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Synergistic Effects of Surfactants on Kid Pregastric Lipase Catalyzed Hydrolysis Reactions† Douglas T. Lai and Charmian J. O’Connor* Department of Chemistry, The University of Auckland, Private Bag 92019, Auckland, New Zealand Received May 19, 1999. In Final Form: September 9, 1999 A combination kinetic model has been used to explain the dual characteristics of kinetic behavior of kid pregastric lipase (PGL) catalyzed hydrolysis of 4-nitrophenyl butyrate and tributyrin in aqueous solution in the presence of surfactants. At low concentrations of surfactant, the activity followed the behavior of an esterase, with a strong preference for catalyzing the hydrolysis of the water-soluble form of the substrate, and the presence of a surfactant served as a solublizer which dissolved and increased the concentration of this solubilized form. However, at high concentrations of surfactant, the PGL acted mainly as a lipase which catalyzed the hydrolysis of substrate aggregates. We have concluded that kid PGL acts as both a lipase and an esterase, and which of these factors is dominant is decided by the reaction conditions. By using this model, it has also been possible to explain the confusion which exists in the literature over the apparent effects of bile salts on the activity of preduodenal lipases. When a bile salt was present, it did not have a molecular interaction with the PGL, but served only as a surfactant in the catalyzed hydrolysis reaction which followed dissolution/encapsulation of the substrate in solutions of increasing bile-salt concentration.
1. Introduction Rennet pastes, prepared from the stomachs of calves, lambs, or kids slaughtered after suckling, were traditionally used in the manufacture of some Italian-style cheese varieties, both to coagulate the milk and to provide the desired “picante” flavor. In 1956 an oral lipase with lipolytic activity was discovered by Ramsey et al.1 They first use the term “pregastric esterase (PGL)” for the lipolytic and esterolytic activity secreted in the oral tissue of calf, a term based on its ability to catalyze the hydrolysis of esters. However, Richardson and Nelson2 found that calf pregastric esterase can catalyze the hydrolysis of triacetin both in aqueous solution and in emulsion media, characteristics which fit neatly into the narrow definition of a lipase. Long usage has established the term “pregastric esterase (PGE)” for any preduodenal lipolytic enzyme in a ruminant, but a more concise description is a “glycerol ester hydrolase of oral origin - which preferentially hydrolyzes short-chain fatty-acids from water insoluble triacylglycerols”.3 Even though the contribution of individual lipases to flavor profiles has been well studied, little research has been carried out on the fundamental kinetics of pregastric lipase catalyzed hydrolysis of esters and lipids. The work of Richardson and Nelson2 did not clarify the debate on terminology, since the hydrolysis of esters or soluble triacetin may have also been due to the presence of an esterase in the enzyme preparation. There is clear evidence for the presence of esterase components in some pregastric lipase extracts, and their presence largely depends on the * To whom correspondence should be addressed. Telephone: +64 9 3737599 ext. 8336. Fax: +64 9 3737422. Email: cj.oconnor@ auckland.ac.nz. † Part of the Special Issue “Clifford A. Bunton: From Reaction Mechanism to Association Colloids; Crucial Contributions to Physical Organic Chemistry.” (1) Ramsey, H. A.; Wise, G. H.; Tove, S. B. J. Dairy Sci. 1956, 39, 1312. (2) Richardson, G. H.; Nelson, J. H. J. Dairy Sci. 1967, 50, 1061. (3) Nelson, J. H.; Jensen, R. G.; Pitas, R. E. J. Dairy Sci. 1977, 60, 327.
methods used for extraction.4,5 The esterolytic component in the enzyme extracts showed those characteristics expected of an esterase, which only catalyzes the hydrolysis of water-soluble esters, e.g., p-nitrophenyl esters, but not monoacid triacylglycerols.6,7 However, the purified lipolytic component in the pregastric extract was capable of catalyzing the hydrolysis of both soluble substrates and water-insoluble aggregates. Thus, classification of an enzyme as a lipase or an esterase should only be made after consideration of both the type of substrate used and the type of reaction medium. Two hydrolysis reactions, hydrolysis of a substrate in solution and in aggregated or micellar form, may take place simultaneously in the same system, and the dominating effect will largely depend on the reaction conditions. In this paper, we summarize the effects of different surfactants on the lipase catalyzed hydrolysis reaction and postulate a mechanism for pregastric lipase catalyzed hydrolysis of soluble substrates and insoluble lipid aggregates. 2. Experimental Section 2.1. Chemicals. Purification of kid pregastric lipase (PGL) from commercial extracts followed the procedure described previously.5 The specific activity was 533 µmol min-1 per mg of enzyme, for the catalyzed hydrolysis of 8.6 mM tributyrin in 1% lecithin emulsion at pH 6.5, 35 °C. Several surfactants were used to study the enzyme-catalyzed reaction: hexadecyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), bis(2-ethylhexyl)sodium sulfosuccinate (AOT), l-R-lecithin (soybean lecithin), polyoxyethylene sorbitan monolaurate (Tween 20), sodium taurocholate (NaTC), isooctylphenoxy-polyethoxyethanol (Triton X-100). Stock solutions of surfactants were (4) Lai, D. T.; MacKenzie, A. D.; O’Connor, C. J.; Turner, K. W. J. Dairy Sci. 1997, 80, 2249. (5) Lai, D. T.; Stanley, R. A.; O’Connor, C. J. J. Am. Oil Chem. Soc. 1998, 75, 411. (6) O’Connor, C. J.; Barton, R. H. Lai, D. T. J. Bioactive Compat. Polym. 1996, 11, 43. (7) O’Connor, C. J.; Lai, D. T.; Sun, C. Q. J. Bioactive Compat. Polym. 1997, 12, 140.
10.1021/la990618q CCC: $19.00 © 2000 American Chemical Society Published on Web 11/17/1999
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prepared in 50 mM Bis-Tris buffer, pH 6.5, 25 °C, and the final pH was adjusted by addition of either dilute HCl or NaOH. 2.2. Enzymatic Hydrolysis of an Ester. The stock solution (500 mM) of substrate was prepared by dissolving p-nitrophenyl butyrate (PNPB) in predried acetonitrile. The PNPB solution (30 µL of different concentrations) and the surfactant solution were placed in a polycarbonate UV cuvette and made up to a final volume of 2970 µL, and the mixture was sonicated for 3 s by using a Kontes-Ultrasonic cell disrupter fitted with a 3 mm sonicator probe (Uniland, NJ). The purified kid PGL solution (0.4 mg mL-1, determined with a Bio-Rad protein assay kit) was diluted 20-fold with 50 mM Mes buffer, and the diluted enzyme solution (30 µL) was then added into the substrate solution and the initial rate of hydrolysis was determined for the first 3 min. The rate of background hydrolysis was similarly determined, but the time required for the assay was increased to 3 h to reduce the experimental error which might otherwise have arisen from the slow rate. It was not necessary to determine the background rate of hydrolysis for every individual condition, since the spontaneous rate of hydrolysis of PNPB (0-1 mM, at 35 °C) was typically ca. (0.6-0.9) × 10-3 A min-1, i.e., 0.5% of the typical enzyme-catalyzed rate of hydrolysis (∼0.2 A min-1). 2.3. Enzymatic Hydrolysis of a Lipid. Various amounts of the substrate, tributyrin, were added into 40 mL of surfactant solution, and the mixture was sonicated with the semi-micro sonicator probe until it became monodispersed. The pH of the emulsion was adjusted by adding NaOH solution (0.1 M) dropwise and allowed to equilibrate to the desired temperature. The enzyme solution (variable volume) was added and the release of butyric acid from tributyrin was measured by titrimetry (Mettler DL 21; Mettler Instruments AG, Greifensee, Switzerland) with 0.01 M NaOH as titrant. The initial rate of the catalyzed reaction was determined by measuring the rate of release of butyric acid for the first 6 min after addition of the enzyme.
3. Enzyme Kinetics In a micellar system, a hydrophobic substrate will be sequestered within the aggregates of surfactant molecules and the effective substrate concentration available for an enzyme-catalyzed hydrolysis reaction will not be equal to the total substrate concentration. The substrate concentration available for interaction with the enzyme will also be dependent upon the nature of the surfactant, the ratio of substrate to surfactant, and other reaction conditions, e.g., pH and temperature. Meanwhile, the active enzyme concentration will also vary in different emulsion media due to adsorption/desorption from the micellar surface. Thus, simple Michaelis-Menten kinetics are not able to explain the kinetics of the enzyme-catalyzed reaction in a micellar system. Two different sets of reaction conditions must be considered, i.e., at concentrations of surfactant above and below the critical micelle concentration (cmc). (a) Surfactant Concentrations Well above the cmc. A general kinetic model for lipolytic enzyme-catalyzed reactions at interfaces has been derived by Verger et al.8 On the basis of the model, the enzymic reaction can be divided into two steps: (1) the enzyme is bound or partitioned onto the interface by adsorption or penetration; (2) the catalyzed reaction then follows pseudo-MichaelisMenten kinetics at the interface. The reaction equation can be expressed as9,10
v)
Vmax[A][B] A
Ks KmB
+
KmB[A]
+ [A][B]
(1)
(8) Verger, R.; Mieras, M. C. E.; de Haas, G. H. J. Biol. Chem. 1973, 248, 4023. (9) Abousalham, A.; Nari, J.; Teissere, M.; Ferte, N.; Noat, G.; Verger, R. Eur. J. Biochem. 1997, 248, 374. (10) Deems, R. A.; Eaton, B. R.; Dennis, E. A. J. Biol. Chem. 1975, 250, 9013.
where the [A], [B], [D]o, and [S]o are the concentration of enzyme adsorbed on the surface of the micelle, the fraction of substrate absorbed on the surface, the total concentration of surfactant, and the total concentration of substrate, respectively. (b) Surfactant Concentrations below the cmc. When the surfactant concentration is less than the cmc, the micelle concentration will be zero, and the surfactant will remain in solution as the free monomer, which is capable of forming a substrate-surfactant complex. Consider that the substrate has only limited solubility in an aqueous medium; then the partitioning constant, KP, between the aqueous phase and the aggregates can be expressed as
KP )
[S] [S] ) [S] + [Sn] [Sn]
if [S] , [Sn]
(2)
where [S] and [Sn] are the concentrations of free substrate monomer and that of substrate aggregates. (1) Formation of Substrate-Surfactant Complex KSD
S + D y\z SD KSD ) [SD]/([S][D])
(3)
[SD] is the concentration of the substrate-surfactant complex, and the total substrate concentration, [S]o, equal to [S]o ) [SD] + [Sn] +[S]. Substitution of [Sn] and [S] into eqs 2 and 3 leads to a value for the concentration of the substrate-surfactant complex, [SD]
[SD] )
[S] )
(
[S]o 1 KP
1+ 1+
KSD[D]
)
[S]o
(
1+
1 + KSD[D] KP
)
(4)
(2) Enzyme-Catalyzed Hydrolysis of SubstrateSurfactant Complex. If the added surfactant does not inhibit the enzyme and the enzyme does not catalyze the hydrolysis of the substrate aggregates, the reaction scheme will then be 1
2
1
2
3
4
3
4
The enzyme-catalyzed reaction for the free substrate and substrate-surfactant complex can be treated by Michaelis-Menten kinetics, since the reaction takes place in a single phase. The total rate of hydrolysis consists of the rate of hydrolysis of the substrate monomer, vS, and that for hydrolysis of the substrate-surfactant complex, vSD.
v ) vS + vSD
(6)
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where
vSD )
Vmax[SD] KmDS + [SD]
)
Vmax[S]o (7) 1 1+ KP 1+ + [S]o KSD[D]
(
KmDS
)
and
vS )
Vmax[S] KmDS
+ [S]
) KmS
Vmax[S]o (8) 1 1+ + KSD[D] + [S]o KP
(
)
KmS is the Michaelis-Menten constant for the free substrate and KmSD the Michaelis-Menten constant for the substrate-surfactant complex. When [D] increases, vSD increases and vS decreases. The net effect on the rate of hydrolysis in the presence of a surfactant is an increase in the effective substrate concentration, provided that KmDS and KmS are of the same order of magnitude. However, if KmDS . KmS, the added surfactant will act as an inhibitor and decrease the total rate of enzymic reaction. For an enzyme-catalyzed reaction in an emulsion system, the effects from eqs 1 and 6 need to be considered, since they will depend on the ratio of surfactant to substrate. At low surfactant concentrations, the effect of eq 6 will be dominant, and the rate of catalysis will increase as the surfactant concentration increases, thereby causing an increase in the effective substrate concentration. On the other hand, the effective substrate concentration will decrease when the concentration of the surfactant further increases to values greater than the cmc, and there will be a decrease in the catalyzed rate in a micellar system with a high surfactant to substrate ratio. 4. Results and Discussion 4.1. Enzyme-Catalyzed Hydrolysis of PNPB. Pregastric lipases show little or no activation at an oil-water interface. The esterase fractions from lamb pregastric enzyme were found to be active only against the free substrate p-nitrophenyl-decanoate, and the rate of the catalyzed hydrolysis remained constant when the substrate concentration was increased beyond its critical micelle concentration.6 On the other hand, purified kid PGL catalyzed both the monomeric and aggregated forms of PNPB, but showed no interfacial activation against PNPB (data not shown). The rate of hydrolysis increased as the substrate concentration increased, and although the rate slightly increased as the concentration of substrate exceeded its solubility limit, the rate of hydrolysis of the monomer was comparable with that of the aggregates. A similar result with non-surface-activated lipases was also found, for example, with Candida antarctica component B (CALB).11 The characteristically strong preference by the pregastric enzyme for soluble esters as substrates was almost certainly the reason for the early terminology of this enzyme as a “pregastric esterase”, but this approach was rather simplistic since it did not seem to take into account the dual roles of the pregastric enzyme as a catalyst for the hydrolysis of both free and aggregated substrate. 4.2. Effect of Surfactants on the Activity of PGL in the Catalyzed Hydrolysis of PNPB. Since PGL has (11) Martinelle, M.; Hult, K. Biochim. Biophys. Acta 1995, 1251, 191.
a preference for catalyzing the hydrolysis of a monomeric substrate rather than its aggregated form, the monomeric pathway should be favored (eq 6) if the concentration of the monomer is increased by the addition of a surfactant to the medium. To reduce the complexity of the enzymesurface interaction, a study of the effect of surfactants was first carried out in the presence of 1 mM PNPB as substrate, i.e., at a concentration below the cmc of the substrate. At this concentration, the effect of an added surfactant would have been restricted to its interaction with the monomeric substrate or with the enzyme itself. Two nonionic surfactants, Triton X-100 and Tween 20, were used to examine the effect of the surfactant on the activity of the enzyme. Parts a and b of Figure 1 show the rate of hydrolysis of PNPB, catalyzed by kid PGL, against the concentration of Triton X-100 and Tween 20, respectively. Both surfactants showed significant enhancement of the rate of hydrolysis of PNPB at low surfactant concentrations and decreasing activity at a higher concentrations. In the presence of Triton X-100, the maximal activity was found at 0.7 mM, at which concentration ∼100% increase in activity was observed. As the concentration of the surfactant was increased beyond its cmc (0.3-0.6 mM), the activation effect decreased, and over 80% of the maximal activity was lost in the presence of 4 mM Triton X-100. This loss in activity is probably due to encapsulation of PNPB within the micelles of the added surfactant. At extremely high concentrations of surfactant, most of the free substrate would have been incorporated into the micellar phase and would thereby have become unavailable to PGL for the catalyzed reaction. Tween 20, however, had less effect on the enzyme-catalyzed hydrolysis reaction. There was approximately only a 35% increase in activity at a concentration close to the cmc (0.049 mM), and the decreasing effect on the activity at high surfactant concentrations was not as marked as it was for Triton X-100. A similar effect was also found when NaTC was used as the surfactant (Figure 1c). The activity was enhanced by 55% in 5.6 mM NaTC, and only 10% of the maximal activity was lost in 25 mM NaTC. The activation effect of surfactants on a lipase-catalyzed reaction has also been found for bile-salt-stimulated human-milk lipase (BSSL)13 and for pancreatic lipase and some other microbial lipases.14 In contrast to the mild interaction seen between the nonionic surfactants and NaTC and kid PGL, some ionic surfactants, e.g., SDS or AOT, bind strongly to most proteins at a concentration less than 25% of their cmc. Evidence of this binding is seen in the large inhibitory effect of these surfactants on the activity of the pregastric enzyme (Figure 2a,b). In the presence of these surfactants, the activity of kid PGL against PNPB quickly decreased as the concentration of the surfactant increased and no improvement in activity was observed. Complete inactivation was observed at concentrations of SDS and AOT equal to 1.0 and 0.7 mM, respectively, values which are approximately 12% and 28% of their cmc values. The effect of the cationic surfactant, CTAB, is different from that of the anionic surfactants, SDS and AOT, and its behavior is very similar to that of NaTC and the nonionic surfactants (Figure 2c). An activation effect was found at a concentration as low as 0.03 mM, when an 80% increase in activity was observed, and then the activity (12) Neugebaur, J. M. Methods Enzymol. 1990, 182, 239. (13) O’Connor, C. J.; Walde, P. Langmuir 1986, 2, 139. (14) Xia, J.; Chen, X.; Nnanna, I. A. J. Am. Oil Chem. Soc. 1996, 73, 115.
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Figure 1. Relative rates of hydrolysis of PNPB catalyzed by purified kid PGL at pH 6.5, 25 °C. Effect of the concentration of different types of surfactants: (a) Triton X-100; (b) Tween 20; (c) NaTC. The cmc value13 for each surfactant is shown as a dotted line.
quickly decreased as the concentration of CTAB increased, until over 90% of the activity was lost at a concentration of 0.5 mM. It is believed that the mechanism of initial activation of the enzyme activity by CTAB is similar to that discussed above for the nonionic surfactants. In contrast, when the phospholipid lecithin was used as the surfactant, no inhibition was observed within the experimental range of concentrations, but rather the rate of hydrolysis was increased almost 3-fold compared with the system without surfactant. Increasing the lecithin concentration further was not possible if the kinetics were assayed by the spectrophotometric method because the solution became turbid. Therefore a further study was made, using a titrimetric assay method. Since activation of PGL-catalyzed hydrolysis was found at concentrations of surfactants below their cmc values and when using a fully solubilized form of the substrate, no interfacial effect occurred in these systems. The activation effect of surfactant on an enzyme may be caused by (1) activation of the enzyme itself by surfactant binding or (2) interaction between the surfactant and the substrate to form a substrate complex which is more favored by the enzyme. However, since inhibition of enzyme activity was generally seen at high concentrations of the surfactants, it is unlikely that the surfactants stimulate the enzyme itself so that it adopts a more highly active state. It is possible that the slightly hydrophilic substrate, PNPB, will become aggregated to a small extract even at concentrations below its solubility limit. The presence of a surfactant will help to break down such aggregates, and thus the rate of hydrolysis will show a small increase. Figure 3 shows the effect of Triton X-100 and NaTC on the spontaneous hydrolysis of PNPB at pH 6.5, 25 °C. The rate of hydrolysis increased with low concentrations of
added surfactants and decreased when the surfactant concentration was greater than its cmc. The maximum rate of hydrolysis showed an increase of ∼30% at 0.33 mM Triton X-100, a concentration which is very close to the literature value of its cmc (0.30 mM).15 In comparison, the enzyme-catalyzed rate (Figure 2a) was ∼100% increased at the same concentration of Triton X-100. Thus, the added surfactant serves to solubilize the substrate, and it also serves to carry the substrate to the enzyme’s active site or to reorient the substrate into a better position for the enzyme-catalyzed reaction. The effect of Triton X-100 on the background rate of hydrolysis of PNPB can best be described by considering the effective water concentration. The spontaneous rate of hydrolysis of PNPB decreased as the concentration of Triton X-100 increased beyond its cmc. The PNPB encapsulated by the micellar Triton X-100 will effectively have restricted the access by water molecules, and thus the rate of hydrolysis will decrease. Restriction of water access into the body of the surfactant micelle can then used to explain why the inhibitory effect of a high surfactant concentration is not the result of a surfactantenzyme interaction but is instead a consequence of limitations imposed on the other substrate, water, and on its interaction with the acyl-enzyme complex. At 5 mM Triton X-100, the rate of spontaneous hydrolysis was 70% of that seen in the absence of surfactant, or 55% of the maximum observed rate. If PGL is a “true” esterase, which does not then have the ability to hydrolyze the substrate in its aggregated micellar form, the decrease in the catalyzed rate should be proportional to the decrease in the free substrate concentration. However, if one (15) Carey, M. C.; Small, D. M. Am. J. Med. 1970, 49, 590.
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Figure 2. Relative rates of hydrolysis of PNPB catalyzed by purified kid PGL at pH 6.5, 25 °C. Effect of the concentration of different types of ionic surfactants: (a) SDS; (b) AOT; (c) CTAB; (d) lecithin.
Figure 3. Effect of Triton X-100 and NaTC on the initial rate of spontaneous hydrolysis of 1 mM PNPB at pH 6.5, 25 °C.
calculates the effective depletion of substrate from the amount of micelle formation, then only a 15% increase of rate should be seen in the presence of PGL. The net gain of activity must imply that the PGL has the ability to act as a lipase, which then has only restricted activity against substrate-surfactant aggregates. The behavior of micelles of the bile salt, NaTC, differs extensively from that of the other surfactants. It has not been possible to define a distinct value of the cmc for NaTC despite using many experimental methods for the assay. cmc values have been reported to lie within the large range of 1.5-15 mM.15 Several authors have suggested that dimeric, trimeric, and tetrameric aggregates are formed in a premicellar solution of the bile salt and that these then serve as building blocks for larger micellar aggregation.16 Given this background, we can then suggest that
the PNPB molecules are adsorbed onto small aggregates at concentrations of bile salt within the range of the determined values of the cmc and that these cause a slight decrease in the rate of either the spontaneous hydrolysis reaction or the kid PGL catalyzed hydrolysis reaction. However, the loose (linear) aggregates formed by NaTC do not prevent penetration of the water molecules into the enzyme-micelle complex, and the rate of hydrolysis, even at 25 mM NaTC (which is much greater than the cmc value) is still significantly higher than that without surfactant (Figure 3). When the concentration of the substrate is greater than its solubility limit, then the addition of surfactant to the solution, at a concentration either close to or above its cmc, will give rise to the formation of larger aggregates between the substrate and the surfactant. The ensuing turbidity, which will increase with increasing concentrations of both surfactant and substrate as a consequence of formation of these aggregates, will interfere with the absorption of released p-nitrophenolate anion and make impossible the measurement of the rate of catalyzed hydrolysis. In order, therefore, to determine the effect of a surfactant in a micellar solution containing a substrate at its saturation concentration, the titrimetric method would be a better candidate for determination of enzymic activity. Such a study is described in the next section. 4.3. Effect of Surfactants on Hydrolysis of Tributyrin. When purified kid and goat PGL were placed into a surfactant-free tributyrin emulsion, then no significant activity was detected; i.e., there was no hydrolysis of tributyrin. There are two main reasons for inactivity of an enzyme in a surfactant-free system. Enzyme stability (16) O’Connor, C. J.; Ch’ng, B. T.; Wallace R. G. J. Colloid Interface Sci. 1983, 95, 410 and references cited therin.
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Figure 4. Effect of surfactant concentration on the apparent saturation rate of hydrolysis of tributyrin catalyzed by purified kid PGL at pH 6.5, 35 °C.
has been found to be very dependent on the concentration of surfactant or protein in the reaction medium, and purified calf pregastric lipase was quickly denatured at an oil-water interface.17 However, since the crude enzyme extract showed marginal activity against tributyrin, even in the absence of added surfactants or proteins, it is believed that the proteins which coexisted in the enzyme extract played an essential role in protecting the enzyme from direct contact with the oil phase. The ability of an enzyme to catalyze a reaction on a pure oil-water interface in the absence of a surfactant is largely dependent on the nature of the enzyme itself, and even lipases in the same family will behave differently. Several pancreatic lipases have been tested for their ability to catalyze the hydrolysis of dicaprin and tributyrin, and some of them were found to be denatured at the interface.19 Since the lipase component is less active against the PNPB aggregate than against the soluble monomeric form, then the addition of surfactant becomes necessary for stabilizing the emulsion, thereby protecting the enzyme from denaturation and providing a solubilized substrate for the enzyme-catalyzed reaction. When low concentrations of surfactants (below their cmc) were added into the tributyrin-water emulsion under conditions of tributyrin saturation (∼8 mM), then the rate of hydrolysis was initially increased as the concentration of the surfactants was increased. However, the rate data derived from the catalyzed hydrolysis were not reproducible in replicate experiments. The scattered results may be due to the instability of the emulsion and denaturation of the enzyme, factors which are the main drawbacks to determination of enzymic activity by the titrimetric method, for which the existence of a consistent, stable emulsion is crucial for reliable detection. Therefore the study of the enzyme-catalyzed reaction in different surfactant media was carried out at concentrations of the surfactants above their cmc values. Since the hydrolysis reaction took place in an aqueous medium and PGL has a strong preference for the solubilized form of the substrate, the dependency of rate on substrate concentration was fitted by eq 8. Figure 4 shows the effect of surfactant concentration on the apparent Vmax (17) De Caro, J.; Ferrato, F.; Verger, R.; De Carol, C. A. Biochim. Biophys. Acta 1995, 1252, 321. (18) Gargouri, Y.; Bensalah, A.; Douchet, I.; Verger, R. Biochim. Biophys. Acta 1995, 1257, 223. (19) Fang, Y.; Dalgleish, D. G. J. Colloid Interface Sci. 1992, 156, 329.
Lai and O’Connor
Figure 5. Effect of surfactant concentration on the apparent Michaelis-Menten constant, Kmapp, for hydrolysis of tributyrin catalyzed by purified kid PGL at pH 6.5, 35 °C.
values for the catalyzed hydrolysis of tributyrin by purified kid PGL in the presence of four different surfactants: lecithin, Tween 20, NaTC, and casein. The saturation rates were found to be unchanged with increasing concentrations of NaTC, Tween 20, and casein, but they increased a little as the concentration of lecithin increased. However, the apparent Michaelis-Menten constant, Kmapp, varied with surfactant concentration (Figure 5). The Kmapp values for the systems with lecithin or casein as surfactant were only slightly changed as the surfactant concentration increased. However, when Tween-20 or NaTC was used in the medium, the Kmapp value changed significantly as the concentration of surfactant was increased. Because of the complex catalytic behavior of pregastric lipase, there were some difficulties in using the MichaelisMenten relationship to explain the enzymic reaction in a micellar system. With respect to the esterase character of PGL, the catalyzed rate of hydrolysis of tributyrin will depend largely on the concentration of the free tributyrinsurfactant complex, which, in turn, is proportional to the surfactant concentration below its cmc but remains constant as the micelle starts to form. The actual effective substrate concentration will be equal to the concentration of the tributyrin-surfactant complex, which will be dependent upon the type of surfactant used. Since the substrate will be partitioned between the free monomer and the micelle, then at a given substrate concentration, the concentration of substrate actually solubilized will decrease as the surfactant concentration is increased and the Kmapp value for a system will represent half the substrate-surfactant concentration at saturation. In this hypothesis, increasing the surfactant concentration over its cmc does not contribute greatly to enhancement of activity, and thus the saturation rate, Vs, is independent of surfactant concentration. At high concentrations of surfactant, the concentration of the free substrate-surfactant complex is close to zero. Thus the substrate concentration at the oil-water interface will dominate the rate of catalyzed hydrolysis and will be influenced by the total surface area or micellar size. If one takes the casein micelle as an example, then the average droplet size of the casein-oil micelle decreases as the concentration increases, and the micellar size remains constant at concentrations of casein greater than 1% (w/v).18 The significant increase in surface area which results from a reduction in the size of the micelle causes
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Figure 6. Double-reciprocal plots of initial rate of hydrolysis of tributyrin against the concentrations of tributyrin in the presence of Tween 20 (w/v).
a boost to the rate of hydrolysis, especially at low concentrations of surfactant. Dependency on surface area is more significant for a “true” lipase, which has little or no activity against the soluble form of the substrate. If the enzyme only reacts with the substrate in the micellar phase, Vmaxapp will depend only on KmB, the affinity between the enzyme and the substrate-micelle complex. For the same surfactant, KmB is independent of concentration, and the true Vmax value may be obtained from a double reciprocal plot of initial rate and surfactant concentration (Figure 6). However, this relationship breaks down when the surfactant concentration approaches the cmc and the contribution from the esterase reaction becomes more dominant. One of the primary assumptions made in using eq 1 to explain the kinetic behavior is that the enzyme-catalyzed hydrolysis reaction follows simple Michaelis-Menten kinetics. This presupposes that the rate of water diffusion through the surfactant layer is infinite. However, hydrolysis of the substrate will then depend on the accessibility of water molecules through the surfactant layer. The spontaneous hydrolysis of PNPB in a Triton X-100 micelle (Figure 3) suggests that (1) the concentration of free monomer of PNPB decreases as the concentration of surfactant increases and (2) the rate of hydrolysis of PNPB in its aggregated form is much less than that of the free monomers, implying that the surfactant molecules actually hinder access of water to the substrate. Thus, at high surfactant concentrations, the aggregated micelle-solubilized form of the substrate is largely excluded from access by water, and the effective water concentration within the micelle must be considered.
Figure 7. Diagrams showing the relationship of the substrate distribution and the rate of hydrolysis catalyzed by PGL against the concentration of added surfactant.
In summary, the interrelationships existing in the pregastric lipase catalyzed hydrolysis of a lipid substrate may be illustrated as shown in Figure 7. The presence of an efficient surfactant is essential for the PGL-catalyzed reaction. At low surfactant concentrations, the rate of hydrolysis is dependent on the soluble form of the lipidsurfactant complex, with little contribution from the aggregated form of the lipid. As the surfactant concentration increases, the effect of hydrolysis at the surface of the micelle will gradually increase and the contribution from the homogeneous catalyzed pathway will slowly disappear. The profile for the net effect of these two mechanisms is similar to that seen in typical Michaelis-Menten kinetics in homogeneous solution and may well account for the original terminology of calling the pregastric enzyme an “esterase” rather than a “lipase”. From the study described above, the pregastric lipase is an enzyme whose behavior reflects its dual functions, which are characteristic of both a lipase and an esterase, and whose dominant character is more likely to be dependent on the environment pertaining to the particular reaction under consideration, than on the enzyme itself. Acknowledgment. We acknowledge financial assistance from the University of Auckland Research Committee and New Zealand Lottery Science. We are especially grateful to Marschall Products, Rho˜ne-Poulenc, for the kind gift of the kid enzyme extract. LA990618Q
