Interaction of Rhizomucor miehei Lipase with an Amphoteric

The interactions of surfactants with the enzyme Rhizomucor miehei lipase were studied using surface tension measurements and ellipsometry. From earlie...
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Interaction of Rhizomucor miehei Lipase with an Amphoteric Surfactant at Different pH Values Britta Folmer,† Krister Holmberg,* and Martin Svensson‡ Institute for Surface Chemistry, Box 5607, SE 11486 Stockholm, Sweden Received December 10, 1996. In Final Form: August 7, 1997X The interactions of surfactants with the enzyme Rhizomucor miehei lipase were studied using surface tension measurements and ellipsometry. From earlier surface tension studies it is known that the cationic surfactant didodecyldimethylammonium bromide forms an aggregate with R. miehei lipase, while anionic and nonionic surfactants do not form such a complex. In this work the influence of surfactant charge on complex formation with the lipase is systematically investigated, using the amphoteric surfactant sodium N-(2-hydroxydodecyl)sarcosinate. The charge of the amphoteric surfactant was varied from anionic via zwitterionic to cationic by adjusting the pH. By this procedure the influence of the surfactant charge on the interaction with the enzyme could be studied while the molecular structure of the surfactant hydrophobe was kept unchanged. It was confirmed that only when the head group is positively charged does the enzyme bind with the surfactant. However, the number of surfactant molecules that bind to each enzyme is dependent on the surfactant structure. A tentative interaction model is presented. Below the critical micelle concentration binding occurs with negatively charged sites on the enzyme interacting with cationic surfactant head groups. It is likely that a hydrophobic domain adjacent to the negatively charged site of the enzyme enables a hydrophobic attraction between the tail of the cationic surfactant and the enzyme.

Introduction Enzymes are used together with surfactants in detergents and other formulated products. In order to make optimum use of both components, they must be largely compatible with each other. Complex formation of surfactant with enzyme can change the activity of the enzyme.1-6 In a recent review article on enzymesurfactant interactions it is stated that surfactants at high concentrations denature enzymes and that anionic surfactants in general are the most deactivating whereas nonionics give the least disruption of enzyme structure.7 It has been shown that for some enzymes the deactivation is irreversible while other enzymes regain their activity upon dilution or salt addition.3 In order to understand the mechanism of the surfactantinduced deactivation of enzymes, the interaction of enzymes with different types of surfactants needs to be systematically studied. Various types of enzyme-surfactant interactions have previously been reported in the literature.2, 7-10 Interactions between lipase and surfactants have recently been investigated by Wannerberger et al., who found complex formation between Humicola lanuginosa lipase and the anionic surfactant sodium dodecyl sulfate (SDS).4,11 Skagerlind et al. showed the * Tel: +46 8 790 9900. Fax: +46 8 20 8998. E-mail: krister. [email protected]. † E-mail: [email protected]. ‡ E-mail: [email protected]. X Abstract published in Advance ACS Abstracts, October 1, 1997. (1) Skagerlind, P. Jansson, M. J. Chem. Tech. Biotechnol. 1992, 54, 277. (2) Borgstro¨m, B.; Donne´r, J. J. Lipid Res. 1976, 17, 491. (3) Schomacker, R.; Robinson, B. H.; Fletcher, P. D. I. J. Chem. Soc., Faraday Trans. 1 1988, 84, 4203. (4) Wannerberger, K.; Arnebrant, T. Colloids Surf. B 1996, 7, 153. (5) Rao, Y. K.; Bahadur, P.; Bahadur, A.; Ghosh, S. Ind. J. Biochem. Biophys. 1989, 26, 390. (6) Skagerlind, P.; Holmberg, K. J. Disp. Sci. Technol. 1994, 16, 317. (7) Rubingh, D. N. Curr. Opinion Colloid Interface Sci. 1996, 1 , 598. (8) Edwards, K.; Chan, R. Y. S.; Sawyer, W. H. Biochemistry 1994, 33, 13304. (9) Jones, M. N.; Manley, P. J. Chem. Soc., Faraday Trans. 1 1979, 75, 1736. (10) Sen, M.; Mitra, S. P.; Chattoray, D. K. Colloids Surf. 1981, 2, 259. (11) Wannerberger, K.; Wahlgren, M.; Arnebrant, T. Colloids Surf. B 1996, 6, 27.

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formation of a complex between Rhizomucor miehei lipase and the cationic surfactant didodecyldimethylammonium bromide.6 The aim of the present study was to extend our knowledge on how the interaction between R. miehei lipase and surfactant is influenced by surfactant charge. By varying the pH of an amphoteric surfactant solution, the surfactant net charge can be changed from positive via neutral to negative. By this approach, the influence of charge on the interaction can be investigated while the surfactant hydrophobe structure remains constant. The techniques used in this work are tensiometry and ellipsometry. Materials and Methods Highly purified lipase from the fungus R. miehei was kindly provided by Novo Nordisk, Denmark. R. miehei lipase (from here on called RM lipase) is a single chain protein consisting of 269 amino acids with a total molecular weight of 29 472 and an isoelectric point of 3.5.12,13 In this study, enzymes of two different batches were used. The first batch, which was obtained as a crystalline powder, gave a surface tension of 45 mN m-1 at the concentration of 0.05 g dm-3. The other enzyme batch was obtained as a solution in a 25 mM Tris-acetate buffer. This enzyme, which was claimed to be purer than the first batch, showed a much lower surface activity, the surface tension at 0.05 g dm-3 being 60 mN m-1. The surface tension measurements reported here of surfactant solutions and of solutions of mixtures of lipase and surfactant were obtained with the first enzyme batch. For comparison, surface tension plots were recorded for two surfactants, one anionic and one cationic, also in the presence and absence of lipase of the second batch. In the ellipsometry studies the purer enzyme of the second batch was used after further purification from Tris-acetate buffer by dialysis and freeze drying. An amphoteric surfactant, sodium N-(2-hydroxydodecyl)sarcosinate, kindly provided by Akzo Nobel Surface Chemistry AB, and a cationic surfactant, didodecyldimethylammonium bromide (DDDMAB) (purchased from Eastman Kodak Co.), were used to study aggregate formation with RM lipase. All measurements were performed in 1 mM sodium phosphate buffer. (12) Brady, L.; Brzozowski, A. M.; Derewenda, Z. S.; Dodson, E.; Tolley, S.; Turkenburg, J. P.; Christiansen, L.; Huge-Jensen,B.; Norskov, L.; Thim, L.; Menge, U. Nature 1990, 343, 767. (13) Personal communication. Per Falholt, Novo Nordisk, Denmark.

© 1997 American Chemical Society

R. miehei Lipase Interaction with a Surfactant

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Figure 1. Amphoteric surfactant sodium N-(2-hydroxydodecyl)sarcosinate carrying a positive, net zero, and negative charge. The critical micelle concentration was determined by measuring the reduction in air-water interfacial tension as a function of surfactant concentration. A KSV Sigma 70 instrument using a du Nou¨y ring was used for the measurements, employing the Zuidema-Waters correction method for the ring. Surfactant addition was done with a Methrom dosimat titration unit. The surface tension measurements of surfactant only were carried out starting with a 1 mM sodium phosphate buffer, to which the surfactant in 1 mM phosphate buffer was added. Measurements in the presence of enzyme were made with a solution of 0.05 g dm-3 lipase in 1 mM phosphate buffer, to which a concentrated solution of surfactant in buffer containing the same concentration of enzyme was added. Surface tension measurements were performed in basic, neutral, and acidic solutions. Ellipsometry is a technique to measure the adsorbed amount and the thickness of a thin film at a solid-liquid interface. The method is based on the measurement of changes of ellipticity of polarized light upon reflection at this interface.14,15 The ellipsometry measurements were performed by means of so-called null ellipsometry.14,15 The instrument used was a Rudolph thinfilm ellipsometer, type 436, controlled by a computer. A xenon lamp, filtered to 4015 Å, was used as the light source. Ellipsometry measurements were performed on hydrophobized silica plates.16 The plates were cleaned by treatment for 5 min in boiling 25% HCl:30% H2O2:H2O ) 1:1:5 (at 80 °C), after which the plates were rinsed five times with millipore water. They were then treated for 5 min in boiling 25% NH3:30% H2O2:H2O ) 1:1:5 (at 80 °C), and rinsed five times with millipore water and four times with absolute ethanol. Subsequently, the plates were rinsed three times with trichloroethylene before they were methylated by immersing in a solution of 0.1 wt % dichlorodimethylsilane in trichloroethylene for 90 min. Finally, the plates were rinsed again five times with trichloroethylene and two times with ethanol. They were stored in absolute ethanol until use. The cuvette was carefully cleaned and filled with buffer. Before the plates were immersed in the cuvette, they were rinsed in ethanol and water and blown dry with nitrogen gas. The ellipsometric angles ∆ and ψ for the clean surface were recorded from the buffer solution before the surfactant was added. Surfactant was added to the buffer solution in the cuvette, until a concentration of 1.5 times the cmc had been reached. The adsorption of surfactant on the hydrophobized silica plate was recorded. After 3600 s lipase solution was added, giving a concentration of 0.05 g dm-3 in the cuvette, and the enzyme adsorption was monitored. Calculation of the adsorbed amount was done with de Feijters formula, using a refractive index increment of 0.15 cm3 g-1, which is a common value for surfactants.17 The refractive index increment for lipase is 0.19 cm3 g-1.18

Results Surface tension vs RM lipase concentration was measured from 0 to 0.1 g dm-3 to find the enzyme concentration at which the surface tension reaches a plateau value. It was found that at a concentration above 0.01 g dm-3 the surface tension varied insignificantly with lipase concentration. Hence, for the measurements a lipase concentration of 0.05 g dm-3 was used. (14) Azzam, R. M. A.; Bashara,N. M. Ellipsometry and Polarized Light; Elsevier: Amsterdam, 1989; Chapter 6. (15) Arnebrant, T. Ph.D. Thesis, Lund University, Lund, Sweden, 1987. (16) Malmsten, M. Colloids Surf. B 1995, 3, 297. (17) van Os, N. M.; Haak, J. R.; Rupert, L. A. M. Physical Chemical Properties of Selected Anionic, Cationic and Nonionic Surfactants; Elsevier: Amsterdam, 1993. (18) Malmsten, M. J. Colloid Interface Sci. 1995, 172, 106.

Figure 2. Surface tension vs log concentration of the amphoteric surfactant sodium N-(2-hydroxydodecyl)sarcosinate at pH 10.5; (O) surfactant only; (b) surfactant + RM lipase.

Figure 3. Surface tension vs log concentration of the amphoteric surfactant sodium N-(2-hydroxydodecyl)sarcosinate at pH 7.5; (O) surfactant only; (b) surfactant + RM lipase.

The surfactant, sodium N-(2-hydroxydodecyl)sarcosinate, is shown in Figure 1. Surface tension reduction of this amphoteric surfactant was measured at three different pH values: 3, 7.5, and 10.5, at which the surfactant carried a positive, net zero, and negative charge, respectively. The surface tension vs logarithmic surfactant concentration graph was compared with that for surfactant in the presence of lipase. At high pH the amphoteric surfactant behaves as an anionic surfactant. With increasing concentration the surface tension curve without enzyme approaches the curve with enzyme, as is shown in Figure 2. At neutral pH, when the surfactant is neutral, the surface tension plots with and without enzyme coincide at a higher concentration (Figure 3). At low pH the amphoteric surfactant is positively charged. As shown in Figure 4, the surface tension in the presence of enzyme remains at an almost constant value until a relatively high surfactant concentration is reached before it decreases steeply. The surface tension plots with and without surfactant cross each other. This behavior is indicative of aggregate formation between the enzyme and the positively charged surfactant at surfactant

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Figure 4. Surface tension vs log concentration of the amphoteric surfactant sodium N-(2-hydroxydodecyl)sarcosinate at pH 3; (O) surfactant only; (b) surfactant + RM lipase.

Figure 6. Surface tension vs log concentration of the cationic surfactant didodecyldimethylammonium bromide at pH 7.5; (O) surfactant only; (b) surfactant + RM lipase.

Figure 5. Surface tension vs log concentration of the cationic surfactant didodecyldimethylammonium bromide at pH 10.5; (O) surfactant only; (b) surfactant + RM lipase.

Figure 7. Surface tension vs log concentration of the cationic surfactant didodecyldimethylammonium bromide at pH 3; (O) surfactant only; (b) surfactant + RM lipase.

concentrations below the critical micelle concentration. Extensive surfactant packing at the air-water interface with a concommitant decrease in surface tension will not occur until the enzyme, both in solution and at the surface, is “saturated” with surfactant molecules. The more surface active surfactants will then remove the enzymesurfactant complexes from the surface. In order to ascertain that the two enzyme batches behaved similarly in the tensiometry experiments, measurements were performed with both lipases in combination with either an anionic surfactant, sodium bis(2ethylhexyl)sulfosuccinate (AOT), or a cationic surfactant, DDDMAB. The two sets of experiments gave very similar results, both showing complex formation between lipase and DDDMAB but not between lipase and AOT, as has been published before.6 To ensure that formation of the enzyme-surfactant complex at low pH, when the surfactant is positively charged, was not due merely to charge variations of the enzyme, surface tension plots of the cationic surfactant didodecyldimethylammonium bromide were also recorded in basic, neutral, and acidic solutions (Figures 5-7). As expected, the surface tension vs log concentration plots of surfactant only, did not show any significant pH dependence. In the curves for the systems containing both enzyme and cationic surfactant, on the other hand, there is a trend toward lower surfactant concentration before the onset of surface tension reduction with decreasing pH. The arrows on the curves indicate the surfactant

concentration at which the enzyme is saturated with surfactant at that specific pH. Adsorption of surfactant on hydrophobized silica was measured by ellipsometry. After stabilization, subsequent addition of RM lipase was recorded. As in the previous surface tension studies, the amphoteric surfactant was used at three pH values, viz., 10.5, 7.5, and 3. In basic and neutral environments the surfactant adsorbs in an amount of approximately 0.70 (0.75 and 0.65) mg m-2, respectively (Figures 8 and 9). Addition of lipase after 3600 s gives no change in adsorbed amount. Under acidic conditions the situation is strikingly different. The adsorbed amount of surfactant was 0.5 mg m-2. Addition of lipase after 3600 s increases the adsorbed amount to 2.55 mg m-2, as can be seen in Figure 10. A control experiment with the cationic surfactant didodecyldimethylammonium bromide was performed at pH 7.5 (Figure 11), again to ascertain that complex formation of the amphoteric surfactant under acidic but not under neutral or alkaline conditions was not merely due to changes in net charge of the enzyme. A plateau value of adsorption of 1.15 mg m-2 is measured with only surfactant; after addition of enzyme the adsorbed amount increased to 6.3 mg m-2. An attempt was made to perform the adsorption measurement in the reverse order, i.e., first adsorb the lipase and then allow the three surfactants to bind to the protein-covered surface. This procedure proved unsuitable, however, since even after 16 h incubation with the enzyme the coverage of the hydrophobic surface was poor.

R. miehei Lipase Interaction with a Surfactant

Figure 8. Adsorption of the amphoteric surfactant sodium N-(2-hydroxydodecyl)sarcosinate at pH 10.5 and the effect of sequential addition of RM lipase (at 3600 s) vs time onto a hydrophobic surface.

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Figure 11. Adsorption of the cationic surfactant didodecyldimethylammonium bromide at pH 7.5 and the effect of sequential addition of RM lipase (at 3600 s) vs time onto a hydrophobic surface.

Discussion and Conclusions

Figure 9. Adsorption of the amphoteric surfactant sodium N-(2-hydroxydodecyl)sarcosinate at pH 7.5 and the effect of sequential addition of RM lipase (at 3600 s) vs time onto a hydrophobic surface.

Figure 10. Adsorption of the amphoteric surfactant sodium N-(2-hydroxydodecyl)sarcosinate at pH 3 and the effect of sequential addition of RM lipase (at 3600 s) vs time onto a hydrophobic surface.

Evidently, the driving force for adsorption of the relatively non-surface-active protein at the hydrophobic surface is not very strong.

The methods used in this work to detect proteinsurfactant interaction, tensiometry and ellipsometry, analyze phenomena at the air-water and solid-water interfaces, respectively. Although the procedures do not give direct information about association in bulk solution, it is reasonable to believe that the results, in qualitative terms, can be extrapolated to illustrate events in solution. Measurements of effects of lipase on heat of micellization of the surfactants would be a method to monitor directly surfactant-enzyme association in the bulk. We have recently initiated this type of study using microcalorimetry. In this work it is clearly shown that RM lipase forms a complex with positively charged surfactants, but not with surfactants carrying a negative or net zero charge. It is also clearly demonstrated that the enzyme-surfactant complex forms in solution at pH values both above and slightly below the isoelectric point of the lipase. Thus, positively charged surfactants associate also with lipase carrying a net positive charge. Negatively charged surfactants, on the other hand, seem not to form a complex with lipase in the pH range studied. The above results imply that the complex formation between RM lipase and positively charged surfactants is due to a combination of electrostatic and hydrophobic interactions. Such combined interaction may not be possible for anionic surfactants. This would be the case if the enzyme contained negatively charged sites in the vicinity of hydrophobic domains but lacked sites of positive charge with such an environment. This issue will be further investigated. Complex formation between cationic surfactant and lipase also below the isoelectric point of the enzyme may be due to the presence of carboxylic groups in the enzyme with very low pKa values situated adjacent to hydrophobic amino acid residues. Alternatively, such complex formation may be the result of deprotonation of protein carboxylic groups during the association with oppositely charged surfactants. Complex formation between cationic surfactant and oppositely charged hydrophobically modified polyelectrolyte at pH values below the pKa of the acid groups has been reported in the literature.19,20 An illustration of the interactions of cationic surfactants with the enzyme in solution and at the air-water interface (19) Shimizu, T.; Seki, M.; Kwak, J. C. T. Colloids Surf. 1986, 20, 289.

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Figure 12. Illustration of the interactions of cationic surfactants with enzyme at the air-water interface: (a) enzyme surfactant complex formed; (b) no enzyme surfactant complex formed.

is shown in Figure 12. When only the enzyme is present, there is an equilibrium between enzyme at the surface, giving rise to surface tension reduction, and enzyme in solution (stage I). When a small amount of the cationic surfactant is added, binding between the enzyme and surfactant will occur as a result of a combination of electrostatic attraction and hydrophobic interaction. Binding of surfactant to the enzyme at this stage does not lead to a significant change in surface tension (stage II). During stage II there is a gradual replacement of enzyme by surfactant at the surface, and when the surface tension starts to drop, at point III of the curve, the surface is almost totally covered by the more surface active surfactant. The slopes of the surface tension vs log concentration plots between points III and IV are the same both in the absence and presence of enzyme (Figures 2-4). This indicates that only surfactant is present at the surface in both instances. With the nonionic or anionic surfactants, stage II does not occur. Hence, the surface tension vs log concentration curve in the presence of enzyme will follow the curve with only surfactant when stage III is reached. This interaction model is largely in accordance with other views on enzyme-surfactant complex formation.2,8-10,21 For instance, Edwards et al.8 claim that the driving force for complex formation is the hydrophobic attraction but that specific binding below the cmc only occurs when an electrostatic attraction takes place in combination with hydrophobic interaction. It is interesting that at high pH, when the sarcosinate surfactant carries a negative net charge and does not form a complex with the enzyme, the combination of surfactant and lipase is considerably more surface active than the surfactant alone. (The surface tension is lower for the combination than for surfactant alone at all surfactant concentrations up to the cmc.) Evidently, the presence of protein at the air-water interface promotes enrichment of surfactant molecules at the surface, possibly due to formation of some kind of mixed surface. Such behavior has been observed for polyelectrolytes with a charge opposite that of the surfactant, e.g., for cationic starch in combination with sodium dodecyl sulfate.22 The surface tension studies of the permanently cationic surfactant DDDMAB together with RM lipase at different (20) Goddard, E. D.; Anathapadmanabhan, K. P. Interactions of Surfactants with Polymers and Proteins; CRC: Boca Raton, FL, 1993; Chapter 5. (21) Treves, C.; Vincenzini, M. T.; Favilli, F.; Vanni, P.; Baccari,V. Can. J. Biochem. Cell. Biol. 1984, 62, 55. (22) Stenius, P.; Merta, J.; Saarinen, T. To be published.

Folmer et al.

pH values cast further light on the surfactant-lipase interaction process. Figures 5-7 show that the surfactant concentration at which the surface tension starts to fall in the presence of enzyme (point III of Figure 12) is lower the lower the pH. Thus, the more positively (and less negatively) charged the enzyme, the fewer surfactant molecules are involved in aggregate formation. This is the expected mode of action. It is noteworthy that also at pH 3.0, i.e., below the isoelectric point of the enzyme, surfactant-enzyme aggregation occurs, as is evidenced by the fact that the two curves in Figure 7 cross each other. This behavior is analogous to that discussed above for the amphoteric surfactant and lends support to the interaction model expressed in Figure 12. Using the ellipsometry results and assuming that a secondary layer of surfactant is not formed on top of the enzyme, the number of surfactant molecules per enzyme can be calculated. For the zwitterionic surfactant (M ) 295) in acidic solution the adsorbed amount of surfactant was 0.5 mg m2, equal to 1.7 × 10-3 mmol m-2. Addition of enzyme (M ) 29 472) gave an additional adsorption of 2.05 mg m-2, equal to 7.0 × 10-5 mmol m-2. Hence, the calculated number of amphoteric surfactants per enzyme is 24. The ellipsometry results do not reveal whether the protein is bound as single molecules distributed over the surfactant surface or whether the enzyme forms small aggregates with surfactants at the surface, the formation of which being induced by sites on the protein that contain both a hydrophobic domain and a negative charge, as discussed above. The values of the adsorbed amount of surfactant that was obtained by ellipsometry, between 0.5 and 0.70 mg m-2, are reasonable for a monolayer. The high amount of lipase that binds to the hydrophobic surface pre-exposed to cationic surfactant is noteworthy. Whereas an enzyme adsorption of 2.05 mg m-2 is obtained on the surface exposed to the amphoteric surfactant at low pH (when the surfactant is effectively cationic in charge), 5.15 mg m-2 adsorbs on the surface exposed to the (permanently) cationic surfactant. A common value for monolayer adsorption of the enzyme is 2-3 mg m-2.18 Thus, the cationic surfactant DDDMAB gives an adsorbed amount almost twice the expected value. The difference in adsorbed amount on the two surfactant-exposed surfaces is most likely caused by the difference in pH at which the experiments have been made. The measurement with the zwitterionic surfactant was performed at pH 3, which is below the lipase isoelectric point of 3.5.13 The measurements with DDDMAB were made at neutral pH where the enzyme carries a surplus of negative charges. In the latter case it is likely that after adsorption of the enzyme on top of the surfactant layer more surfactant will bind to the negatively charged protein layer, giving rise to a surfactant-enzyme-surfactant sandwich structure. One should keep in mind that in the ellipsometry experiments the enzyme is added to the cell containing adsorbed surfactant in equilibrium with surfactant in solution. Thus, there are surfactants available in the bulk solution for such a second adsorption step. The present study does not address the question of the structure of the lipase-surfactant complex. The literature contains several suggestions for the models of such aggregates.23-26 Some of these, such as the “necklace (23) Reynolds, J. A.; Tanford, C. Proc. Natl. Acad. Sci. U.S.A. 1970, 66, 1002. (24) Shirama, K.; Tsujii, K.; Takagi, T. J. Biochem. 1974, 75, 309. (25) Lundahl, P.; Greijier, E.; Sandberg, M.; Cardell, S.; Eriksson, K. O. Biochim. Biophys. Acta 1986, 837, 20. (26) Ibel, K.; May, R. P.; Kirschner, K.; Szadkowski, H.; Mascher, E.; Lundahl, P. Eur. J. Biochem. 1990, 190, 311.

R. miehei Lipase Interaction with a Surfactant

model” 23 and the “flexible helix model”,24 involve protein unfolding to such an extent that the biological activity is likely to be severely affected. We have earlier reported that the lipase activity is much lower in the presence of a cationic surfactant than in the presence of an anionic or nonionic surfactant with similar hydrophobic structure.6 This observation may be seen as an indication that formation of the complex involves some kind of protein denaturation. However, the reduced activity need not be due to conformational changes of the protein. If one or more binding sites for the cationic surfactant is situated in the vicinity of the active site of the lipase, surfactant

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binding will most likely lead to a reduced biological activity, as has been found for other enzyme-amphiphile systems.9 Thus, no firm conclusion about the structure of the lipase-cationic surfactant complex can be drawn from this work. Acknowledgment. We are greatful to Dr. P. Falholt, Novo Nordisk A/S for the gift of the lipase and to Dr. K. Bergstro¨m, Akzo Nobel Surface Chemistry AB for the amphoteric surfactant. LA962094P