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Activity of acetone-treated Chromobacterium viscosum lipase in AOT reverse micelles in the presence of low molecular weight polyethylene glycol
Biochemical Engineering Journal 29 (2006) 46–54
Activity of acetone-treated Chromobacterium viscosum lipase in AOT reverse micelles in the presence of low molecular weight polyethylene glycol M.M. Zaman a , Y. Hayashi a,∗ , M.M.R. Talukder b , T. Kawanishi a a b
Division of Material Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma Machi, Kanazawa 920-1192, Japan Institute of Chemical and Engineering Sciences, 1 Pesek Road, Jurong Island, Singapore 627833, Singapore Received 15 October 2004; accepted 23 February 2005
Abstract The activity of Chromobacterium viscosum lipase (glycerol-ester hydrolase, EC 3.1.1.3) entrapped in AOT/isooctane/water reverse micelles was significantly enhanced by pretreatment with acetone, using the hydrolysis of olive oil as a model reaction. The activity of acetone treated lipase was further enhanced when low molecular weight polyethylene glycol (PEG 400) was introduced into AOT reverse micelles. To know the effects of acetone treatment on the lipase activity in simple AOT and mixed AOT/PEG 400 reverse micelles, the influence of various parameters, such as W0 (molar ratio of water to surfactant), pH, surfactant concentration, ionic strength and reaction temperature were investigated and compared with those for native lipase in simple AOT reverse micelles. The optimal activities of treated lipase in AOT reverse micelles with and without PEG 400 were 2.0 and 1.6 times higher respectively than that of native lipase in AOT reverse micelles. A kinetic model that considers substrate adsorption equilibrium between the bulk phase of organic solvent and the micellar phase was successfully used to understand the improvement of the lipase activity. The Michaelis constant (Km ) and substrate adsorption equilibrium constant (Kad ) were obviously reduced compared with those for native lipase in AOT reverse micelles. The stability of the lipase in reverse micelles was also studied, and the values of half-life time (t1/2 ) were determined from residual activity profiles. © 2005 Elsevier B.V. All rights reserved. Keywords: Acetone treatment; PEG 400; Reverse micelles; Chromobacterium viscosum lipase; AOT; Hydrolysis
1. Introduction In industrial processes, the non-enzymatic hydrolysis of oils and fats is operated at high temperature and pressure, and such extreme conditions demand high energy consumption. For the sake of energy conservation and minimizing thermal degradation of products, enzymatic hydrolysis has been introduced during the last decade. Enzyme-catalyzed hydrolysis possesses advantages such as mild conditions, high substrate specificity and less environmental pollution compared with conventional chemical reactions. Enzymatic hydrolysis in organic solvents has resulted in important advances in enzyme technology [1–3]. Lipases in general are known to act ∗
Corresponding author. Tel.: +81 76 234 4806; fax: +81 76 234 4811. E-mail address: [email protected] (Y. Hayashi).
1369-703X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2005.02.031
only at or near the oil/water interface [4,5]. It is thus necessary to increase the interfacial area for rapid lipase-catalyzed reaction rate. On the various approaches used to carry out the reaction in interfacial media, reverse micelles appear to be one of the most effective systems [6,7]. Compared to other organic interfacial media, reverse micelles provide a larger interfacial area that promotes contact between enzyme and substrate [6]. The anionic double-tailed surfactant AOT [sodium bis-(2-ethyl 1-hexyl) sulfosucccinate] is frequently used in micellar technologies due to its optical clarity and ease of preparation. However, activities of enzymes entrapped in AOT reverse micelles are adversely affected by strong interactions with surfactant molecules [8–11] as well as the distinctive properties of micellar water, which usually inhibits the accessibility of the lipase active site to the micellar interface [12].
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To overcome these problems, one approach is to modify the reverse micellar systems by using additives such as low molecular weight polyethylene glycols (PEGs). Our previous studies [13,14] have shown that PEG 400 is very effective in enhancing the lipase activity in AOT reverse micelles. PEG 400 can be dissolved inside the water pool and reduces the lipase–AOT interaction by burying charges at the micellar interface and enzyme surface. PEG 400 also strongly interacts with enzymes by noncovalent linkage, which may cause a change in lipase conformation entrapped in reverse micelles. In addition, FTIR results [14] suggest that the PEG 400 molecule can change the properties of micellar water. As a consequence, the active site of lipase may be oriented more easily toward the micellar interface. In this study, we wish to report that pretreatment of lipase with acetone increases the activity of lipase entrapped in simple AOT and mixed AOT/PEG 400 reverse micellar systems. The pretreatment of enzymes with acetone enhances the interaction between hydrophobic substrate and lipase active site by facilitating the exposure of hydrophobic amino acids to the lipase surface, which causes a change in lipase conformation from closed form (lid covering the active site is in closed state) to open form (lid is in open state) at the micellar interface. A similar phenomenon has also been reported by other researchers [15–18]. Since the active site of Chromobacterium viscosum lipase is located in the hydrophobic amino acids region, it can easily access the micellar interface and comes into contact with hydrophobic substrate more effectively. Furthermore, enhanced hydrophobic amino acids on the enzyme surface may contribute to enzyme activation by making lipase environmentally compatible inside the water pool that usually inhibits the access of the lipase active site to the micellar interface. It can be assumed that pretreatment of lipase with acetone, and then the coexistence effect of the interaction between acetone treated lipase with PEG 400 molecules, lead to a change in lipase conformation so that the combination between active site of the treated lipase and substrate is not hindered. In the present study, the initial activity of acetone treated lipase entrapped in simple AOT and AOT/PEG 400 reverse micelles was characterized by optimizing various physical parameters. Kinetics and stability studies were then undertaken to clarify the effect of acetone pretreatment as well as PEG 400 addition on lipase activity. Finally, the change in lipase conformation due to acetone pretreatment was investigated by tryptophyl fluorescence spectroscopy.
2. Materials and methods
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weight of 30 kDa (isoelectric point 7.3). Lipase B is the main portion of the mixture with a weight content over 80% [19]. Surfactant AOT [sodium bis- (2-ethyl 1-hexyl) sulfosuccinate], isooctane, polyethylene glycol (PEG 400), benzene, pyridine, olive oil, copper (II) acetate and acetone were purchased from Wako Pure Chemical Industries Co. The average molecular weight of the olive oil was 875 g mol−1 and density was 0.91 g cm−3 . All chemicals were analytical grade and used without further purification. 2.2. Treatment of lipase with acetone The pretreatment was performed by adding 10 mg of lipase into a solution of 50% (volume) cold acetone in Brinton buffer [20], followed by agitating for a desired time by a magnetic stirrer at 500 rpm in an incubator at 4 ◦ C. After freezing the solution at −80 ◦ C for about 12 h, the sample was dried under a freeze dryer (EYALA, FDU-506) at a pressure approximately 8 Pa and a condensed temperature of −50 ◦ C. Lyophilized lipase powders were stored in a freezer at −20 ◦ C. 2.3. Preparation of reverse micelles and encapsulation of treated lipase in reverse micelles Reverse micellar solutions were prepared by dissolving AOT in isooctane with a limited amount of water. Freeze dried lipase was encapsulated in reverse micelles by the dissolution method. The treated lipase powder was directly added to the reverse micellar solution and agitated by a magnetic stirrer at 500 rpm in an incubator at 25 ◦ C for 15 min. After centrifuging (HIMAC centrifuge, Hitachi, model CR15) at 1000 rpm for 5 min, the upper phase was separated carefully. The lipase content in the upper phase was determined by UV method. The absorbance was detected at 278 nm by double beam Spectrophotometer and the lipase concentration was calculated according to the standard curve. Final reverse micelles containing lipase (2 mg dm−3 ) were then prepared by adding an appropriate amount of reverse micellar solution in which the freeze dried lipase had been dissolved in advance. Water–AOT molar ratio W0 was adjusted by adding the desired amount of buffer solution. Reverse micelles containing native lipase were prepared with the same procedures as pretreated lipase. The systems containing PEG 400 were prepared by directly mixing reverse micelles solution in a glass bottle in which the appropriate amount of PEG had been added in advance. A magnetic stirrer was used to agitate the mixture until a clear and optically transparent solution was obtained. The micellar solution containing lipase was placed in an incubator at 25 ◦ C to initiate the hydrolysis reaction.
2.1. Materials 2.4. Determination of lipase activity Purified C. viscosum lipase (glycerol-ester hydrolase, EC 3.1.1.3) was provided by Asahi Chemical Industry Co. Ltd. It consists of a mixture of lipase A with a molecular weight of 120 kDa (isoelectric point 3.7) and lipase B with a molecular
In this study, lipase activity was measured as the initial reaction rate, V [mol dm−3 s−1 ], using olive oil as a water insoluble substrate. The reaction was initiated by adding an
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appropriate amount of olive oil into a pre-incubated micellar solution containing lipase. The reaction mixture was agitated by a magnetic stirrer at 500 rpm in an incubator at constant temperature of 25 ◦ C for exactly 20 min as it was found that within this time range, the production of free fatty acid was linearly dependent on time [21]. The resulting fatty acid was analyzed by the Lowry technique [22]. Details of activity determination have been described elsewhere [21]. All data are the average of five replicated experiments and are reproducible within ±5%. In this study, all concentration terms are based on total volume of the reverse micellar systems unless otherwise specified. 3. Results and discussion 3.1. Optimization of pretreatment parameters The effect of various relevant pretreatment parameters such as acetone content in water–acetone solution, pH of water–acetone solution, lipase agitation time in water–acetone solution and freeze-drying time on the catalytic activity of enzymes were optimized and are listed in Table 1. The effect of each operational parameter was investigated by varying only the parameter considered, the others being kept constant. Our findings (data not shown) reveal the surprising dependence of catalytic activity of acetone treated lipase in AOT reverse micelles on the pretreatment conditions. 3.2. Effect of PEG 400 concentration
Fig. 1. Effect of PEG 400 concentration on the activity of acetone treated lipase in AOT reverse micelles. Experimental conditions: Clipase = 2 mg dm−3 , Colive oil = 0.055 mol dm−3 , CAOT = 0.05 mol dm−3 , CNaCl (in buffer) = 0.3 mol dm−3 , buffer pH = 8, W0 = 8 and reaction temperature = 25 ◦ C.
shaped profile observed in simple AOT micellar systems for native lipase. The optimum W0 of acetone treated lipase for simple AOT and mixed AOT/PEG 400 systems are 10 and 8, respectively, similar to those for native lipase in simple AOT and AOT/PEG 400 systems [13]. It has been reported that the optimum value of W0 is commonly related to a situation where the inner diameter of the empty micelle corresponds to
Fig. 1 shows that with the increase in PEG 400 concentrations, the enzyme activity increases to a maximum value at a PEG concentration of 12.5–15 [mmol dm−3 ] and then begins to decrease. The PEG 400 molecule buries the charges on the micellar interface through interaction with AOT head groups, and PEG clouds are formed around the lipase molecules. At lower PEG concentrations, the linkage of PEG to the AOT head groups as well as the enzyme surface may not develop sufficiently. At higher PEG concentrations, the reverse micellar solution becomes turbid, and is in fact, found to be broken. The results in Fig. 1 show this clearly. 3.3. Effect of W0 Fig. 2 indicates that the dependence of acetone treated enzyme activity on W0 is not recognized as a classic bellTable 1 Optimum value of parameters related to the pretreatment of Chromobacterium viscosum lipase with acetone Pretreatment condition
Optimum value
Acetone content in water–acetone solution Water–acetone solution pH Agitation time of lipase in water–acetone solution Freeze drying (lyophilization) time
50% (volume) 7.2 1h 20 h
Fig. 2. Effect of W0 on the activity of treated and native lipases in individual reverse micelles. () Treated lipase in AOT/PEG 400 systems, () treated lipase in AOT systems and () native lipase in AOT systems. Experimental conditions: Clipase = 2 mg dm−3 , Colive oil = 0.055 mol dm−3 , CAOT = 0.05 mol dm−3 , CPEG 400 = 12.5 mmol dm−3 , CNaCl (in buffer) = 0.3 mol dm−3 , pH = 8 and reaction temperature = 25 ◦ C.
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the size of the encapsulated enzymes [23–25]. It seems that the above conclusion holds true in AOT and AOT/PEG 400 reverse micellar systems for treated lipase. It was seen that the lipase activity at higher W0 values was improved by the pretreatment with acetone. At higher W0 values, the size of reverse micelles is higher than the size of lipase, leading the decrease in lipase immobilization inside the water-pool, and it becomes difficult to expose its active site at the micellar interface to combine with substrate molecules [26,27]. Since the surface of treated lipase is more hydrophobic than that of native lipase, it exists relatively near to the micellar interface. As a result, the active site of treated lipase can easily access the micellar interface, and its interaction with substrate becomes less inhibited compared to native lipase. Once again the results support our conjecture that acetone pretreatment may cause a change in lipase conformation, which favors the exposure of the hydrophobic binding site in the open conformation. It should be noted that in the presence of PEG 400, lipase activity is enhanced appreciably at lower and higher W0 values compared to that in simple AOT systems for native and treated lipases. This enhancement is attributed to the decrease in the interaction between treated lipase and surfactant molecules [13,28]. PEG 400 also participates in the redistribution of water and binds to the enzyme surface by noncovalent linkage [29]. Hence, the enzyme can be protected in an unfavorable environment by PEG molecules at its surface [30]. In addition, in the presence of PEG 400, the shifting of optimum W0 to a lower value and the improvement in lipase activity at lower W0 values are due to the increase in micellar size by PEG 400 molecules, which promotes the lipase encapsulation into the micellar inner core [13]. It is worth mentioning that at values of W0 lower than 6, reverse micelles could not solubilize all the PEG 400 molecules at a concentration of 12.5 mmol dm−3 .
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Fig. 3. Dependence of treated and native lipase activity on pH in individual reverse micelles. () Treated lipase in AOT/PEG 400 systems for W0 8, () treated lipase in AOT systems for W0 10 and () native lipase in AOT systems for W0 10. Other experimental conditions are the same as those in Fig. 2 except pH.
Although the optimal pH of native and treated lipases is the same, the activity of acetone treated lipase is higher than that of native lipase. This result suggests that acetone pretreatment would tend to convert the closed form of lipase into the open one. It should be noted that in the presence of PEG 400, the activity of acetone treated lipase at higher pH increases: the activity at pH 10 is similar to that at pH 7. The effective protection of the surrounding PEG 400 molecules may be responsible for the alkali resistance of treated lipase [13]. At lower or higher pH values, lipase suffers from denaturation, and activity decreases. 3.5. Effect of surfactant concentration
3.4. Effect of pH The effect of pH on the activity of treated lipase in AOT and AOT/PEG 400 reverse micellar systems is shown in Fig. 3 and compared with the results for native lipase in AOT reverse micellar systems. It is found that the optimal pH for treated lipase in AOT or AOT/PEG 400 reverse micellar systems is almost the same as that for native lipase in AOT reverse micellar systems. The optimal pH value is found to be 8, which is quite close to the isoelectric point (pI) of C. viscosum lipase (pH 7.3). Since, the net charge on the enzyme surface is zero when the value of pH is equal to pI, the ionic interaction between enzymes and negatively charged head groups of the surfactant (AOT) would be minimized. From Fig. 3, it is evident that the optimum pH is slightly higher than pI. It is worth mentioning that there is some decrease in pH when bulk water of a certain pH is transferred in the reverse micellar phase [31,32]. Therefore, the effective pH inside reverse micelles may be somewhat lower than that of buffer pH that is injected in the reverse micellar phase.
Fig. 4 shows that although the activity of native and treated lipases in simple AOT reverse micelles decreases with the increase in AOT concentration, in the presence of PEG 400 the activity of treated lipase goes through a maximum value with the increase in surfactant concentration and then decreases. However, the tendency of a linear decrease in lipase activity with AOT concentration observed in simple AOT reverse micelles, disappeared with pretreatment. In general, enzyme activity drops sharply with an increase in surfactant concentration [33–36]. In AOT/PEG 400 reverse micelles at constant molar ratios of PEG 400 to AOT, the overall PEG concentration increases with the increase in AOT concentration, leading to the increase in treated lipase activity. Additionally, with the increase in AOT concentration, both micelle number and total amount of substrate adsorbed on the micellar surfactant tails increases, leading to the decrease in free substrate concentration taking part in the hydrolysis reaction. Hence, the optimal value of surfactant concentration is found to be 0.05 mol dm−3 .
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Fig. 4. Effect of AOT concentration on activity of treated and native lipases in individual reverse micelles. () Treated lipase in AOT/PEG 400 systems for W0 8 and CPEG 400 /CAOT = 0.25, () treated lipase in AOT systems for W0 10 and () native lipase in AOT systems for W0 10. Other experimental conditions are the same as those in Fig. 2 except AOT concentration.
Fig. 5. Effect of ionic strength on the activity of treated and native lipases in individual reverse micelles. () Treated lipase in AOT/PEG 400 systems for W0 8, () treated lipase in AOT systems for W0 10 and () native lipase in AOT systems for W0 10. Other experimental conditions are the same as those in Fig. 2 except NaCl concentration.
This study also revealed that the improvement of treated lipase activity in both simple AOT and mixed AOT/PEG 400 systems is more significant at higher AOT concentrations than at lower concentrations. The rate of lipase-catalyzed hydrolysis of olive oil in AOT reverse micelles may decreases due to two main reasons, i.e., the interaction of lipase with AOT molecules and the adsorption of substrates on the micellar surfactant surface. However, the pretreatment of lipase has no effect on the substrate adsorption. Therefore, improvements in lipase activity can be obtained by reducing the interaction between lipase and AOT molecules due to the change in lipase conformation. In addition, PEG 400 forms clouds around the lipase molecule, which also suppresses the interaction of treated lipase with surfactant molecules.
the lipase surface properties. In fact, the surface of treated lipase is enhanced with hydrophobic amino acids, which are unfavorable for electrostatic interaction. Moreover, PEG 400 inhibits the cationic Na+ approaching the micellar interface, and the effect of ionic strength on micellar size becomes less effective [37]. Therefore, activity seems to be unaffected by ionic strength up to CNaCl = 0.6 M. At higher ionic strengths, treated lipase shows deactivation due to the salting out effect.
3.6. Effect of buffer ionic strength (CNaCl in buffer) It is obvious from Fig. 5 that the activity of treated lipases in simple AOT and PEG 400 modified reverse micelles is almost independent of ionic strength up to 0.3 and 0.6 mol dm−3 , respectively. Generally, enzyme activity increases with an increase in ionic strength up to an optimum value as the increase in ionic strength causes a reduction in the electrostatic interaction between lipase and AOT head groups. Further an increase in ionic strength decreases enzyme activity for two reasons. First, high ionic strength decreases the solubility and activity of the enzyme due to the salting out effect. Secondly, the size of the micelle significantly decreases at high ionic strength [37]. This is evident in AOT systems for native lipase. The enhanced treated lipase activity at lower ionic strength may be attributed to the suppression of the electrostatic interaction between lipase and AOT molecules due to changes in
3.7. Effect of reaction temperature Fig. 6 shows that the optimum reaction temperature for all three systems was around 25–30 ◦ C. The ascending portion of the temperature curve (Fig. 6) reflects the general effect of increasing temperature on the rate of chemical reactions. Above 30 ◦ C the enzyme is denatured rapidly: the descending portion of the curve reflects the loss of catalytic activity as the enzyme is denatured at higher temperatures. 3.8. Reaction kinetics A kinetic model based on the adsorption equilibrium of substrate between the organic and micelle phases is used to understand the hydrolysis reaction catalyzed by acetone treated lipase entrapped in AOT and AOT/PEG 400 reverse micellar systems. This model has been reported in the literature [21,33], and is expressed in the following formula: v=
Vmax [ST ] Km (1 + Kad [CS ]) + [ST ]
(1)
Vmax [ST ] Km.a + [ST ]
(2)
or v=
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Fig. 6. Dependence of treated and native lipase activity on reaction temperature in individual reverse micelles. () Treated lipase in AOT/PEG 400 systems for W0 8, () treated lipase in AOT systems for W0 10 and () native lipase in AOT systems for W0 10. Other experimental conditions are the same as those in Fig. 2 except reaction temperature.
where Km.a = Km (1 + Kad [CS ]) and is called the apparent Michaelis constant. Eq. (2) can be rearranged 1 Km.a 1 1 + = v Vmax Vmax [ST ]
(3)
For the treated lipase in simple AOT and AOT/PEG 400 systems, the Lineweaver–Burk and Km.a versus [CS ] plots are linear. From the slope and intercepts of the plot of 1/v versus1/[ST ] (Eq. (3)) for different AOT concentrations, the corresponding values of Km.a could be determined. From the slope and intercept of the plot Km.a versus [CS ], the values of Km and Kad could also be calculated. The values of kinetic parameters of native lipase are taken from literature [33]. The determined kinetic parameters are listed in Table 2. Maximum enzyme activity (Vmax ) is shown to be almost the same for treated and native lipase in AOT and AOT/PEG 400 reverse micellar systems. However, the Michaelis constant (Km ) and the adsorption equilibrium constant (Kad ) decrease significantly for the acetone treated lipase in AOT and AOT/PEG 400 reverse micellar systems compared with those for native lipase in simple AOT reverse micellar systems.
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The low Km value indicates a high affinity for the substrate. Because of the increase in the exposure of hydrophobic amino acids on the treated lipase surface, it can be imagined that the lipase molecule is expanded over the micellar surface with better orientation, leading to higher affinity for hydrophobic substrates as well as more available active sites at the micellar interface. In AOT/PEG 400 systems, the decrease in Km implies that the treated lipase conformation is changed by the direct interaction with PEG molecules, while the combination between the substrate and lipase active site becomes much easier [13]. The Kad value reflects two factors [28]: the adsorption of substrate on the surfactant tails, and the interaction of enzyme with AOT molecules. Since both lipase and PEG 400 molecules exist in the micellar water pool, the substrate adsorption on the micellar surfactant surface is not affected by either the pretreatment of lipase or the addition of PEG 400 to the micellar systems. Therefore, the significant decrease in Kad indicates that the interaction of lipase with AOT molecules is reduced by acetone pretreatment as well as the addition of PEG 400 to the AOT reverse micellar system. Fig. 7 shows that the activity of acetone treated lipase in simple AOT and AOT/PEG 400 systems, calculated using the model equation (1), agrees well with the experimental results. The results show that the kinetic model for lipase-catalyzed hydrolysis of olive oil in AOT reverse micellar systems [33] can also be applied in the enzymatic reaction catalyzed by the acetone treated lipase in AOT and AOT/PEG 400 reverse micellar systems. 3.9. Stability of acetone treated lipase The stability of acetone treated lipase in AOT and AOT/PEG 400 reverse micellar systems was also investigated, as this is a crucial factor in industrial applications. Stability was examined by assaying residual activity after incubation without substrate at 25 ◦ C for a required period. Residual activity was calculated as a percentage of original activity (considered 100%), obtained at t = 0 min incubation. Lipase half-life (t1/2 ) was calculated directly from the residual activity profiles. Fig. 8 shows that acetone treated lipase is very stable when entrapped in simple AOT and mixed AOT/PEG 400 reverse micellar systems, retaining approximately 92.2 and 94% respectively of initial activity after 15 days of incubation. In contrast, native lipase retains only 75% initial activity in AOT systems over the same duration.
Table 2 Comparison of kinetics parameters in individual reverse micellar systems for treated and native lipases Kinetic parameters
Treated lipase in AOT/PEG 400 systems
Treated lipase in AOT systems
Native lipase in AOT systems, Ref. [33]
Vmax [ mol dm−3 s−1 ] Km [mol dm−3 ] Kad [mol−1 dm3 ] Vmax /Km [s−1 ]
29.5 0.0333 5.018 8.85 × 10−4
26.8 0.0365 7.98 7.34 × 10−4
27 0.083 16.2 3.25 × 10−4
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enzyme against denaturation in AOT reverse micelles (a similar phenomenon has been previously reported in the literature [39]). Moreover, the observed improvement in the lipase activity in AOT/PEG 400 reverse micellar systems is due to the suppression of the interaction between lipase and AOT molecules, since charges on the micellar interface and enzyme surface are buried by neutral PEG molecules [30]. To understand the change in lipase conformation by pretreatment, fluorescence properties of native and treated lipase was investigated (details have been described in the next section). The variation of maximum fluorescence intensity versus incubation time at 25 ◦ C indicates that treated lipase retains 95% of its initial maximum fluorescence intensity after 10 days (data not shown). Therefore, it can be concluded that the change in lipase conformation by acetone pretreatment is stable. 3.10. Fluorescence spectroscopy of lipase Fig. 7. Comparison of activity values obtained experimentally with those predicted by the model Eq. (1). () Treated lipase in AOT/PEG 400 systems for W0 = 8 and CPEG 400 = 12.5 mmol dm−3 and () treated lipase in AOT systems for W0 = 10. Curved lines are predicted by model equation. Other experimental conditions: Clipase = 2 mg dm−3 , CNaCl (in buffer) = 0.3 mol dm−3 , pH = 8, reaction temperature = 25 ◦ C.
The half-life of treated lipase in AOT and AOT/PEG 400 systems is 120 and 160 days respectively, whereas half-life of native lipase in AOT systems is only 38 days, comparable with results reported elsewhere [8,38]. Increased stability from pretreatment with acetone may be attributed to enhanced hydrophobic residues on the lipase surface that stabilize the
Fig. 8. Stability of lipase in individual reverse micelles at 255 ◦ C. () Treated lipase in AOT/PEG 400 systems for W0 = 8 and CPEG 400 = 12.5 mmol dm−3 , () treated lipase in AOT systems for W0 = 10 and () native lipase in AOT systems for W0 10. Storage conditions: Clipase = 2 mg dm−3 , CAOT = 0.05 mol dm−3 , CNaCl =0.03 mol dm−3 , pH = 8. Activity assay conditions: Colive oil = 0.055 mol dm−3 , reaction time = 20 min, reaction temperature = 25 ◦ C.
Proteins contain several chromophores that absorb light in the ultra-violet and infrared regions. Many chromophores called fluorophores can also display fluorescence. Fluorescence is a useful probe to know the structure and structural changes in enzymes [40,41]. The fluorescence emission spectra for native and treated lipases in AOT and AOT/PEG 400 reverse micellar systems were recorded at 25 ◦ C (Fig. 9). The emission spectra were recorded from 300 to 400 nm with a Hitachi F-3010 fluorescence spectrophotometer at an excitation wavelength of 280 nm, the selective excitation wavelength for tryptophan residues [42,43]. Emission and excitation slit widths were 5 nm. Spectra were uncorrected for instrument sensitivity, but the emission of blank reverse
Fig. 9. Fluorescence spectra of lipase in individual reverse micelles. () Treated lipase in AOT/PEG 400 systems for W0 = 8 and CPEG −3 400 = 12.5 mmol dm , () treated lipase in AOT systems for W0 = 10 and () native lipase in AOT systems for W0 10. Experimental conditions: Clipase = 10 mg dm−3 , CAOT = 0.05 mol dm−3 , CNaCl = 0.03 mol dm−3 , pH = 8.
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micelles (without lipase) was subtracted. Three dimensional crystal structure [44] and amino acid sequence indicate that C. viscosum lipase contains 3 tryptophan, 10 tyrosine and 8 phenylalanine residues as fluorophores whose excitation wavelength are 280, 257 and 270 nm, respectively. However, only tryptophan residues are sensitive in water or AOT/water/isooctane systems. From Fig. 9, it is evident that the fluorescence intensity of treated lipase both in simple AOT and AOT/PEG 400 systems is higher than that of the native lipase in AOT systems. The increase in fluorescence intensity indicates that treated lipase may have a better orientation, which led to more tryptophan (hydrophobic) residues on the lipase surface. Grauper et al. [45] have reported that tryptophan residues as well as the active site of C. viscosum lipase in native form are located in the hydrophobic region, and their approach to the lipase surface is very difficult. Therefore, only a conformational change would result in the enhancement of fluorescence intensity. The addition of PEG 400 further enhances this intensity, and this is attributed to the change in lipase conformation due to the interaction with the PEG molecules.
4. Conclusions The activity of acetone-treated C. viscosum lipasecatalyzed hydrolysis of olive oil was carried out in AOT/isooctane and AOT/PEG 400/isooctane reverse micellar systems. The favorable effects of both pretreatment with acetone and the addition of PEG 400 molecules enhanced the activity of lipase significantly in AOT systems. The improvement in lipase activity at higher W0 as well as at higher AOT concentrations indicated that the acetone treated lipase might possess a better orientation for enzymatic reactions than that of native lipase. When PEG 400 was added to the AOT reverse micellar systems, the interaction between the anionic AOT head groups and the treated lipase was markedly suppressed, and the active site of treated lipase was also protected from unfavorable action of AOT molecules. The lower value of the Michaelis constant suggested that the affinity between lipase and substrate increased by pretreatment with acetone as well as the addition of PEG 400. The stability study revealed that the treated lipase entrapped in AOT and AOT/PEG 400 reverse micellar systems was very stable. Finally, fluorescence studies supported the hypothesis of a change in lipase conformation.
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Activity of acetone-treated Chromobacterium viscosum lipase in AOT reverse micelles in the presence of low molecular weight polyethylene glycol M.M. Zaman a , Y. Hayashi a,∗ , M.M.R. Talukder b , T. Kawanishi a a b
Division of Material Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma Machi, Kanazawa 920-1192, Japan Institute of Chemical and Engineering Sciences, 1 Pesek Road, Jurong Island, Singapore 627833, Singapore Received 15 October 2004; accepted 23 February 2005
Abstract The activity of Chromobacterium viscosum lipase (glycerol-ester hydrolase, EC 3.1.1.3) entrapped in AOT/isooctane/water reverse micelles was significantly enhanced by pretreatment with acetone, using the hydrolysis of olive oil as a model reaction. The activity of acetone treated lipase was further enhanced when low molecular weight polyethylene glycol (PEG 400) was introduced into AOT reverse micelles. To know the effects of acetone treatment on the lipase activity in simple AOT and mixed AOT/PEG 400 reverse micelles, the influence of various parameters, such as W0 (molar ratio of water to surfactant), pH, surfactant concentration, ionic strength and reaction temperature were investigated and compared with those for native lipase in simple AOT reverse micelles. The optimal activities of treated lipase in AOT reverse micelles with and without PEG 400 were 2.0 and 1.6 times higher respectively than that of native lipase in AOT reverse micelles. A kinetic model that considers substrate adsorption equilibrium between the bulk phase of organic solvent and the micellar phase was successfully used to understand the improvement of the lipase activity. The Michaelis constant (Km ) and substrate adsorption equilibrium constant (Kad ) were obviously reduced compared with those for native lipase in AOT reverse micelles. The stability of the lipase in reverse micelles was also studied, and the values of half-life time (t1/2 ) were determined from residual activity profiles. © 2005 Elsevier B.V. All rights reserved. Keywords: Acetone treatment; PEG 400; Reverse micelles; Chromobacterium viscosum lipase; AOT; Hydrolysis
1. Introduction In industrial processes, the non-enzymatic hydrolysis of oils and fats is operated at high temperature and pressure, and such extreme conditions demand high energy consumption. For the sake of energy conservation and minimizing thermal degradation of products, enzymatic hydrolysis has been introduced during the last decade. Enzyme-catalyzed hydrolysis possesses advantages such as mild conditions, high substrate specificity and less environmental pollution compared with conventional chemical reactions. Enzymatic hydrolysis in organic solvents has resulted in important advances in enzyme technology [1–3]. Lipases in general are known to act ∗
Corresponding author. Tel.: +81 76 234 4806; fax: +81 76 234 4811. E-mail address: [email protected] (Y. Hayashi).
1369-703X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2005.02.031
only at or near the oil/water interface [4,5]. It is thus necessary to increase the interfacial area for rapid lipase-catalyzed reaction rate. On the various approaches used to carry out the reaction in interfacial media, reverse micelles appear to be one of the most effective systems [6,7]. Compared to other organic interfacial media, reverse micelles provide a larger interfacial area that promotes contact between enzyme and substrate [6]. The anionic double-tailed surfactant AOT [sodium bis-(2-ethyl 1-hexyl) sulfosucccinate] is frequently used in micellar technologies due to its optical clarity and ease of preparation. However, activities of enzymes entrapped in AOT reverse micelles are adversely affected by strong interactions with surfactant molecules [8–11] as well as the distinctive properties of micellar water, which usually inhibits the accessibility of the lipase active site to the micellar interface [12].
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To overcome these problems, one approach is to modify the reverse micellar systems by using additives such as low molecular weight polyethylene glycols (PEGs). Our previous studies [13,14] have shown that PEG 400 is very effective in enhancing the lipase activity in AOT reverse micelles. PEG 400 can be dissolved inside the water pool and reduces the lipase–AOT interaction by burying charges at the micellar interface and enzyme surface. PEG 400 also strongly interacts with enzymes by noncovalent linkage, which may cause a change in lipase conformation entrapped in reverse micelles. In addition, FTIR results [14] suggest that the PEG 400 molecule can change the properties of micellar water. As a consequence, the active site of lipase may be oriented more easily toward the micellar interface. In this study, we wish to report that pretreatment of lipase with acetone increases the activity of lipase entrapped in simple AOT and mixed AOT/PEG 400 reverse micellar systems. The pretreatment of enzymes with acetone enhances the interaction between hydrophobic substrate and lipase active site by facilitating the exposure of hydrophobic amino acids to the lipase surface, which causes a change in lipase conformation from closed form (lid covering the active site is in closed state) to open form (lid is in open state) at the micellar interface. A similar phenomenon has also been reported by other researchers [15–18]. Since the active site of Chromobacterium viscosum lipase is located in the hydrophobic amino acids region, it can easily access the micellar interface and comes into contact with hydrophobic substrate more effectively. Furthermore, enhanced hydrophobic amino acids on the enzyme surface may contribute to enzyme activation by making lipase environmentally compatible inside the water pool that usually inhibits the access of the lipase active site to the micellar interface. It can be assumed that pretreatment of lipase with acetone, and then the coexistence effect of the interaction between acetone treated lipase with PEG 400 molecules, lead to a change in lipase conformation so that the combination between active site of the treated lipase and substrate is not hindered. In the present study, the initial activity of acetone treated lipase entrapped in simple AOT and AOT/PEG 400 reverse micelles was characterized by optimizing various physical parameters. Kinetics and stability studies were then undertaken to clarify the effect of acetone pretreatment as well as PEG 400 addition on lipase activity. Finally, the change in lipase conformation due to acetone pretreatment was investigated by tryptophyl fluorescence spectroscopy.
2. Materials and methods
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weight of 30 kDa (isoelectric point 7.3). Lipase B is the main portion of the mixture with a weight content over 80% [19]. Surfactant AOT [sodium bis- (2-ethyl 1-hexyl) sulfosuccinate], isooctane, polyethylene glycol (PEG 400), benzene, pyridine, olive oil, copper (II) acetate and acetone were purchased from Wako Pure Chemical Industries Co. The average molecular weight of the olive oil was 875 g mol−1 and density was 0.91 g cm−3 . All chemicals were analytical grade and used without further purification. 2.2. Treatment of lipase with acetone The pretreatment was performed by adding 10 mg of lipase into a solution of 50% (volume) cold acetone in Brinton buffer [20], followed by agitating for a desired time by a magnetic stirrer at 500 rpm in an incubator at 4 ◦ C. After freezing the solution at −80 ◦ C for about 12 h, the sample was dried under a freeze dryer (EYALA, FDU-506) at a pressure approximately 8 Pa and a condensed temperature of −50 ◦ C. Lyophilized lipase powders were stored in a freezer at −20 ◦ C. 2.3. Preparation of reverse micelles and encapsulation of treated lipase in reverse micelles Reverse micellar solutions were prepared by dissolving AOT in isooctane with a limited amount of water. Freeze dried lipase was encapsulated in reverse micelles by the dissolution method. The treated lipase powder was directly added to the reverse micellar solution and agitated by a magnetic stirrer at 500 rpm in an incubator at 25 ◦ C for 15 min. After centrifuging (HIMAC centrifuge, Hitachi, model CR15) at 1000 rpm for 5 min, the upper phase was separated carefully. The lipase content in the upper phase was determined by UV method. The absorbance was detected at 278 nm by double beam Spectrophotometer and the lipase concentration was calculated according to the standard curve. Final reverse micelles containing lipase (2 mg dm−3 ) were then prepared by adding an appropriate amount of reverse micellar solution in which the freeze dried lipase had been dissolved in advance. Water–AOT molar ratio W0 was adjusted by adding the desired amount of buffer solution. Reverse micelles containing native lipase were prepared with the same procedures as pretreated lipase. The systems containing PEG 400 were prepared by directly mixing reverse micelles solution in a glass bottle in which the appropriate amount of PEG had been added in advance. A magnetic stirrer was used to agitate the mixture until a clear and optically transparent solution was obtained. The micellar solution containing lipase was placed in an incubator at 25 ◦ C to initiate the hydrolysis reaction.
2.1. Materials 2.4. Determination of lipase activity Purified C. viscosum lipase (glycerol-ester hydrolase, EC 3.1.1.3) was provided by Asahi Chemical Industry Co. Ltd. It consists of a mixture of lipase A with a molecular weight of 120 kDa (isoelectric point 3.7) and lipase B with a molecular
In this study, lipase activity was measured as the initial reaction rate, V [mol dm−3 s−1 ], using olive oil as a water insoluble substrate. The reaction was initiated by adding an
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appropriate amount of olive oil into a pre-incubated micellar solution containing lipase. The reaction mixture was agitated by a magnetic stirrer at 500 rpm in an incubator at constant temperature of 25 ◦ C for exactly 20 min as it was found that within this time range, the production of free fatty acid was linearly dependent on time [21]. The resulting fatty acid was analyzed by the Lowry technique [22]. Details of activity determination have been described elsewhere [21]. All data are the average of five replicated experiments and are reproducible within ±5%. In this study, all concentration terms are based on total volume of the reverse micellar systems unless otherwise specified. 3. Results and discussion 3.1. Optimization of pretreatment parameters The effect of various relevant pretreatment parameters such as acetone content in water–acetone solution, pH of water–acetone solution, lipase agitation time in water–acetone solution and freeze-drying time on the catalytic activity of enzymes were optimized and are listed in Table 1. The effect of each operational parameter was investigated by varying only the parameter considered, the others being kept constant. Our findings (data not shown) reveal the surprising dependence of catalytic activity of acetone treated lipase in AOT reverse micelles on the pretreatment conditions. 3.2. Effect of PEG 400 concentration
Fig. 1. Effect of PEG 400 concentration on the activity of acetone treated lipase in AOT reverse micelles. Experimental conditions: Clipase = 2 mg dm−3 , Colive oil = 0.055 mol dm−3 , CAOT = 0.05 mol dm−3 , CNaCl (in buffer) = 0.3 mol dm−3 , buffer pH = 8, W0 = 8 and reaction temperature = 25 ◦ C.
shaped profile observed in simple AOT micellar systems for native lipase. The optimum W0 of acetone treated lipase for simple AOT and mixed AOT/PEG 400 systems are 10 and 8, respectively, similar to those for native lipase in simple AOT and AOT/PEG 400 systems [13]. It has been reported that the optimum value of W0 is commonly related to a situation where the inner diameter of the empty micelle corresponds to
Fig. 1 shows that with the increase in PEG 400 concentrations, the enzyme activity increases to a maximum value at a PEG concentration of 12.5–15 [mmol dm−3 ] and then begins to decrease. The PEG 400 molecule buries the charges on the micellar interface through interaction with AOT head groups, and PEG clouds are formed around the lipase molecules. At lower PEG concentrations, the linkage of PEG to the AOT head groups as well as the enzyme surface may not develop sufficiently. At higher PEG concentrations, the reverse micellar solution becomes turbid, and is in fact, found to be broken. The results in Fig. 1 show this clearly. 3.3. Effect of W0 Fig. 2 indicates that the dependence of acetone treated enzyme activity on W0 is not recognized as a classic bellTable 1 Optimum value of parameters related to the pretreatment of Chromobacterium viscosum lipase with acetone Pretreatment condition
Optimum value
Acetone content in water–acetone solution Water–acetone solution pH Agitation time of lipase in water–acetone solution Freeze drying (lyophilization) time
50% (volume) 7.2 1h 20 h
Fig. 2. Effect of W0 on the activity of treated and native lipases in individual reverse micelles. () Treated lipase in AOT/PEG 400 systems, () treated lipase in AOT systems and () native lipase in AOT systems. Experimental conditions: Clipase = 2 mg dm−3 , Colive oil = 0.055 mol dm−3 , CAOT = 0.05 mol dm−3 , CPEG 400 = 12.5 mmol dm−3 , CNaCl (in buffer) = 0.3 mol dm−3 , pH = 8 and reaction temperature = 25 ◦ C.
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the size of the encapsulated enzymes [23–25]. It seems that the above conclusion holds true in AOT and AOT/PEG 400 reverse micellar systems for treated lipase. It was seen that the lipase activity at higher W0 values was improved by the pretreatment with acetone. At higher W0 values, the size of reverse micelles is higher than the size of lipase, leading the decrease in lipase immobilization inside the water-pool, and it becomes difficult to expose its active site at the micellar interface to combine with substrate molecules [26,27]. Since the surface of treated lipase is more hydrophobic than that of native lipase, it exists relatively near to the micellar interface. As a result, the active site of treated lipase can easily access the micellar interface, and its interaction with substrate becomes less inhibited compared to native lipase. Once again the results support our conjecture that acetone pretreatment may cause a change in lipase conformation, which favors the exposure of the hydrophobic binding site in the open conformation. It should be noted that in the presence of PEG 400, lipase activity is enhanced appreciably at lower and higher W0 values compared to that in simple AOT systems for native and treated lipases. This enhancement is attributed to the decrease in the interaction between treated lipase and surfactant molecules [13,28]. PEG 400 also participates in the redistribution of water and binds to the enzyme surface by noncovalent linkage [29]. Hence, the enzyme can be protected in an unfavorable environment by PEG molecules at its surface [30]. In addition, in the presence of PEG 400, the shifting of optimum W0 to a lower value and the improvement in lipase activity at lower W0 values are due to the increase in micellar size by PEG 400 molecules, which promotes the lipase encapsulation into the micellar inner core [13]. It is worth mentioning that at values of W0 lower than 6, reverse micelles could not solubilize all the PEG 400 molecules at a concentration of 12.5 mmol dm−3 .
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Fig. 3. Dependence of treated and native lipase activity on pH in individual reverse micelles. () Treated lipase in AOT/PEG 400 systems for W0 8, () treated lipase in AOT systems for W0 10 and () native lipase in AOT systems for W0 10. Other experimental conditions are the same as those in Fig. 2 except pH.
Although the optimal pH of native and treated lipases is the same, the activity of acetone treated lipase is higher than that of native lipase. This result suggests that acetone pretreatment would tend to convert the closed form of lipase into the open one. It should be noted that in the presence of PEG 400, the activity of acetone treated lipase at higher pH increases: the activity at pH 10 is similar to that at pH 7. The effective protection of the surrounding PEG 400 molecules may be responsible for the alkali resistance of treated lipase [13]. At lower or higher pH values, lipase suffers from denaturation, and activity decreases. 3.5. Effect of surfactant concentration
3.4. Effect of pH The effect of pH on the activity of treated lipase in AOT and AOT/PEG 400 reverse micellar systems is shown in Fig. 3 and compared with the results for native lipase in AOT reverse micellar systems. It is found that the optimal pH for treated lipase in AOT or AOT/PEG 400 reverse micellar systems is almost the same as that for native lipase in AOT reverse micellar systems. The optimal pH value is found to be 8, which is quite close to the isoelectric point (pI) of C. viscosum lipase (pH 7.3). Since, the net charge on the enzyme surface is zero when the value of pH is equal to pI, the ionic interaction between enzymes and negatively charged head groups of the surfactant (AOT) would be minimized. From Fig. 3, it is evident that the optimum pH is slightly higher than pI. It is worth mentioning that there is some decrease in pH when bulk water of a certain pH is transferred in the reverse micellar phase [31,32]. Therefore, the effective pH inside reverse micelles may be somewhat lower than that of buffer pH that is injected in the reverse micellar phase.
Fig. 4 shows that although the activity of native and treated lipases in simple AOT reverse micelles decreases with the increase in AOT concentration, in the presence of PEG 400 the activity of treated lipase goes through a maximum value with the increase in surfactant concentration and then decreases. However, the tendency of a linear decrease in lipase activity with AOT concentration observed in simple AOT reverse micelles, disappeared with pretreatment. In general, enzyme activity drops sharply with an increase in surfactant concentration [33–36]. In AOT/PEG 400 reverse micelles at constant molar ratios of PEG 400 to AOT, the overall PEG concentration increases with the increase in AOT concentration, leading to the increase in treated lipase activity. Additionally, with the increase in AOT concentration, both micelle number and total amount of substrate adsorbed on the micellar surfactant tails increases, leading to the decrease in free substrate concentration taking part in the hydrolysis reaction. Hence, the optimal value of surfactant concentration is found to be 0.05 mol dm−3 .
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Fig. 4. Effect of AOT concentration on activity of treated and native lipases in individual reverse micelles. () Treated lipase in AOT/PEG 400 systems for W0 8 and CPEG 400 /CAOT = 0.25, () treated lipase in AOT systems for W0 10 and () native lipase in AOT systems for W0 10. Other experimental conditions are the same as those in Fig. 2 except AOT concentration.
Fig. 5. Effect of ionic strength on the activity of treated and native lipases in individual reverse micelles. () Treated lipase in AOT/PEG 400 systems for W0 8, () treated lipase in AOT systems for W0 10 and () native lipase in AOT systems for W0 10. Other experimental conditions are the same as those in Fig. 2 except NaCl concentration.
This study also revealed that the improvement of treated lipase activity in both simple AOT and mixed AOT/PEG 400 systems is more significant at higher AOT concentrations than at lower concentrations. The rate of lipase-catalyzed hydrolysis of olive oil in AOT reverse micelles may decreases due to two main reasons, i.e., the interaction of lipase with AOT molecules and the adsorption of substrates on the micellar surfactant surface. However, the pretreatment of lipase has no effect on the substrate adsorption. Therefore, improvements in lipase activity can be obtained by reducing the interaction between lipase and AOT molecules due to the change in lipase conformation. In addition, PEG 400 forms clouds around the lipase molecule, which also suppresses the interaction of treated lipase with surfactant molecules.
the lipase surface properties. In fact, the surface of treated lipase is enhanced with hydrophobic amino acids, which are unfavorable for electrostatic interaction. Moreover, PEG 400 inhibits the cationic Na+ approaching the micellar interface, and the effect of ionic strength on micellar size becomes less effective [37]. Therefore, activity seems to be unaffected by ionic strength up to CNaCl = 0.6 M. At higher ionic strengths, treated lipase shows deactivation due to the salting out effect.
3.6. Effect of buffer ionic strength (CNaCl in buffer) It is obvious from Fig. 5 that the activity of treated lipases in simple AOT and PEG 400 modified reverse micelles is almost independent of ionic strength up to 0.3 and 0.6 mol dm−3 , respectively. Generally, enzyme activity increases with an increase in ionic strength up to an optimum value as the increase in ionic strength causes a reduction in the electrostatic interaction between lipase and AOT head groups. Further an increase in ionic strength decreases enzyme activity for two reasons. First, high ionic strength decreases the solubility and activity of the enzyme due to the salting out effect. Secondly, the size of the micelle significantly decreases at high ionic strength [37]. This is evident in AOT systems for native lipase. The enhanced treated lipase activity at lower ionic strength may be attributed to the suppression of the electrostatic interaction between lipase and AOT molecules due to changes in
3.7. Effect of reaction temperature Fig. 6 shows that the optimum reaction temperature for all three systems was around 25–30 ◦ C. The ascending portion of the temperature curve (Fig. 6) reflects the general effect of increasing temperature on the rate of chemical reactions. Above 30 ◦ C the enzyme is denatured rapidly: the descending portion of the curve reflects the loss of catalytic activity as the enzyme is denatured at higher temperatures. 3.8. Reaction kinetics A kinetic model based on the adsorption equilibrium of substrate between the organic and micelle phases is used to understand the hydrolysis reaction catalyzed by acetone treated lipase entrapped in AOT and AOT/PEG 400 reverse micellar systems. This model has been reported in the literature [21,33], and is expressed in the following formula: v=
Vmax [ST ] Km (1 + Kad [CS ]) + [ST ]
(1)
Vmax [ST ] Km.a + [ST ]
(2)
or v=
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Fig. 6. Dependence of treated and native lipase activity on reaction temperature in individual reverse micelles. () Treated lipase in AOT/PEG 400 systems for W0 8, () treated lipase in AOT systems for W0 10 and () native lipase in AOT systems for W0 10. Other experimental conditions are the same as those in Fig. 2 except reaction temperature.
where Km.a = Km (1 + Kad [CS ]) and is called the apparent Michaelis constant. Eq. (2) can be rearranged 1 Km.a 1 1 + = v Vmax Vmax [ST ]
(3)
For the treated lipase in simple AOT and AOT/PEG 400 systems, the Lineweaver–Burk and Km.a versus [CS ] plots are linear. From the slope and intercepts of the plot of 1/v versus1/[ST ] (Eq. (3)) for different AOT concentrations, the corresponding values of Km.a could be determined. From the slope and intercept of the plot Km.a versus [CS ], the values of Km and Kad could also be calculated. The values of kinetic parameters of native lipase are taken from literature [33]. The determined kinetic parameters are listed in Table 2. Maximum enzyme activity (Vmax ) is shown to be almost the same for treated and native lipase in AOT and AOT/PEG 400 reverse micellar systems. However, the Michaelis constant (Km ) and the adsorption equilibrium constant (Kad ) decrease significantly for the acetone treated lipase in AOT and AOT/PEG 400 reverse micellar systems compared with those for native lipase in simple AOT reverse micellar systems.
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The low Km value indicates a high affinity for the substrate. Because of the increase in the exposure of hydrophobic amino acids on the treated lipase surface, it can be imagined that the lipase molecule is expanded over the micellar surface with better orientation, leading to higher affinity for hydrophobic substrates as well as more available active sites at the micellar interface. In AOT/PEG 400 systems, the decrease in Km implies that the treated lipase conformation is changed by the direct interaction with PEG molecules, while the combination between the substrate and lipase active site becomes much easier [13]. The Kad value reflects two factors [28]: the adsorption of substrate on the surfactant tails, and the interaction of enzyme with AOT molecules. Since both lipase and PEG 400 molecules exist in the micellar water pool, the substrate adsorption on the micellar surfactant surface is not affected by either the pretreatment of lipase or the addition of PEG 400 to the micellar systems. Therefore, the significant decrease in Kad indicates that the interaction of lipase with AOT molecules is reduced by acetone pretreatment as well as the addition of PEG 400 to the AOT reverse micellar system. Fig. 7 shows that the activity of acetone treated lipase in simple AOT and AOT/PEG 400 systems, calculated using the model equation (1), agrees well with the experimental results. The results show that the kinetic model for lipase-catalyzed hydrolysis of olive oil in AOT reverse micellar systems [33] can also be applied in the enzymatic reaction catalyzed by the acetone treated lipase in AOT and AOT/PEG 400 reverse micellar systems. 3.9. Stability of acetone treated lipase The stability of acetone treated lipase in AOT and AOT/PEG 400 reverse micellar systems was also investigated, as this is a crucial factor in industrial applications. Stability was examined by assaying residual activity after incubation without substrate at 25 ◦ C for a required period. Residual activity was calculated as a percentage of original activity (considered 100%), obtained at t = 0 min incubation. Lipase half-life (t1/2 ) was calculated directly from the residual activity profiles. Fig. 8 shows that acetone treated lipase is very stable when entrapped in simple AOT and mixed AOT/PEG 400 reverse micellar systems, retaining approximately 92.2 and 94% respectively of initial activity after 15 days of incubation. In contrast, native lipase retains only 75% initial activity in AOT systems over the same duration.
Table 2 Comparison of kinetics parameters in individual reverse micellar systems for treated and native lipases Kinetic parameters
Treated lipase in AOT/PEG 400 systems
Treated lipase in AOT systems
Native lipase in AOT systems, Ref. [33]
Vmax [ mol dm−3 s−1 ] Km [mol dm−3 ] Kad [mol−1 dm3 ] Vmax /Km [s−1 ]
29.5 0.0333 5.018 8.85 × 10−4
26.8 0.0365 7.98 7.34 × 10−4
27 0.083 16.2 3.25 × 10−4
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enzyme against denaturation in AOT reverse micelles (a similar phenomenon has been previously reported in the literature [39]). Moreover, the observed improvement in the lipase activity in AOT/PEG 400 reverse micellar systems is due to the suppression of the interaction between lipase and AOT molecules, since charges on the micellar interface and enzyme surface are buried by neutral PEG molecules [30]. To understand the change in lipase conformation by pretreatment, fluorescence properties of native and treated lipase was investigated (details have been described in the next section). The variation of maximum fluorescence intensity versus incubation time at 25 ◦ C indicates that treated lipase retains 95% of its initial maximum fluorescence intensity after 10 days (data not shown). Therefore, it can be concluded that the change in lipase conformation by acetone pretreatment is stable. 3.10. Fluorescence spectroscopy of lipase Fig. 7. Comparison of activity values obtained experimentally with those predicted by the model Eq. (1). () Treated lipase in AOT/PEG 400 systems for W0 = 8 and CPEG 400 = 12.5 mmol dm−3 and () treated lipase in AOT systems for W0 = 10. Curved lines are predicted by model equation. Other experimental conditions: Clipase = 2 mg dm−3 , CNaCl (in buffer) = 0.3 mol dm−3 , pH = 8, reaction temperature = 25 ◦ C.
The half-life of treated lipase in AOT and AOT/PEG 400 systems is 120 and 160 days respectively, whereas half-life of native lipase in AOT systems is only 38 days, comparable with results reported elsewhere [8,38]. Increased stability from pretreatment with acetone may be attributed to enhanced hydrophobic residues on the lipase surface that stabilize the
Fig. 8. Stability of lipase in individual reverse micelles at 255 ◦ C. () Treated lipase in AOT/PEG 400 systems for W0 = 8 and CPEG 400 = 12.5 mmol dm−3 , () treated lipase in AOT systems for W0 = 10 and () native lipase in AOT systems for W0 10. Storage conditions: Clipase = 2 mg dm−3 , CAOT = 0.05 mol dm−3 , CNaCl =0.03 mol dm−3 , pH = 8. Activity assay conditions: Colive oil = 0.055 mol dm−3 , reaction time = 20 min, reaction temperature = 25 ◦ C.
Proteins contain several chromophores that absorb light in the ultra-violet and infrared regions. Many chromophores called fluorophores can also display fluorescence. Fluorescence is a useful probe to know the structure and structural changes in enzymes [40,41]. The fluorescence emission spectra for native and treated lipases in AOT and AOT/PEG 400 reverse micellar systems were recorded at 25 ◦ C (Fig. 9). The emission spectra were recorded from 300 to 400 nm with a Hitachi F-3010 fluorescence spectrophotometer at an excitation wavelength of 280 nm, the selective excitation wavelength for tryptophan residues [42,43]. Emission and excitation slit widths were 5 nm. Spectra were uncorrected for instrument sensitivity, but the emission of blank reverse
Fig. 9. Fluorescence spectra of lipase in individual reverse micelles. () Treated lipase in AOT/PEG 400 systems for W0 = 8 and CPEG −3 400 = 12.5 mmol dm , () treated lipase in AOT systems for W0 = 10 and () native lipase in AOT systems for W0 10. Experimental conditions: Clipase = 10 mg dm−3 , CAOT = 0.05 mol dm−3 , CNaCl = 0.03 mol dm−3 , pH = 8.
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micelles (without lipase) was subtracted. Three dimensional crystal structure [44] and amino acid sequence indicate that C. viscosum lipase contains 3 tryptophan, 10 tyrosine and 8 phenylalanine residues as fluorophores whose excitation wavelength are 280, 257 and 270 nm, respectively. However, only tryptophan residues are sensitive in water or AOT/water/isooctane systems. From Fig. 9, it is evident that the fluorescence intensity of treated lipase both in simple AOT and AOT/PEG 400 systems is higher than that of the native lipase in AOT systems. The increase in fluorescence intensity indicates that treated lipase may have a better orientation, which led to more tryptophan (hydrophobic) residues on the lipase surface. Grauper et al. [45] have reported that tryptophan residues as well as the active site of C. viscosum lipase in native form are located in the hydrophobic region, and their approach to the lipase surface is very difficult. Therefore, only a conformational change would result in the enhancement of fluorescence intensity. The addition of PEG 400 further enhances this intensity, and this is attributed to the change in lipase conformation due to the interaction with the PEG molecules.
4. Conclusions The activity of acetone-treated C. viscosum lipasecatalyzed hydrolysis of olive oil was carried out in AOT/isooctane and AOT/PEG 400/isooctane reverse micellar systems. The favorable effects of both pretreatment with acetone and the addition of PEG 400 molecules enhanced the activity of lipase significantly in AOT systems. The improvement in lipase activity at higher W0 as well as at higher AOT concentrations indicated that the acetone treated lipase might possess a better orientation for enzymatic reactions than that of native lipase. When PEG 400 was added to the AOT reverse micellar systems, the interaction between the anionic AOT head groups and the treated lipase was markedly suppressed, and the active site of treated lipase was also protected from unfavorable action of AOT molecules. The lower value of the Michaelis constant suggested that the affinity between lipase and substrate increased by pretreatment with acetone as well as the addition of PEG 400. The stability study revealed that the treated lipase entrapped in AOT and AOT/PEG 400 reverse micellar systems was very stable. Finally, fluorescence studies supported the hypothesis of a change in lipase conformation.
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