Effect of Lipase on Different Lipid Liquid Crystalline Phases Formed by

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Effect of Lipase on Different Lipid Liquid Crystalline Phases Formed by Oleic Acid Based Acylglycerols in Aqueous Systems Johanna Borne´, Tommy Nylander,* and Ali Khan Physical Chemistry 1, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden Received April 18, 2002. In Final Form: August 8, 2002 This study deals with the interplay between the interfacial structure of lipid liquid crystalline (lc) substrates and the lipolysis rate. Thermomyces (formerly Humicola) lanuginosa lipase (TLL) was added to lamellar (LR), reversed bicontinuous cubic (C), and reversed hexagonal (HII) lc phases, based on monoolein (MO), MO-sodium oleate (NaO), MO-oleic acid (OA), and MO-diolein (DO) with water. The changes in self-assembled structure and lipid composition during lipolytic processes were followed by polarizing microscopy, small-angle X-ray diffraction, and high-performance liquid chromatography (HPLC). Indeed, the observed changes in self-assembled structures could be predicted from either the MO-OA-2H2O ternary phase diagram, where the lipolysis gives rise to a transition of C f HII f micellar cubic (Cmic) f reversed micellar phase + dispersion, or the MO-NaO-2H2O ternary phase diagram, where the corresponding sequence is LR f HI. These observations are discussed in terms of the degree of protonation of the fatty acid. The specific activity of TLL on the CD and OA-HII samples as determined from the lipolysis rate was found to be the similar under the employed experimental conditions. The HPLC data showed that the ratio between the substrate (MO/DO) and final product (OA) approached about the same values regardless of the initial substrate composition and structure.

Introduction Lipases, which are water soluble, act at an oil-water interface during lipolysis of a triglyceride. During lipolysis, the hydrolysis products such as glycerol, free fatty acids di- and monoglycerides, and the added surfactant molecules will alter the lipid self-assembled structure of the substrate and the characteristics of the lipid-aqueous interface. Lipase-catalyzed lipolysis is a complex reaction involving many steps, for example, binding, orientation and activation of the lipase, binding of the substrate molecule into the active site, and the catalytic reaction itself (cf. refs 1 and 2). These steps, as well as the solubility of the products formed, will be determined by the selfassembled structures formed by the amphiphiles present. Detailed knowledge of the phase behavior of the relevant lipid-aqueous system is necessary in order to predict the lipid self-assembly structures expected to form during lipolysis and to understand how these substrates (vesicles, micelles, emulsions, gels, and liquid crystals) will affect the lipase activity. We have previously studied the phase behavior of the ternary systems monoolein (MO)-diolein (DO),3 MO-oleic acid (OA) and MO-sodium oleate (NaO)4 with water. The phase diagrams determined helped us to prepare substrates of well-defined structure and composition. They can also serve as maps to navigate through the changes in the lipid self-assembled structures that occur during the lipolytic process. The cubic and lamellar liquid crystalline (lc) phases, composed of monoglycerides and fatty acids, have been * Corresponding author. Fax Int: + 46 46 222 4413. Phone Int: + 46 46 222 8158. E-mail: [email protected]. (1) Svendsen, A. Biochim. Biophys. Acta 2000, 1543, 223. (2) Panaiotov, I.; Verger, R. Enzymatic reactions at interfaces: Interfacial and temporal organization of enzymatic lipolysis. In Physical Chemistry of Biological Interfaces; Baszkin, A., Norde, W., Eds.; Marcel Dekker: New York, 2000; p 359. (3) Borne´, J.; Nylander, T.; Khan, A. Langmuir 2000, 16, 10044. (4) Borne´, J.; Nylander, T.; Khan, A. Langmuir 2001, 17, 7742.

reported to occur during the lipolysis of triolein (TO) in vivo and in vitro.5-8 The TO-aqueous system becomes increasingly complex as the lipolytic process proceeds, involving up to five additional components (DO, MO, OA, oleate, and glycerol). In this study, we have focused on the product end of the process, that is, MO, OA, and oleate, to facilitate the interpretation of the data. The effect of lipase action on (1) MO-2H2O-based bicontinuous cubic lc phases,9-12 (2) MO-2H2O-based cubic dispersions,13,14 and (3) MO-2H2O-based vesicles14 in relation to lipid selfassembly has been reported previously. The bicontinuity and the ability to incorporate other molecules are important features of the monoglyceride-based cubic phases that have been suggested to facilitate the lipolysis process.7 In addition, a cubic phase creates a large effective area compared, for instance, with an oil-aqueous interface, which can lead to a much faster lipase-catalyzed lipolytic process.11 In the present study, we have investigated the action of lipase on different MO-2H2O-based lc phases. The objectives were as follows: 1. To determine the lipid self-assembly structures formed during lipolysis in relation to initial substrate (5) Patton, J. S.; Carey, M. C. Science 1979, 204, 145. (6) Lindstro¨m, M.; Ljusberg-Wahren, H.; Larsson, K.; Borgstro¨m, B. Lipids 1981, 16, 749. (7) Patton, J. S.; Vetter, R. D.; Hamosh, B.; Borgstro¨m, B.; Lindstro¨m, M.; Carey, M. C. Food Microstruct. 1985, 4, 29. (8) Staggers, J. E.; Hernell, O.; Stafford, R. J.; Carey, M. C. Biochemistry 1990, 29, 2028. (9) Luzzati, V.; Gulik, A.; DeRosa, M.; Gambacorta, A. Chem. Scr. 1987, 27B, 211. (10) Mariani, P.; Luzzati, V.; Delacroix, H. J. Mol. Biol. 1988, 204, 165. (11) Wallin, R.; Arnebrant, T. J. Colloid Interface Sci. 1994, 164, 16. (12) Caboi, F.; Borne´, J.; Nylander, T.; Khan, A.; Svendsen, A.; Patkar, S. Colloids Surf., B 2002, 26, 159. (13) Zhao, L.; Landh, T.; Sternby, B.; Nilsson, Å. Prog. Colloid Polym. Sci. 2002, 120, 92. (14) Borne´, J.; Nylander, T.; Khan, A. J. Phys. Chem. B 2002, 106, 10492.

10.1021/la020377d CCC: $22.00 © 2002 American Chemical Society Published on Web 10/17/2002

Effect of Lipase on Lipid Liquid Crystalline Phase

structure. The structural information obtained from smallangle X-ray diffraction (SAXD) is correlated to changes in the component composition as determined with highperformance liquid chromatography (HPLC) and phase diagrams of the relevant systems. 2. To determine the effect of the initial substrate selfassembly structure on the lipase activity. The change in sample composition due to lipase action was determined by HPLC to obtain the initial enzyme kinetics. The kinetic data were analyzed in relation to the changes in the selfassembled structure. For this purpose, we added Thermomyces (formerly Humicola) lanuginosa lipase (TLL) to lamellar (LR), reversed bicontinuous cubic (C), and reversed hexagonal (HII) lc phases formed in the aqueous systems of MONaO, MO, MO-OA, and MO-DO. These selected structures, which represent increasing negative curvature of the aqueous-lipid interface, are the dominating lc phases observed during lipolysis of triglycerides. Experimental Section Materials. MO, Rylo Mg 90-glycerol monooleate (TS-ED 173) (lot no. 1876-88), and DO, glycerol dioleate (TS-ED 175), were kindly provided by Danisco Ingredients (Braband, Denmark). The monoolein samples consisted of 95.7 wt % monoglycerides, 3.8 wt % diglycerides, 0.4 wt % free fatty acids, and 0.1% free glycerol. The fatty acid composition was 90.0 wt % oleic acid, 5.0 wt % linoleic acid, 2.7 wt % stearic acid, 1.0 wt % palmitic acid, 0.3 wt % linolenic acid, and 1.0 wt % other fatty acids. The phase behavior of the MO batch and the commercial samples from the same source is similar to that obtained with pure (and much more expensive) samples, although the exact phase boundaries are slightly different.15,16 The DO sample consisted of 98.3 wt % diglycerides, 1.3 wt % triglycerides, and 0.4 wt % monoglycerides. Sodium oleate (purity > 99%) was obtained from Nu-Chek-Prep Inc. and oleic acid (O1008, 11280-1) from Sigma (purity, 99%) with the following fatty acid compositions: C18:1, 92%; C18:2, 6%; saturated acids, 2%. Glycerol (purity, 99%) was purchased from Merck. The deuterated water (>99.8%) was obtained from Dr. Glaser AG (Basel). Acetonitrile, dichloromethane, acetic acid, chloroform, and ethyl acetate for HPLC were also obtained from Merck. All these chemicals were used as received. The lipase, TLL, was obtained from Novozymes A/S, Denmark, and had a molecular weight of 32 000 g/mol. The lipase stock solution (1 mg/mL) was dialyzed against a 0.1 M sodium phosphate buffer at pH 8, for 36 h. Sample Preparation. The samples for the ternary systems MO-2H2O-additive (third component) were prepared by weighing the components into glass ampules, ∼0.5 cm (i.d.). After being flame-sealed, the samples were mixed by repeated centrifugation at 2900g, where the sample tubes were inverted every other cycle. Samples containing OA and DO were centrifuged at 25 °C and those with NaO at 37 °C (above the Krafft temperature for NaO17). Centrifugation was performed 40 min every day for 1 week. The samples were then stored at 25 °C in the dark for 2 weeks or until the samples appeared homogeneous by visual inspection, before the lipase was added. Enzyme solutions of two different concentrations were prepared by adding 20, 10, and 5 µL of TLL stock solution (1 mg/mL) to 1200 µL of 0.1 M phosphate buffer at pH 8, giving final lipase concentrations of 512, 256, and 128 nM, respectively. The lipolytic process was started by adding 20 µL of lipase solution to 30 mg of the liquid crystalline phase under mixing with a spatula to deliver the enzyme to the whole sample as homogeneously as possible. For the polarizing microscopy study, a lipase concentration of 0.0164 mg/mL (512 nM) was used, while for the small-angle X-ray diffraction (SAXD) and HPLC studies all concentrations of lipase solutions were used. (15) Larsson, K.; Fontell, K.; Krog, N. Chem. Phys. Lipids 1980, 27, 321. (16) Landh, T. J. Phys. Chem. 1994, 98, 8453. (17) Small, D. M. The Physical chemistry of Lipids: From Alkanes to Phospholipids. Handbook of Lipid Research; Plenum Press: New York, 1986; Vol. 4.

Langmuir, Vol. 18, No. 23, 2002 8973 To freeze the concentration for HPLC analysis, 1 mL of ethyl acetate-chloroform (9:1, v/v) mixture was added to the sample. No further changes in the sample composition could be detected after this dilution, indicating that the lipolytic reaction was inhibited. Standard mixtures of triolein, diolein, monoolein, oleic acid, and sodium oleate were prepared by dissolving 0.25-2 mg of each component in the mixed solvent. Methods. Optical Polarizing Microscopy. Polarizing microscopy is a well-established technique to identify liquid crystalline phases by observing their textures.18-20 Isotropic phases are seen as a dark background in the microscope, while anisotropic phases show characteristic textures in polarized light. Large vesicle structures and emulsion droplets can also be easily investigated with the technique, using normal light. An Axioplan Universal polarizing light microscope from Carl Zeiss, equipped with a differential interference contrast (DIC) unit to enhance the contrast between object and background, was used down to achieve a resolution of 0.5 µm. A small amount of the sample was applied to a microscope slide, and a cover glass was gently pressed on top of it. The edges between the cover glass and the slide were sealed with transparent nail polish to prevent evaporation. Photographic images of the textures were recorded with an MC 100 Olympus camera. Small-Angle X-ray Diffraction (SAXD). The SAXD data were recorded with a Kratky compact small-angle system equipped with a position-sensitive detector (OED 50M from Mbraun, Austria) containing 1024 channels of width 53.0 µm. A monochromator with a nickel filter was used to select the Cu KR radiation (λ ) 1.542 Å) provided by a Seifert ID-300 X-ray generator, operating at 50 kV and 40 mA. A few milligrams of the sample was enclosed in a stainless steel sample holder with mica windows. The distance between the sample and detector was 277 mm. The diffraction patterns were recorded at 25 and 37 °C. The temperature was maintained within (0.1 °C by a Peltier element. The optics and the sample cell were both kept under vacuum to minimize scattering caused by the air. High-Performance Liquid Chromatography. The lipid composition was analyzed in parallel with the SAXD measurements, using reverse-phase chromatography on a Supelcosil LC-8 column end-capped with Si-(CH3)3, fitted into a Shimadzu SCL-10AVP system equipped with an evaporative light scattering mass detector, ELSD (Sedex 55). The mobile phase was a ternary gradient system composed of dichloromethane/acetonitrile (30%/ 70% v/v), acetonitrile, and acetic acid (0.1% in ultrapure water) as described.21 The flow rate was 1.3 mL/min, and the column and detector temperature was 30 °C, giving retention times of 5.9 and 6.5 min for MO and NaO/OA, respectively. We noted that it was difficult to analyze samples that contained large amounts of NaO, due to limited solubility in the solvent system employed. Hence we could not accurately determine the composition in the lamellar sample.

Results Lipid Self-Assembly Structures Formed during the Lipolysis in Relation to Initial Substrate Structure. We studied the effect of T. lanuginosa lipase on several thermodynamically stable MO-based liquid crystalline phases in the ternary MO-DO-2H2O, MO-NaO2 H2O, and MO-OA-2H2O phase diagrams. The phase equilibria have been studied in detail previously.3,4,22 On the basis of these studies, we chose to study the effect on (1) the bicontinuous diamond type of cubic phase (CD), which exists in an excess of water in the MO-2H2O system, (2) the reversed hexagonal (HII) phases which are formed when DO, OA, or NaO is added to the binary MO-2H2O system, and (3) the lamellar phase (LR), which dominates the MO-NaO-2H2O system and gives vesicles at high (18) Rosvear, F. B. J. Am. Oil Chem. Soc. 1954, 31, 628. (19) Rosvear, F. B. J. Soc. Cosmet. Chem. 1968, 19, 581. (20) Ekwall, P. In Advances in Liquid Crystals, Vol. 1; Brown, G. H., Eds.; Academic Press: New York, 1975; p 1. (21) Marcato, B.; Cecchin, G. J. Chromatogr., A 1996, 730, 83. (22) Borne´, J.; Nylander, T.; Khan, A. J. Colloid Interface Sci., accepted.

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Table 1. Effect of Adding 20 µL of 512 nM TLL Solution to 30 mg of Cubic (CD) Phase with an Initial Composition of 63 and 37 wt % MO and 2H2O, Respectively, Giving a Final TLL Concentration of 205 nMa time (min) 0 10 20 45 180 270 300 480 960 1440

R (Å) 87.8 ( 0.3 91.7 ( 0.2 87.0 ( 0.4 60.5 ( 0.04 50.0 ( 0.03 ≈149 149.0 ( 0.6 49.1 ( 0.09 145.3 ( 0.9

phase (space group)

MO (wt %)

OA (wt %)

CD (Pn3m) CD (Pn3m) CD (Pn3m) HII HII HII Cmic (Fd3m) Cmic (Fd3m) HII Cmic (Fd3m) + dispersion dispersion dispersion

63.0 61.0 60.2 54.9 44.2

0 1.40 2.21 6.0 14.2

MO/OA (wt %/wt %)

39.7

17.7

2.24

29.9 21.6 19.7

24.2 28.8 29.8

1.23 0.75 0.66

43.7 27.3 9.10 3.12 2.83b

a The SAXD data indicating the change in the self-assembled structure and the changes in the composition measured by HPLC are summarized. b Calculated based on data extrapolated from Figure 5a.

Table 2. Effect of Adding 20 µL of 512 nM TLL Solution to 30 mg of HII Phases in the MO-OA-2H2O and MO-DO-2H2O Ternary Systems, Giving a Final TLL Concentration of 205 nMa The MO-OA-2H2O Substrate with 65.4 wt % MO, 15.6 wt % OA, and 19 wt % H2O time (min)

R (Å)

phase (space group)

MO (wt %)

OA (wt %)

MO/OA (wt %/wt %)

0 10 300 465 600 765 1440

52.3 + 0.08 53.9 + 0.2 49.0 + 0.02 145.3 ( 0.9

HII HII HII Cmic (Fd3m) Cmic (Fd3m) + dispersion dispersion dispersion

65.4 62.8 45.9

15.6 17.0 29.7

30.7

41.2

16.9

51.5

4.2 1.6 1.5 1.1b 0.74 0.68b 0.33

The

MO-DO-2H2O

Substrate with 68 wt % MO, 18 wt % DO, and 14 wt % H2O

time (min)

R (Å)

phase (space group)

MO (wt %)

DO (wt %)

OA (wt %)

MO/OA (wt %/wt %)

0 300 480 1440

61. 7 ( 0.03 49.1 ( 0.1 145.1 ( 0.7

HII HII Cmic (Fd3m) dispersion

68.0 46.6 29.1 15.4

18.0 8.6 4.6 0

0 24.2 40.8 54.8

16.6 1.9 0.71 0.28

a The SAXD data indicating the change in the self-assembled structure and the changes in the composition measured by HPLC are summarized. b Calculated based on data extrapolated from Figure 5a.

Table 3. Effect of Adding 20 µL of 512 nM TLL Solution to 30 mg of Lr Phase with an Initial Composition of 10 wt % MO, 85 wt % 2H2O, and 5 wt % NaO at 25 °C, Giving a Final TLL Concentration of 205 nMa time (min)

R (Å)

phase

0 20

140 132.5 ( 2.5 65.8 120 57 (170, 100)b 127.5 ( 2.5 56.6 ( 0.3 (175, 100)b 56.4 ( 0.4 (150)b 56.4 ( 0.4 (157)b

LR LR HI LR HI X LR HI X HI X HI X

95 240 720 960

a The SAXD data on the change of the self-assembly structure are summarized. b Lattice spacing from unknown phases, X.

water content. The pH of the CD and HII samples was about 6.5, while it was about 10 in the LR sample. We noted that the pH remained fairly constant during lipolysis. To maintain the properties of the lc phase when adding the lipase solution, we chose compositions such that they exist in equilibrium with water (Tables 1-3). Lipase Action on a CD Phase. Changes in the Microscopic Morphology. To determine the appropriate lipase concentration and estimate the time dependence of the structural changes, optical polarizing microscopy was performed (see Figure 2). These studies were performed on the isotropic CD phase as it is easy to monitor

transformations to anisotropic phases. Within 25 min of lipase addition, giving a final TLL concentration of 205 nM, a dramatic change in the microstructure was observed. The cubic phase (to the right in Figure 1a) was found to coexist with an anisotropic phase (to the left in Figure 1a), with a texture typical for an HII phase. The images recorded after about 300 min suggested that the HII phase undergoes a phase transition as the texture gradually disappears (Figure 1b). After 330 min, an isotropic phase, which appeared to be less viscous than the initial bicontinuous cubic phase, was observed (Figure 1c). As observed for the micellar cubic (Cmic) phase in the ternary MO-OA-2H2O system, the viscosity decreases when the isotropic phase is exposed to shearing and increased temperature.4 The sample appears more and more milky as lipolysis proceeds, and finally after 570 min we observed only a dispersion of droplets (Figure 1d). SAXD Analysis of the Structure. The SAXD data recorded after lipase action on the CD phase are summarized in Table 1, and representative SAXD spectra are shown in Figure 2. The corresponding change in sample composition, as determined by HPLC, is also included in Table 1 and will be discussed in relation to the phase diagrams in Figure 7. The initial cubic phase (Figure 2a) persisted 10 min after lipase addition (Figure 2b), but the addition of sodium phosphate buffer containing lipase led to a slight swelling of the CD phase (Table 1). Despite the observed decrease in MO/OA weight ratio from 44 to 27 after 20 min, the CD phase remained (Table 1), which is consistent with the


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Figure 1. Polarizing microscopy images of lipolysis of the CD phase (63 wt % MO, 37 wt % 2H2O) after adding TLL to a final concentration of 205 nM. The images are recorded after (a) 25 min, where the cubic phase coexists with a hexagonal phase; (b) 300 min, when the hexagonal phase is destabilized; (c) 330 min, when a cubic micellar phase dominates; and (d) 570 min, when the sample features a dispersion of droplets.

Figure 2. SAXD data recorded after adding 20 µL of (512 nM) TLL solution to 30 mg of CD phase (63 wt % MO, 37 wt % 2H2O) at 25 °C, giving a final TLL concentration of 205 nM. The intensity is plotted versus the wave vector, q ) 2*π/d, where d is the spacing between the lattice planes. The sequence of phase transitions observed is (a) CD (0 min), (b) CD (10 min), (c) CD + HII (25 min), (d) HII (45 min), (e) Cmic + HII (300 min), and (f) L2 (960 min). The d values for the reflections corresponding to the HII phase have been underlined in the figure.

phase behavior of the ternary MO-OA-2H2O system (Figure 7a). After 25 min, we observed a change in structure, as determined with both SAXD and microscopy (Figure 1a). Apart from the pattern associated with the CD phase,

additional reflections appeared at 53.5, 30.7, and 26.9 Å, that is, the 1:x3:2 ratio typical of a hexagonal phase (here an HII phase) (Figure 2c). After 45 min, only an HII phase was observed (Figure 2d). HPLC data showed that the MO/OA weight ratio had

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decreased to 9.1. At this composition, the ternary MOOA-2H2O system is expected to give HII (Figure 7), with a lattice parameter RHII of about 57.7 Å.4 This is consistent with RHII ) 60.5 Å, extracted from the spectra in Figure 2d. Since the ternary MO-NaO-2H2O system (Figure 7b) shows a pure gyroid type of cubic phase (CG) at this composition, we concluded that the reaction path is better described by the MO-OA-2H2O phase diagram. The HII phase was still observed 255 min after lipase addition, but RHII had decreased from 60.5 to 50.0 Å, indicating decreased water content in the phase. Again, this is consistent with the MO-OA-2H2O phase diagram, where the HII phase can take up less water with increased OA content (Figure 7a). The RHII of the HII phase in equilibrium with water decreases from 60.2 to 48.5 Å within its stability range.4 After 270 min, we again observed a significant change in structure as determined with SAXD (Table 1). This is probably the same change as was observed with microscopy after 300 min (5 h) (Figure 1c). The HII phase has been partly replaced by another cubic phase, which can be indexed as belonging to the space group Fd3m. It has been suggested that the space group Fd3m is a micellar cubic phase, Cmic, where disjointed reversed micelles embedded in a three-dimensional hydrocarbon matrix are organized in a cubic symmetry.23 The formation of Cmic phases has previously been reported for aqueous systems containing MO and OA23-25 and for aqueous mixtures of NaO and OA.26 We also identified this structure in the ternary MO-OA-aqueous system.4 The Cmic phase is even more apparent after 300 min when several reflections from the cubic phase (Fd3m) can be identified in the SAXS spectra (Figure 2e, Table 1). This cubic phase remains stable up to 480 min (8 h) after lipase addition. The HPLC measurements showed that the MO/OA weight ratio after 300 and 480 min was 2.2 and 1.2, respectively. From the MO-OA-2H2O system, we know that a single Cmic phase exists at a MO/OA ratio of about 1.5 and that the single HII phase is stable until the MO/OA ratio has decreased to a value of 2.5. Thus, the HPLC results and the phase changes indicated in Table 1 are in complete agreement with the ternary phase diagram of the MO-OA-2H2O system. In addition, the lattice parameter, RCmic, of 149.0 Å recorded for the Cmic phase (Table 1) is similar to the value (146.1 Å) recorded for the same phase in the MO-OA-2H2O system at maximum swelling. After 570 min, the Cmic phase had partly disappeared and the SAXD spectra are characterized by a broad peak (similar to Figure 2e). This is typical of a fluid isotropic phase (reversed micellar phase, L2), which was observed in the MO aqueous system27 and in the ternary systems with OA.4 Indeed, the microscopy study indicated dispersion of an isotropic phase. After 20 h, the SAXD peak was even broader and the intensity had decreased significantly. The MO/OA weight ratio was 0.7, and in the MO-OA2 H2O system this corresponds to a multiphase region. The experiments were repeated at lower lipase concentrations (51 and 102 nM TLL) at 25 °C, and the same sequence of phase transitions was observed. However, the lipolysis rate decreased, as expected (see Figure 5a), and the phase transitions therefore occurred over a longer time (23) Luzzati, V.; Vargas, R.; Gulik, A.; Mariani, P.; Seddon, J. M.; Rivas, E. Biochemistry 1992, 31, 279. (24) Seddon, J. M.; Bartle, E. A.; Mingins, J. J. Phys.: Condens. Matter, 2 1990, SA285. (25) Eriksson, P. O.; Lindblom, G. Biophys. J. 1993, 64, 129. (26) Mills, R. J. Phys. Chem. 1973, 77, 685. (27) Qui, H.; Caffrey, M. Biomaterials 2000, 21, 223.

Borne´ et al.

scale. An increase in temperature to 37 °C also gave the same sequence of phase transitions, but at a higher rate, as expected from the higher hydrolysis rate (Figure 5a). For instance, the CD f HII and HII f Cmic phase transitions at 37 °C occurred after only 10 and 150 min, respectively. Lipase Action on an HII Phase. The effect of lipase on two types of HII phases, one based on the MO-OA2 H2O system and the other on the MO-DO-2H2O system, was investigated. Here we noted that DO is also a substrate for TLL. The SAXD data are summarized in Table 2, and representative SAXD spectra are shown in Figure 3. The corresponding change in sample composition as determined by HPLC is also included in Table 2 and will be discussed in relation to the phase diagrams in Figure 7. The MO-OA-2H2O Substrate. As for the CD phase substrate, a slight increase (of about 1 Å) in the lattice parameter, RHII, was observed immediately after diluting the sample with the TLL solution (Figure 3b). The HII phase remained for the first 300 min after lipase addition (Figure 3b-d), although the MO/OA weight ratio decreased to about 1.5 (Table 2). A slight decrease in RHII to 49 Å was observed. These findings are in agreement with those from the ternary MO-OA-2H2O system, where we observed RHII ) 48.5 Å at the maximum OA content that can give the HII phase in an excess of water. The HII phase was more or less replaced by the Cmic phase (space group Fd3m) after 390 min when the MO/ OA weight ratio was found to be 1.3. A better resolved SAXD spectrum of the Cmic phase was observed after 465 min with R ) 145.3 Å, when the MO/OA weight ratio was determined to be 1.1. The value of R agrees well with the value of 145.7 recorded for Cmic in the ternary MO-OA2 H2O system with a similar sample composition.4 After 600 min at a MO/OA weight ratio of 0.8, the SAXD spectra featured a broad peak together with the reflections from the Cmic phase (Figure 3f). Further hydrolyses, after about 765 min, gave an almost identical structure to that observed for the CD phase, that is, an isotropic dispersion appeared. The MO-DO-2H2O Substrate. The lipase action on the HII phases based on the MO-DO-2H2O system gave an identical sequence of phase transitions to that observed for the corresponding phase from the MO-OA-2H2O system (Table 2). The transitions also seem to appear after similar times, and the recorded values for RHII and for R for the Cmic phase are close. This is not surprising as the ternary MO-OA-2H2O and MO-DO-2H2O systems are quite similar, although no Cmic is observed in the MODO-2H2O system. Here we noted that most of the DO had been hydrolyzed when the Cmic phase appeared (Table 2). Lipase Action on an Lr Phase. The SAXD spectra recorded for the lipolysis of the initial vesicle phase at 37 °C are shown in Figure 4, and the SAXD data are summarized in Table 3. Here we noted that this sample contained significantly more water than the other samples studied. The recorded diffractograms are therefore less resolved (Figure 4). This makes it difficult to assign the reflections to a certain structure. As mentioned in the Experimental Section, it was not possible to determine the sample composition by HPLC. The initial spectrum shows a peak at 140 Å and shoulder at about 70 Å, which indicates an LR phase with an interlayer spacing of 140 Å (Figure 4a). This was found in our previous cryogenic transmission electron microscopy (cryo-TEM) study of a similar sample, in which multilamellar vesicles with an interlayer spacing of 120-150 Å were observed.4 The LR phase was still present after 20 min, although an additional reflection at 57 Å was apparent and could be


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Figure 3. SAXD data recorded after adding 20 µL of (512 nM) TLL solution to 30 mg of HII phase (65.4 wt % MO, 15.6 wt % OA, 19 wt % 2H2O) at 25 °C, giving a final TLL concentration of 205 nM. The intensity is plotted versus the wave vector, q ) 2*π/d, where d is the spacing between the lattice planes. The sequence of phase transitions observed is (a) HII (0 min), (b) HII (10 min), (c) HII (45 min), (d) HII (285 min), and (e) Cmic (465 min), and panel f features a broad peak from the isotropic dispersion together with reflections from the Cmic phase (765 min).

assigned to an HI phase as will be further discussed below (Figure 4b). After 95 min, the reflections corresponding to the LR phase have almost disappeared, while the intensity of the 57 Å reflection had increased (Figure 4c). New peaks at 170 and 100 Å, with a ratio of 1:x3, became visible. We were unable to assign these reflections to a particular structure. However, we noted that phase separation into vesicles of different sizes with different interlayer spacings was pronounced in this domain of the ternary MO-NaO2 H2O system.22 After 240 min (4 h) (Figure 4d), two new reflections were observed at 32.7 and 28.1 Å. These are likely to be second and third reflections of the HI phase with a primary reflection at 57 Å. From the MO-NaO-2H2O system, we know that a normal hexagonal phase can coexist with vesicles.4,22 In the ternary system, we determined the lattice parameter, RHI, to be 70 Å, which is slightly larger than the 66 Å obtained from Figure 4d. The RHII of the HII phase in equilibrium with water in the ternary MO-OA2 H2O system was found to be between 60.2 and 48.5 Å at a maximum water content of 18 wt %.4 We noted that the pH and water content are significantly higher, about 10 and 85 wt %, respectively, for the sample studied. This makes the HII phase less likely, and the lipolysis of the LR sample is expected to give structures according to the ternary MO-NaO-2H2O system. The HI phase becomes more and more dominating as lipolysis proceeds, and after 960 min (Figure 4f), the additional reflections, which we were unable to assign to any specific structure, disappeared and were not visible after 1440 min.

The Effect of the Initial Self-Assembled Structure of the Substrate on the Lipase Activity. One of the main purposes of this study was to investigate how the lipase activity is affected by the self-assembled structures of liquid crystalline phases in MO-based aqueous systems. To the best of our knowledge, this has not been done previously. The change in sample composition due to lipase action was determined by HPLC, and the results are shown in Figure 5a as the OA concentration versus time for the different systems. The data presented represent the mean of at least three independent measurements, with a maximum deviation of about 5%. The OA concentration at a given time increases, as expected, with the TLL concentration (CD phase and the HII phase containing OA) and with temperature (CD phase). The substrate (MO/DO)/product (OA) molar ratio nS/nP after 1440 min (24 h), which assumes values of 1.01.9 for the investigated systems, is also inserted in Figure 5a. For the system containing DO, we used twice the molar concentration as both chains on DO can be hydrolyzed. The obtained nS/nP values suggest that the lipolytic reaction has not yet reached complete steady state after 24 h as the values decrease with increasing TLL concentration. However, we noted that the largest increase in OA concentration took place during the first 600 min (10 h). It is difficult to compare the data for the different systems in terms of TLL activity, based on Figure 5a, as the OA-HII sample initially contained the lipolysis product (OA). One way of circumventing this problem is to analyze the reaction rate under different conditions. A complete kinetic analysis is very complex and difficult in these systems (cf. ref 2) and is beyond the scope of this work.

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Figure 4. SAXD data recorded after adding 20 µL of (512 nM) TLL solution to 30 mg of LR phase (10 wt % MO, 5 wt % OA, 85 wt % 2H2O) at 25 °C, giving a final TLL concentration of 205 nM. The intensity is plotted versus the wave vector, q ) 2*π/d, where d is the spacing between the lattice planes. The sequence of phase transitions observed is (a) LR (0 min), (b) LR + HI (20 min), (c) LR + HI + X (95 min), (d) HI + X + LR (240 min), (e) HI + X (720 min), and (f) HI + X (960 min). Some of the observed reflections could not be unambiguously assigned to a structure, and the unidentified structure is denoted X. Table 4. Initial Kinetics of TLL-Catalyzed Lipolysis of MO in C and HII Liquid Crystalline Phases in an Excess of Watera ∆[OA]/∆t × 1/cTLL from initial slope (Figure 5a) (µmol min-1 mg-1)b

a

∆[OA]/∆t × 1/cTLL from initial rate (Figure 5b,c) (µmol min-1 mg-1)

sample

based on [OA]

based on [MO]

based on [OA]

based on [MO]

C: 51 nM TLL 25 °C C: 102 nM TLL 25 °C C: 205 nM TLL 25 °C C: 205 nM TLL 37 °C OA-HII: 102 nM TLL, 25 °C OA-HII: 205 nM TLL, 25 °C

405 ( 1 983 ( 3 693 ( 9 1134 ( 10 1133 ( 2c 735 ( 1c

415 ( 1 1018 ( 3 723 ( 10 1175 ( 9 1164 ( 3c 899 ( 3c

458 1303 753 1995 1263 763

475 1354 870 2062 1319 1032

See text for details. b ( Standard deviation from fitted line. c Fit based on only three values.

We have therefore applied the simplest approach when analyzing our kinetic data, that is, to calculate a global initial reaction rate expressed as a specific activity (SA):

SA )

1 ∆[OA] cTLL ∆t

Here, as is common when discussing the SA, cTLL is the enzyme concentration in mg/mL, and ∆[OA] is the change in OA (or MO) concentration in µmol/mL during ∆t min. The values of SA obtained are summarized in Table 4 and were calculated from the initial reaction rate normalized to the TLL concentration. The initial reaction rate was determined from the initial slope of the curves shown in Figure 5a. The slope was calculated from values obtained when less than 10% of the substrate was consumed. The values of the initial rate obtained from Figure 5b,c are also inserted. Figure 5b,c shows the reaction rate versus time obtained by differentiating the curve in Figure 5a. No significant difference was found in the reaction rates determined from the substrate (MO) and product (OA)

concentrations, as illustrated in Table 4 and Figure 5b,c. This verifies the accuracy of the HPLC analyses. The SA values obtained by differentiation (Figure 5b,c) are quite scattered, due to the few data points available, although SA values showed a tendency to decrease with time. The initial values from Figure 5b,c show the same trends as the data obtained from the initial slope in Figure 5a (Table 4). The most remarkable result is that the specific activity of TLL in CD and HII phases is the same, independent of the TLL concentration (Table 4, Figure 5b,c). Thus it seems that the SA does not depend on the initial self-assembly structure under the conditions investigated. Furthermore, the SA obtained at 102 nM TLL is higher than that for 205 nM TLL for the lypolysis of both the CD and the HII phases. Therefore, additional experiments were performed using the CD phase and 51 nM TLL, and the results are given in Table 4. From these results, it can be concluded that the highest SA under the given conditions is observed within a narrow range of TLL concentration of about 102


Langmuir, Vol. 18, No. 23, 2002 8979

Figure 6. Schematic representation of the change in structure during lipolysis of MO (or DO) in different lc phases: (a) CD phase (63 wt % MO, 37 wt % 2H2O), (b) OA-HII phase (65.4 wt % MO, 15.6 wt % OA, 19 wt % 2H2O), (c) DO-HII phase (68 wt % MO, 18 wt % DO, 14 wt % 2H2O), and (d) LR phase (10 wt % MO, 5 wt % NaO, 85 wt % 2H2O). The main lc phases as determined by SAXD are indicated. These may exist in an excess of water or in the presence of minor amounts of other phases. Some of the observed reflections in the diffractograms, obtained by SAXD, could not be unambiguously assigned to a structure. This unidentified structure is denoted X.

Figure 5. Kinetics of lipolysis catalyzed by TLL of MO (or DO) in different lc phases: CD phase (63 wt % MO, 37 wt % 2H2O), OA-HII phase (65.4 wt % MO, 15.6 wt % OA, 19 wt % 2H2O), and DO-HII phase (68 wt % MO, 18 wt % DO, 14 wt % 2H2O) at selected enzyme concentrations and temperatures. Panel a shows the change in OA concentration versus time. The substrate (MO or DO)/product (OA) molar ratio nS/nP after 1440 min (24 h) is also inserted in the figure. Panels b and c show the specific hydrolysis rates (∆[OA]/∆t × 1/cTLL) versus time for the CD and OA-HII phases, respectively. Two values are shown at each time, one of which is based on the measured amount of OA and the other is determined from the amount of hydrolyzed MO.

nM. Finally, we noted that the SA is higher at 37 °C than at 25 °C on the CD phase, as expected. Discussion Lipid Self-Assembly Structures during Lipolysis. A schematic representation of the changes in structure during lipolysis of MO (or DO) in different lc phases is shown in Figure 6. In Figure 7, arrows indicate the suggested paths of the lipolytic reaction in the ternary systems. It is obvious that except for the LR sample, the investigated samples seem to follow the same sequence of phase transitions, which is best described by the ternary MO-OA-2H2O system (Figure 7b). This is not surprising as the pH of the CD, OA-HII, and DO-HII samples is

about 6.5 before and after lipolysis. As discussed previously,14 the pKa of OA in a self-assembled structure can be significantly higher than 5.0, as reported for isolated fatty acids in aqueous solution.28 In fact, the complex titration curve of potassium oleate in an aqueous dispersion shows three plateaus at pH 7.0, 7.5, and 9.0,29 indicating that deprotonation can occur at a pH as high as 9.0. The sequence of phase transitions during the lipolysis of the LR sample, which had a pH of about 10, is consequently best described by the ternary MO-NaO2 H2O system (Figure 7a). This is verified by the presence of the LR phase, the absence of the Cmic phase, and the fact that the final recorded structure is suggested to be an HI structure. The presence of the HI phase and not the HII phase was shown by the larger lattice parameter, which remained constant during lipolysis, as expected from the ternary MO-NaO-2H2O system (Figure 7a). However, it was not possible to determine the composition of the LR phase as a function of time by HPLC, due to the presence of the dissociated acid in this sample. We observed that the cubic phase disappeared almost immediately after lipase addition. This is consistent with the ternary MO-OA-2H2O phase diagram, which shows that the MO-based aqueous cubic phase can take up only about 0.5 wt % OA at 37 wt % water. Due to the higher water content in the initially cubic sample, the HII phase formed initially has a larger lattice parameter than the HII samples (Tables 1 and 2). This is also expected from the phase diagram (Figure 7b). The observed phase transitions of the two HII samples, prepared from OA and DO, are almost identical. This is expected as the phase (28) Ptka, M.; Egret-Charlier, M.; Sanson, A.; Boulossa, O. Biochim. Biophys. Acta 1980, 600, 387. (29) Cistola, D. P.; Hamilton, J. A.; Jackson, D.; Small, D. M. Biochemistry 1988, 27, 1881.

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Figure 7. The ternary phase diagrams of (a) the MO-NaO-2H2O system and (b) the MO-OA-2H2O system at 25 °C. Phase notations: C, cubic phase; LR, lamellar phase; L2, reversed micellar solution phase; hydr lip cryst, hydrated lipid crystals; HII, reversed hexagonal phase; HI, normal hexagonal phase; Cmic, cubic micellar phase. Compositions are given in wt %. The arrows indicate the expected path of the phase transitions caused by the lipolytic reaction from the initial sample composition of the CD, HII, and LR samples. For the DO-HII sample, this is indicated as if DO had been replaced by OA.

diagrams of the ternary MO-OA-2H2O and MO-DO2 H2O are quite similar. In addition, the DO content is reduced relatively fast during lipolysis (Table 2). As is clear from Figure 6, the phase transitions induced by the lipase action on cubic and HII phases seem to follow the same pathway and the transitions occur at about the same time (and composition). For instance, the Cmic phase occurs in all these systems after about 300 min. The lattice parameter (about 145 Å) for this phase is similar in all three systems. The one-phase region of Cmic in the investigated systems is rather small. In fact, it was very difficult to prepare a pure single-phase sample of this phase.4 It is proposed that the Cmic phase is important for the progress of the lipolytic process as it makes the substrate available to the lipase and helps to solubilize the product. 9,10,30 The bicontinuous cubic phase has been assigned the same role during the lipolytic process.6,11 Our previous self-diffusion measurements, using NMR, showed that the self-diffusion coefficient, Dlip, of the lipid in a bicontinuous cubic phase is significantly higher (10 × 10-12 m2/s) than in the Cmic phase (6 × 10-12 m2/s) at the same water content of about 15 wt %.12 This means that if the lipase activity were limited by the transport of the product, the lipolytic process would be favored in the bicontinuous cubic phase compared with the Cmic phase. On the other hand, Cmic can exist at much higher OA concentrations. When comparing the results obtained from the hydrolysis experiments with the ternary phase diagrams, it should be borne in mind that the structures resulting from hydrolysis are not necessarily the equilibrium structures. Furthermore, glycerol is generated as a product of hydrolysis, and thus the system is no longer a ternary system. To estimate these effects, glycerol and oleic acid were added to the CD sample, corresponding to some of the compositions given in Table 2. For the composition corresponding to 20 min of hydrolysis, the SAXD spectrum is dominated by an HII phase with RHII ) 59 Å with traces of a cubic phase. No traces of cubic phase were observed after 30 min and RHII ) 60 Å. These structures appeared during lipolysis after 45 min, with the same lattice parameter. This slight time difference indicates that the (30) Mariani, P.; Luzzati, V.; Rivas, E.; Delacroix, H. Biochemistry 1990, 29, 6799.

structures observed during lipolysis are not at complete equilibrium. However, the structures as such do not seem to be affected by the presence of glycerol. In a previous investigation, we studied the lipolysis of a CG phase with initial compositions of 50.2, 42.5, and 7.3 wt % of MO, H2O, and NaO, respectively (45.7, 47.7, and 6.6 wt %, respectively, after adding TLL).12 The reaction sequence in terms of phase transitions closely followed that shown in Figure 6, although the rate was slower as the TLL concentration was 47 nM. The pH of this sample was also about 6.5. Hence, the phase transitions could be described by the ternary MO-OA-2H2O system. In parallel with the present study, we have investigated the effect of TLL on dispersed cubic and vesicle samples at a water content of 98 wt %.14 The dispersed cubic (or cubosome) sample had a pH of about 6.5, and changes in morphology, studied by cryo-TEM, were as expected from the ternary MO-OA-2H2O system. The vesicle sample was prepared from a MO-NaO mixture, which gave a pH of about 10. Therefore, the changes in morphology, as observed for the LR sample in the present study, corresponded to the ternary MO-NaO-2H2O system. Does the Self-Assembly Structure Affect the Lipase Activity? The effect of the substrate’s self-assembled structure on the lipase activity has, to our knowledge, not been investigated. It has been suggested that certain structures, for example, different types of cubic phases, would facilitate lipase-catalyzed hydrolysis.6,9-11,30 This would then be observed as a change in the specific activity of the lipase. One of the objectives of the present study was to investigate the effect of the substrate structure, in terms of different lc phases, on the lipase activity. We are well aware of the difficulties in measuring the lipase activity on these types of insoluble substrates. The effective substrate concentration is not well-defined, as the area that is accessible to the lipase cannot be accurately determined. Therefore, the data presented should not be taken as absolute values. They do, however, give an estimate of the activity of TLL on a liquid crystal substrate and make it possible to compare different substrate lc structures. One way to overcome these difficulties is to spread the substrate over an interface as has been done successfully by Verger et al. (cf. refs 2 and 31). However, such studies are limited to substrates that can form stable


monolayers at an interface, and it is difficult to interpret the results for more complex substrate mixtures. To obtain a well-defined surface of triglycerides, gum arabic is often used as an emulsifier. However, gum arabic has recently been shown to affect the activity.32 Figure 5a shows that independent of the initial composition and structure of the substrate, the ratio between the substrate (MO/DO) and final product (OA) seems to approach about the same value. The initial hydrolysis rates were determined and are expressed as the specific lipase activity in Table 4. We noted that the specific activity was dependent on the TLL concentration. For the CD sample, the activity at 51 nM TLL was about 2-3 times lower than at 102 nM. Here it is important to take into account the fact that the lipase adsorbed onto the different surfaces, for example, the glass walls of the vials. At sufficiently low lipase concentration, this will reduce the effective lipase concentration. The plateau value of TLL adsorption on a hydrophilic silica surface was found to be 0.5 mg/m2.33 The total volume of the TLL/substrate mixture in the present study was about 50 µL. This means that the total amount of TLL in the vial is about 8 × 10-5 mg, which is just enough to cover 1.6 cm2 with a monolayer of TLL. In fact, this is about the same order of magnitude as the surface of the vial exposed to the TLL solution. Therefore, adsorption onto the vial surfaces can explain the lower specificity at the lower lipase concentration. When increasing the lipase concentration from 102 nM to 205 nM, we again observed a decrease in the lipase activity. This was also observed for the OA-HII sample. At the higher TLL concentration, the reaction is probably too fast to observe with the sampling procedure employed. This is even more evident at the higher temperature (37 °C) and 205 nM TLL, where the specific activity decreased sharply with time (Figure 5b). Upon comparing the data for the specific activity on the CD and OA-HII samples, we concluded that there was no significant difference under the employed experimental conditions. This is quite remarkable, considering the different nanostructures of the two substrates. Moreover, the obtained specific activity of about 1000 µmol min-1 mg-1 is comparable to the 2000 µmol min-1 mg-1 observed for TLL on triolein.32 As mentioned above, it has been suggested that cubic phases facilitate lipase-catalyzed hydrolysis. However, we again stress that the bicontinuous cubic phase can take up only a limited amount (∼0.5 wt %) of OA. Thus, the CD phase is expected to rapidly

Langmuir, Vol. 18, No. 23, 2002 8981

transform into an HII phase as depicted in Figure 6. The initial reaction rate might therefore be affected by this phase transition. On the other hand, the HII sample used as a substrate for TLL contains about 16 wt % OA, which is the product of lipolysis. Lipase activity is normally considered to be hampered by the accumulation of products (cf. ref 34). However, it is possible that OA is solubilized within the nonpolar domain of the HII phase and is not accessible to the enzyme. The lipolytic reaction seemed to produce the same steady-state composition regardless of the initial substrate composition and self-assembled structure. Here it is important to stress that the action of lipase only decreases the time taken to reach equilibrium and does not affect the equilibrium composition as such. Thus, the changes in structure and composition of the lipid dispersions reported in this work would have occurred without the lipase if given enough time. Conclusion Here, phase diagrams have been used as maps to navigate through the changes in the self-assembly structures of the substrate and product. This approach has been demonstrated to be invaluable in the interpretation and prediction of phase changes when lipids in glyceride systems are subjected to lipolysis. Such knowledge also enables the correlation between lipase activity and substrate structure to be determined. Although we observed similar specific activity of TLL on the CD samples as on the HII samples, significant differences can occur for other self-assembly structures, sample compositions, and/ or experimental conditions. Insight into which structures may form during the lipolysis of triolein and how the selfassembled structure affects the activity of the lipase helps us to understand the physicochemical events that take place in nature. In addition, the increased knowledge about possible substrates and products will hopefully make it possible to design new lipases with higher activity. Acknowledgment. We are grateful to Allan Svendsen and Shamkant Patkar, Novozymes, A/S Denmark, for the lipase sample and stimulating discussions and to Karl Hult for his generous help and suggestions. Financial support was obtained from the EU Biotech Shared Cost Project, Contract No. BIO4-97-2365. LA020377D

(31) Beisson, F.; Tiss, A.; Rivie`re, C.; Verger, R. Eur. J. Lipid Sci. Technol. 2000, 133. (32) Tiss, A.; Carrie`re, F.; Verger, R. Anal. Biochem. 2001, 294, 36. (33) Wannerberger, K.; Arnebrant, T. J. Colloid Interface Sci. 1996, 177, 316.

(34) Brockman, H. L. General features of lipolysis. In Lipases; Borgstro¨m, B., Brockman, H. L., Eds.; Elsevier Science: Amsterdam, 1984; p 1.

Effect of Lipase on Different Lipid Liquid Crystalline Phases Formed by

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