, and these systems therefore show promise for combining the advantages of organic- and aqueous-phase enzyme systems.
Description of Reversed Micelles Reversed micelles are spontaneously formed aggregates of amphiphilic molecules in organic solvents. The hydrophilic head groups associate to form structures with polar cores, and the hydrophobic tails extend outward into the bulk organic solvent. Water can be solubilized within the reversed micelles, leading to optically transparent, thermodynamically stable water-in-oil microemulsions. The molar ratio of the water solubilized within the reversed micelles to the amount of surfactant present is denoted by woand is an indication of the size of the reversed micelles. Differing amounts of water can be solubilized in an organic solvent at the same zoo by adjusting the amount
Ind. Eng. Chem. Fundam., Vol. 25, No. 4, 1986 605 Enzyme
f
Hydrophilic
Hydrophobic
Head GrouD
Tail
v
h
e \
Figure 1. Dioctyl sodium sulfosuccinate (Aerosol OT or AOT).
Figure 2. Schematic representation of an AOT reversed micelle.
of surfactant present. Up to 60% water can be solubilized in organic solvents by incorporation in these “water pools” (103), but most research involving enzymes in reversed micelles has been a t 1-590 water, corresponding to wg)s of 5-40. In systems solubilizing enzymes within reversed micelles, the surfactant dioctyl sodium sulfosuccinate (Aerosol OT or AOT) is frequently used (Figure 1). A conceptual rendering of an AOT reversed micelle is shown in Figure 2. Some of the water solubilized within a reversed micelle hydrates the hydrophilic head groups of the surfactant. This water is tightly bound and is much less mobile than bulk water. For AOT reversed micelles in a hydrocarbon solvent, approximately seven water molecules are required to hydrate each surfactant molecule. Additional water in excess of that required to hydrate the amphiphilic head groups is not as tightly constrained; as the water content of the reversed micelle increases, the properties of this water approach those of bulk water (111). Since the water in the intracellular environment in which most enzymes evolved is thought to be less mobile than bulk water, the environment experienced by an enzyme within a reversed micelle may be a closer representation of its natural intracellular conditions than a bulk aqueous solution. Additionally, intracellular protein concentrations (- 100 mg/mL (99)) can be approached inside the micellar core (79, 101). The structural features of reversed micelles are such that an enzyme molecule can be solubilized in a small, localized aqueous environment within the bulk organic solvent. Figure 3 is a schematic representation of an enzyme solubilized in this manner within a reversed micelle. The
Organic Solvent
\
,Surfactant
‘Aqueous Buffer
Figure 3. Idealized representation of an enzyme completely encapsulated within the water pool of a reversed micelle.
most prevalent method of introducing enzymes into reversed micelles involves injecting an aqueous enzyme solution into the surfactant-containing organic solvent followed by agitation until an optically clear solution is formed (73). This method permits excellent control over the contents and size of the aqueous core of the reversed micelle. Phase transfer of the enzyme from a bulk aqueous solution into an organic/surfactant solution is a second means of forming enzyme-laden reversed micellar solutions (68). Higher enzyme concentrations a t low wo are possible than with the injection method (101). The ionic strength, the p l of the enzyme, and the pH of the bulk aqueous phase affect the transfer, and the conditions of the reversed micellar phase (for example w,) must be measured. The amount of enzyme denaturation caused by both injection and phase-transfer methods of introducing enzymes into reversed micelles is governed by the length of the enzyme’s exposure to the denaturing effects of the organic solvent and by the extent of agitation upon mixing. Enzymes have also been introduced into reversed micelles by agitating a reversed micellar solution over solid enzymes (37, 81). This technique is no longer favored because more time is required and more denaturation occurs compared to the above methods. Another difficulty of the method is that only a limited number of enzymes are available in solid form. However, repeated contact of a reversed micellar system with dry protein has been used to determine 25 mg/mL as the wo-independent limit of solubility of a-chymotrypsin in 0.1 M AOT in octane (55, 79). Preliminary results suggest that reversed micelles provide a system that allows a protein to find the environment most compatible to its native one. Membrane-bound enzymes with hydrophobic regions can position themselves in the surfactant layer, mimicking their natural configuration. Hydrophilic proteins can exist centered in the aqueous core and be “unaware” of the bulk hydrocarbon solution (76). X-ray scattering evidence indicates that different types of enzymes are solubilized by these different means in reversed micelles (92). Some enzymes might demonstrate increased stability when solubilized within reversed micelles. In aqueous solutions, the protease a-chymotrypsin is known to cleave itself, resulting in an activity loss over time. In a reversed micellar system, the enzymes are better protected from one another and activity can be retained longer (73). Levashov has claimed that a-chymotrypsin can retain catalytic activity for over 2 years in an AOT/water/octane system (61). However, in similar experiments, Fletcher found a-chymotrypsin activity to degrade more rapidly in reversed micelles than in aqueous solutions (31). Martinek reported that trypsin activity in reversed micelles can retain activity from a week to only a few minutes depending on wo,the nature of the buffer, and the type of substrate (75). These results suggest that the character-
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T a b l e I. E n z y m a t i c S t u d i e s i n Reversed Micelles" author year significance of work Misiorowski 1974 influence of water and cations on activity of phospholipase A? in phosphatidylcholine/diethyl ether reversed micelles 1977 n-chymotrypsin-catalyzed hydrolysis Martinek reaction in AOTjisooctane reversed micelles Luisi 1977 transfer of tu-chymotrypsin into TOMAC/cyclohexane reversed micelles Menger 1979 a-chymotrypsin-catalyzedhydrolysis in AOT/heptane reversed micelles Douzou 1979 cryoenzymology: trypsin-catalyzed hydrolysis in AOT/silicone oil reversed micelles Luisi 1979 transfer of enzymes into organic solvents with retention of activity Pileni 1981 photoreduction of cytochrome c in AOT isooctane reversed micelles 1981 activity and specificity studies of various Martinek enzymes in reversed micellar systems Laane 1983 keto steroid conversions by dehydrogenase in CTAB/octane reversed micelles Fletcher 1984 kinetics of a-chymotrypsin hydrolysis in AOT/heptane reversed micelles 1984 ik-chymotrypsin-catalyzed peptide Luisi synthesis in AOT/isooctane reversed micelles 1985 enzyme separation via solubilization in Hatton AOTiisooctane reversed micelles
ref 82
73 69
81
21 68 91
75 41 31 71
36
a Abbreviations: AOT, aerosol OT; CTAB, cetyltrimethylammonium bromide; TOMAC, trioctylmethylammonium chloride.
istics of the reversed micellar system have a profound effect on the retention of enzymatic activity.
History Early accounts of the solubilization of enzymes within reversed micelles were made by biochemical researchers examining methods to isolate proteins. In 1952, Hanahan reported the formation of an enzyme/surfactant complex that retained enzymatic activity in diethyl ether solutions (39). In this system, phosphatidylcholine was used as both the surfactant and the substrate in a reaction catalyzed by phospholipase. Other biochemists have used reversed micellar systems with natural and synthetic surfactants to study various proteins (20,94,96). It was not until the work of Misiorowski and Wells in 1972 (82) that the influence of conditions within reversed micelles on enzyme activity was studied. The direction of research over the past years on enzymes in reversed micelles is detailed in Table 1. Systems incorporating synthetic surfactants possess superior reversed micelle formation qualities and seem to be applicable to a wider range of reactions and enzymes. Martinek in 1976 demonstrated a-chymotrypsin activity in reversed micelles of AOT in octane (73). Since then, a variety of other enzymes, surfactants, substrates, and solvents have been used for studying enzymes in reversed micelles (Table 11). AOT in heptane or octane, however has been the preferred system and is much better characterized than other possible solvent/surfactant configurations. The majority of kinetic studies in enzyme-containing reversed micellar reaction media have emphasized hydrolases, especially the protease a-chymotrypsin. The predominant reaction carried out has been the hydrolysis of model compounds such as N-glutaryl-L-phenylalanine p-nitroaiiilide (6, 31. 7Ei) or N-benzoy1-D.L-arginine p -
nitroanilide (75). Other hydrolysis reactions have also been studied, such as lysozyme-mediated degradation of oligosaccharides (37). However, the low-water environment of a reversed micellar system may be inappropriate for nianv hydrolysis reactions in an industrial process. The field of synthetic applications with reversed micelles using lipophilic substrates or products has barely been tapped; only a few enzymatic reactions other than hydrolyses have been reported in reversed micelles. Three reports are of particular interest. The first involves the synthesis of a tripeptide from a protected dipeptide and an amino acid, yielding alcohol as a byproduct, performed by Luthi and Luisi (71). The peptide product preferentially partitioned out of the reversed micelles, driving the reaction equilibrium toward completion. In the second report, Luisi presented initial studies of the lipoxygenase-catalyzed oxidation of linoleic acid, a regiospecific oxidation which has potential for opening a route to prostaglandins and leukotrienes (67). Finally, the reduction of keto steroids in reversed micelles has been investigated by Hilhorst et al. (41, 4 2 ) . The steroids progesterone and prednisone were reduced by a multienzyme system using a cofactor regeneration scheme where either hydrogen gas or the cathode of a bioelectrochemical cell functioned as the ultimate electron acceptor (43). In none of the above cases was a reversed micellar system compared with other aqueous/organic configurations for the solution of synthesis problems. Therefore, it is premature to judge quantitatively the merit of a reversed micellar reaction medium vs. alternative types of aqueous/organic media. However, because of the large number of interesting reactions that are difficult to carry out in aqueous systems, a reversed micellar system consisting primarily of an organic solvent but having the benefit of a hospitable enzymatic environment is likely to be an important process alternative.
Potential Applications for Reversed Micellar Processes The possible synthesis applications for enzymes in organic media are extensive, and reversed micellar systems demonstrate potential advantages over alternative means of exploiting enzymatic catalyzed synthesis. A few of the possible applications are noted here. Enantiomeric Resolution of Amino Acids. Chemical synthesis is often the least expensive method to produce amino acids, but the product is normally a racemic mixture. Since usually only one enantiomer is desired, an enantiomeric resolution step has to follow. Currently, immobilized acylase is used commercially to hydrolyze only the L isomer of acyl-D,L-amino acid mixtures (18). With reversed micelles, proteases could be used to selectively esterify only the L isomer of an amino acid, permitting resolution to become a separation of an acid from an ester (14).
Peptide Synthesis. Oligopeptides are being manufactured for a number of applications including artificial sweeteners, pesticides, and pharmaceuticals. In the pharmaceutical area there is a wide variety of synthetic peptides currently on the market or under active development (51). The market for synthetic peptides is growing rapidly, both in the breadth of products made and in the quantities required. However, current synthesis methods are small-volume batch processes. Enzymes solubilized in organic solvents may provide the larger scale, more efficient processes needed for the cost-effective synthesis of these peptides. Oxidation or Reduction of Steroids. A multitude of steroids are pharmacologically active, many of which can
Ind. Eng. Chem. Fundam., Vol. 25, No. 4, 1986 607 Table 11. Enzymes Solubilized in Reversed Micellar Systemsa enzyme a-amylase a-chymotrypsin
catalase cytochrome c cytochrome P450 dehydrogenase, BOP-hydroxy steroid dehydrogenase, lipoamide dehydrogenase, horse liver alcohol dehydrogenase, lactate hydrogenase lipase
lipoxygenase lysozyme pepsin peroxidase phosphatase
phospholipase A pyrophosphate pyruvate kinase ribonuclease trypsin
surfactant TOMAC AOT AOT AOT AOT Brij 56 C12E4 CTAB TOMAC AOT AOT AOT AOT AOT CTAB + AOT CTAB CTAB CTAB AOT AOT Brij 56 CTAB CTAB AOT AOT AOT CTAB PTC AOT AOT AOT TOMAC AOT AOT AOT AOT PTC PTEA PTA PTC Brij 56 Brij 56 AOT AOT AOT AOT CTAB CTAB
organic solvent isooctane octane octane isooctane heptane cyclohexane/ hexanol heptane chloroform/octane cyclohexane silicone oil silicone oil isooctane heptane n-heptane octane hexanol hexanol/octane hexanol/octane isooctane octane cyclohexane chloroform/octane hexanol/octane isooctane heptane octane heptane/chloroform n-hexane octane octane isooctane cyclohexane octane silicone oil heptane n-heptane n-heptane n-heptane n-heptane ether/methanol cyclohexane cyclohexane octane isooctane silicone oil octane chloroform/octane cyclohexane
DrinciDal researcher van't Reit (105) Martinek (73, 75),Fletcher (31) Balasubramanian ( 3 ) ,Levashov (60, 62) Luisi (6, 711,Levashov (65, 66), Pileni (92) Menger (81) Balasubramanian (3) Fletcher (31) Martinek (75) Luisi (68, 69) Douzou ( 4 ) Douzou (4, 21) Goklen (35, 36), Pileni (91, 92) Visser (107) Douzou ( 4 ) Metelitza (28) Laane (42) Laane (41,59) Laane (41,59) Martinek (76),Luisi (8, 80) Levashov (65, 66) Martinek (75) Laane (40) Laane (41, 59) Rhee (38) Fletcher (32) Levashov (72) Fletcher (32) Morita (84) Levashov (58) Levashov (65, 66) Luisi (8, 37), Goklen (35) Luisi (68) Martinek (73, 76),Levashov (53) Douzou (4, 22) Metelitza (27) Ohshima (86) Ohshima (86) Ohshima (86) Ohshima (86) Misiorowski (82) Martinek (54) Martinek (75) Luisi (110) Luisi (8), Goklen (35) Douzou (21) Martinek (75),Levashov (65, 66) Martinek (75) Luisi (68)
Abbreviations: AOT, aerosol OT; CTAB, cetyltrimethylammonium bromide; TOMAC, trioctylmethylammonium chloride; CI2E4,tetraethyleneglycol mono-n-dodecyl ether; PTC, phosphatidylcholine; PTEA, phosphatidylethanolamine; PTA, phosphatidic acid.
be obtained by modification of a few precursor steroid molecules to a variety of active compounds. Such modifications are difficult to carry out with traditional synthesis, because of the large number of potential reaction sites in a steroid molecule. Many enzymes, however, are sitespecific redox catalysts. Steroid conversions are preferably carried out using organic solvents because of low steroid solubilities in water (41, 42). Selective Modification of Ester Bonds of Triglycerides or Diesters. There is an abundance of oils and fats available in the biosphere for potential use as raw materials. These mostly lipophilic compounds can often be modified for use as chemical intermediates in the food and consumer products industries. Enzymes in organic solvent systems function as selective catalysts for transesterification and hydrolysis reactions, whereas in aqueous systems, undesired, nonspecific reactions can occur (38, 84).
Selective Oxidation of Isomeric Mixtures of Aromatics. Industrially produced di- or trisubstituted aromatic compounds normally contain a spectrum of regioisomers. Usually, only certain isomers are desired, and
separation of the original mixture is often material and energy intensive. Oxidation of specific isomers by enzymes alters the physical properties of the modified compound so that a separation can be easily accomplished. Such conversions might open new routes to specific aromatic products. The oxidation of aromatic aldehydes to carboxylic acids by xanthine oxidase is a case in point, where ortho-substituted substrates react much more slowly than other isomers (90).
Enzymatic Catalysis within Reversed Micelles The initial-rate kinetics of enzyme-catalyzed reactions commonly follow a substrate saturation mechanism, well-known as Michaelis-Menten kinetics. For the enzymatic reaction E + S G ES -.+ E + P (1) let E, S, ES, and P represent the enzyme, substrate, enzyme/substrate complex, and product, respectively. If the ES complex concentration is constant and small relative to the substrate concentration, the initial-rate kinetics can be represented by
where u is the reaction velocity, dF'/dt; KM is the Michaelis constant; and kcat[EIOis the maximum velocity attained at saturating substrate concentrations. Here the subscript 0 refers to the total concentration in the system, and the subscript f refers to free, unbound concentration. The terms k,,, and I
