3082
Langmuir 2000, 16, 3082-3092
An Integrated Model for Enzymatic Reactions in Reverse Micellar Systems: Nominal and Effective Substrate Concentrations C. M. L. Carvalho, M. R. Aires-Barros, and J. M. S. Cabral* Centro de Engenharia Biolo´ gica e Quı´mica, Instituto Superior Te´ cnico, 1049-001 Lisboa, Portugal Received July 27, 1999. In Final Form: December 27, 1999 The distribution of hexanol in a reversed micellar system of AOT/isooctane/buffer was quantitatively described by a model based on the volume fractions of micellar components and partition coefficients of the alcohol. A recombinant cutinase was used as a model biocatalyst since its activity is strongly dictated by the presence of short to medium alcohol chain lengths. It was assumed that hexanol would be accessed by cutinase if it is either in the water pool or in the surfactant layer (micellar) since the micelles are very small and resized upon protein microencapsulation. The integrated model takes into account the partitioning, the diffusion, and the adsorption of hexanol to AOT hydrocarbon tails and the effectiveness factors calculated to model the kinetic experiments. Thermostability data also support the model generated. Therefore, the model defines the reversed micelles composition in terms of hexanol concentration integrating the process globally.
Introduction Reverse micelles present advantages over other organic systems, especially due to their capacity to incorporate enzymes in the aqueous core and to the ability to dissolve hydrophobic and hydrophilic compounds. They offer the additional advantage of allowing a judicious control of the water content, which is beneficial for synthetic purposes. Commonly, reversed micelles are referred as a pseudobiphasic system forming an isotropic phase. However, this is an oversimplification as the oil-in-water (o/w) or waterin-oil (w/o) microemulsions are not entirely homogeneous. Several authors attempted to explain the role of alcohols as cosurfactants. On the basis of kinetic data, Hayes and Gulari1 classified alcohols into three groups according to their degree of polarity. The more polar belong to group 1 (C1-C3), the amphiphilic ones constitute group 2 (C4C6), and the more hydrophobic ones constitute group 3 (C7-C10). Group 1 remains mainly in the micelles aqueous phase causing significant changes on reverse micellar size. On the contrary, groups 2 and 3 may partition between organic and aqueous phase. The classification is also dependent on the effect of the alcohols in the micellar interface. The former group promotes more fluid interfaces while the later makes them more rigid.1 Hou et al.2 used quasi-elastic light scattering (QELS) and static light scattering (SLS) to study the interdroplet interactions and verified the increase in attractive interaction strength with propanol and the opposite effect with heptanol. It was also verified that the addition of alcohol affects the elasticity and the curvature of the interface, the effects being dependent on the alcohol chain length.3 Following the same trend Førland et al.4 studied the influence of 1-propanol, 1-butanol, and 1-pentanol on the * Corresponding author. Phone: 351-21-8419065. Fax: 351-218419062. E-mail:
[email protected]. (1) Hayes D. G.; Gulari, E. Biotechnol. Bioeng. 1990, 35, 793. (2) Hou, M. J.; Kim, M.; Shah, D. O. J. Colloid Interface Sci. 1988, 123, 398. (3) Leung, R.; Shah, D. O. J. Colloid Interface Sci. 1987, 120, 330.
behavior of sodium dodecyl sulfate (SDS) (anionic surfactant) micelles by means of small angle neutron scattering (SANS) and Fourier transform pulsed field gradient spin-echo (FT-PGSE). Being a polar alcohol, propanol is mainly dissolved in the aqueous core phase and increases the self-diffusion coefficient of SDS indicating a progressive breakdown of the micelles. Pentanol, as a medium chain alcohol, is preferentially solubilized in the micellar layer between the surfactant chains and tends to build large wormlike micelles. The effect of pentanol was gradual in the range studied (3.5-112 mM).4 The main characteristic of the sodium bis(2-ethylhexyl) sulfosuccinate (AOT) phase diagram considering the ternary system water-oil-AOT system is the existence of a w/o microemulsion (L2 phase) over a wide range of concentrations and temperatures. Sager5 mimicked the effect of AOT manufacturing impurities adding 1-octanol to the salt-free system water-AOT-hexane. The addition of octanol led to drastic shifts on the phase boundaries especially in the phases L2 and L2 + W and L2 and L2 + LR. The transition to L2 was conditioned by the addition of a smaller quantity of water as there was a boundary shift for lower temperatures.5 The separation of a homogeneous L2 phase into two phases L2 + W was accomplished by octanol partitioning between the interface and the bulk-oil phase that influenced the properties of the system components. Henceforth, the surfactant mixture in the interfacial film becomes more hydrophobic upon alcohol addition and the bulk-oil phase becomes less hydrophobic.5 Similarly, it may be extrapolated that a similar phenomenon occurs upon alcohol addition to the reversed micelles even considering there is no phase change. As a consequence, the addition of hexanol would favor its own dissolution in the micellar and in the aqueous phases. This synergy may account for the stabilization process exhibited by cutinase.6-8 (4) Førland, G. M.; Samseth, J.; Gjerde, M. I.; Høiland, H.; Jensen, A. ø.; Mortensen, K. J. Colloid Interface Sci. 1998, 203, 328. (5) Sager, W. F. C. Langmuir 1998, 14, 6385. (6) Carvalho, C. M. L.; Serralheiro, M. L. M.; Cabral, J. M. S.; AiresBarros, M. R. Enzyme Microb. Technol. 1997, 21, 117.
10.1021/la991008t CCC: $19.00 © 2000 American Chemical Society Published on Web 02/19/2000
Modeling Substrate Distribution in Reverse Micelles
Hayes and Marchio9 found that protein exclusion increased with cosurfactant chain length for the homologous series of 1-alkanols starting at 1-butanol. The addition of a long-chain alcohol (1-hexanol or larger) appears to expel protein, water, and other kinds of solubilizates. These alcohols increase the interfacial curvature and might also change the position of L2 phase boundary. The consequences would be the rigidification of microemulsion interface and a reduction on percolation, attractive interactions, and exchange rates. These authors verified that protein exclusion is improved by increasing the amount of alcohol, by reducing the W0 value below 10, or by increasing the fractional occupancy. Electron paramagnetic resonance (EPR) studies performed with lecithin microemulsions, in the presence of a spin probe (5-DSA), allowed verification that the amount of water and alcohol and also the type of alcohol greatly influenced the mobility of the probe.10 The increase in the alcohol chain length increases the rigidity of the micellar layer from ethanol to hexanol. Short-chain alcohols as ethanol and propanol have more affinity to the aqueous dispersed phase, while longer alcohols such as 1-pentanol and 1-hexanol prefer the organic continuous phase and the interface, where they act as cosurfactants. This was supported by following the esterification of the probe with different alcohols, the rate with 1-butanol being 17-fold higher than that with 1-hexanol.10 All these works focused on the effects of alcohols in the micelle structure, but they do not explain either its distribution in micellar systems or the biocatalyst behavior in such systems. Although, they gave important contributions for the micellar research field, they do not interpret the experimental data assembled with microencapsulated enzymes. The absence of specific and readily feasible techniques to determine directly the chemical species in contact with the enzyme imply different kinetic treatments for enzymes in different microenvironments. The very few works developed up to now fail in their lack of embrace. For instance, Maestro and Walde11 developed a simple diffusion model to describe the R-chymotrypsin activity in reversed micelles. The authors considered two consecutive diffusion steps: the intermicellar diffusion (between micelles containing enzyme and micelles with substrate) and the intramicellar diffusion. This theoretical approach presented very low correlations with experimental data and did not consider the substrate partitioning. Verhaert et al.12 also attempted to explain enzyme kinetics based on the pseudomicellar phase composition. The authors raised the problem of “real” substrate concentrations for microencapsulated enzymes and distinguished between overall and aqueous substrate concentrations combining an exchange process between filled and nonfilled micelles. Their approach focused on the exchange rates due to the use of hydrophilic substrates that preferentially locate in the aqueous phase of the reverse micelles, although in most cases these rates are much faster than the turnover rate of enzymes. The distribution concentration of alcohols is of great interest in the micellar systems due to their properties as (7) Carvalho, C. M. L.; Cabral, J. M. S.; Aires-Barros M. R. Enzyme Microb. Technol. 1999, 24, 569. (8) Sebastia˜o, M. J.; Cabral, J. M. S.; Aires-Barros, M. R. Biotechnol. Bioeng. 1993, 42, 326. (9) Hayes, D. G.; Marchio, C. Biotechnol. Bioeng. 1998, 59, 557. (10) Avramiotis, S.; Cazianis, C. T.; Xenakis, A. Langmuir 1999, 15, 2375. (11) Maestro, M.; Walde, P. J. Colloid Interface Sci. 1992, 154, 298. (12) Verhaert, R. M. D.; Hilhorst, R.; Vermue¨, M.; Schaafsma, T.; Veeger, C. Eur. J. Biochem. 1990, 187, 59.
Langmuir, Vol. 16, No. 7, 2000 3083
cosurfactants. Besides these properties, alcohols also act as substrates and stabilizing agents, which give them further interest to model. To examine this issue, a recombinant cutinase from Fusarium solani pisi was microencapsulated in AOT/ isooctane reversed micelles to catalyze the transesterification of butyl acetate with hexanol. Cutinase was a suitable enzyme since its activity and stability severely depend on the presence of hexanol, which plays a multiple role as substrate and as a cosurfactant and strongly improves cutinase stability.6-8 Our aim was to integrate different experimental data sources to achieve a model to describe the hexanol distribution and consequently the kinetic behavior of microencapsulated cutinase and the stabilization attained with different hexanol concentrations. Materials and Methods Enzyme and Chemicals. Fusarium solani pisi cutinase was cloned and expressed in Escherichia coli WK-6 recombinant strain, a kind gift of Corvas International (Ghent, Belgium). Cutinase was produced and purified in our laboratory, with a purity over 99%, following the procedure of Lauwereys et al.13 Surfactant bis(2-ethylhexyl) sodium sulfosuccinate, AOT (99%) and butyl acetate (99.7%) were purchased from Sigma. The AOT was used without further purification. 1-Hexanol (98%) and isooctane (99.8%) were obtained from Merck. All other chemicals including eluents and salts were of analytical reagent grade. Preparation of Reversed Micelles. Reversed micelles containing the enzyme were prepared by the injection method adding appropriate amounts of buffer solution with the enzyme into a solution of AOT in isooctane with mixing. The total reaction volume was 5 mL and the concentrations mentioned are all referred to that volume, except the buffer molarity, which is only considered in the aqueous phase. The pH values mentioned are those of the buffer in which cutinase is dissolved to prepare the aqueous phase, which is further used to form the reversed micelles. Thermostability Assays. Cutinase microencapsulation in AOT/isooctane was followed by the addition of hexanol (unless otherwise stated) and maintained in capped flasks in a thermostated water bath at 30 °C. A total volume of 5 mL was prepared for incubation. Cutinase was microencapsulated (0.3 mg/mL) in reversed micelles of AOT/isooctane using the waterto-surfactant ratio (W0) of 2.7 (0.72% v/vtotal of buffer and 150 mM AOT). The aqueous phase was 200 mM phosphate buffer pH 8. The temperature was kept at 30 °C. At defined intervals of time the activity retention was analyzed, adding 1 M of the second substrate (butyl acetate) to 4.54 mL of the incubated solution and following the reaction for 20 min. The activity assays were performed in a stirred batch reactor with a water jacket connected to a thermostated bath (T ) 30 °C). All experiments were done using magnetic stirring at 600 rpm. The determination of hexyl acetate concentration was carried out using HPLC analysis (see Analytical methods). The enzyme deactivation was described considering a firstorder decay. The deactivation constant obtained was used to obtain the half-life through the relation t1/2 ) (ln 2)/kd. Kinetic Experiments. The experimental conditions (W0, pH, buffer molarity, temperature, agitation, and enzyme concentration) were similar to the ones used in thermostability assays. The reactions were performed in a batch stirred tank reactor (BSTR) and followed for 24 h. The kinetic constants were attained by the Hanes method. Initial velocity was defined as the amount of product, hexyl acetate, in millimoles per liter formed per minute. The initial velocity measurements were made in the absence of products and the concentration of hexanol was varied from 100 up to 1200 mM, whereas the butyl acetate concentration ranged from 100 to 3000 mM. (13) Lauwereys, M.; De Geus, P.; De Meutter, J.; Stanssens, P.; Mathyssens, G. In Lipases-Structure, function and genetic engineering; VCH: Weinheim, 1991; pp 243-251.
3084
Langmuir, Vol. 16, No. 7, 2000
Carvalho et al.
Figure 1. Dependence of the effectiveness factor on hexanol concentration. The values are based on the comparison of initial velocities experimentally measured and those expected by Michaelis-Menten reversible kinetics. Experimental conditions: [cutinase] ) 0.3 mg/mL; [AOT] ) 150 mM; W0 ) 2.7; T ) 30 °C; 200 mM phosphate buffer pH 8.0. The concentration of butyl acetate was 1 M. Table 1. Kinetic Parameters Determined for a Reversible Two-Substrate Reaction Vmax KM
hexanol butanol butyl acetate hexyl acetate
70 mm/min 254 mM 119 mM 3110 mM 4148 mM
The Hanes graphical representation was chosen plotting s/v against s.14 For the substituted-enzyme mechanism, the initial rate, v, in the absence of products is
v)
V[H][BAc] KBAc[H] + KH[BAc] + [H][BAc]
where V is the limiting velocity and KBAc and KH are the Michaelis constants for butyl acetate (BAc) and hexanol (H), respectively. Analytical Methods. The time course reaction was followed through the analysis of hexyl acetate in samples. The HPLC analysis was carried out in Waters equipment associated to a 3390A integrator from Hewlett-Packard. A RP column Lichrocart 250-4 (RP-select B, 5 µm, Lichrospher 60) was used in isocratic mode with an eluent composed by 40% water and 60% acetonitrile (flow rate of 1 mL/min). The hexyl acetate formation was followed by UV at 220 nm, using a Waters model 481 LC spectrophotometer.
Results and Discussion Kinetic and Stability Results/Evaluation. The nonhomogeneity of microemulsions reflects on the analysis of kinetics performed, since the quantities of substrate accessed by the biocatalyst were not straightforward related with the substrate amount added to the reactional media. The addition of hexanol up to 300 mM led to reaction velocities that do not follow the Michaelis-Menten kinetics. Nevertheless, the deviation was overcome by calculating an effectiveness factor, which was related to overall substrate concentration (Figure 1). The effectiveness factors (η) were calculated using the kinetic parameters determined for a reversible twosubstrate reaction14,15 (see Table 1) and are defined as
η ) Vef/Vkin
(1)
(14) Cornish-Bowden, A. Fundamentals of Enzyme Kinetics; Butterworths: London, 1981. (15) Carvalho, C. M. L. Ph.D. Thesis, Instituto Superior Te´cnico, Universidade Te´cnica de Lisboa, 1999.
Figure 2. Effect of hexanol concentration on cutinase thermostability. Experimental conditions: [cutinase] ) 0.3 mg/mL; [AOT] ) 150 mM; W0 ) 2.7; T ) 30 °C; 200 mM phosphate buffer pH 8.0. 1 M butyl acetate was added to start the activity evaluation.
considering Vef to be the observed velocity of reaction and Vkin the kinetic velocity expected for a Michaelis-Menten kinetics. At first sight this could be a consequence of diffusional effects before equilibrium was reached, since the low substrate concentrations tend to yield diffusion limitations. The short time (40-45 s) that mediated the addition of hexanol and the starting of reaction could explain the noncompleteness of the process. With low hexanol concentrations (
