Physicochemical Aspects of Lipase B from Candida antarctica in

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Physicochemical Aspects of Lipase B from Candida antarctica in Bicontinuous Microemulsions Mireia Subinya,† Anne K. Steudle,§ Bettina Nestl,‡ Bernd Nebel,‡ Bernhard Hauer,‡ Cosima Stubenrauch,† and Sandra Engelskirchen*,§ †

Institut für Physikalische Chemie, Universität Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart Institut für Technische Biochemie, Universität Stuttgart, Allmandring 31, 70569 Stuttgart § Department of Chemistry, Durham University, South Road DH1 3LE, United Kingdom ‡

ABSTRACT: Biotechnology involves applying enzymes in organic synthesis to convert non-natural substrates into enantiomerically pure products under mild reaction conditions. Non-natural substrates are often lipophilic molecules that can hardly be accessed and converted by enzymes in their natural aqueous environment. Bicontinuous microemulsions provide a spongelike nanostructure with a large interfacial area between aqueous and oil domains, which makes them valuable alternative reaction media. In the present study, we introduced lipase B from Candida antarctica into a bicontinuous microemulsion of composition H2O/NaCl− n-octane−pentaethylene glycol monodecylether (C10E5). Phase behavior, partitioning studies, and pulsed-field-gradient NMR measurements revealed that the lipase is mostly adsorbed at the microemulsions interface. Phase diagrams showed a maximum in efficiency with increasing amount of lipase added to the water phase of the microemulsion. It was observed that the ratio between the mass of lipase that is introduced into the system and the mass of lipase that is located at the interface stays constant. Self-diffusion coefficients of all components showed that the presence of the lipase is not influencing the bicontinuity of the microemulsion.

1. INTRODUCTION Nature’s catalysts have excellent selectivity toward substrates under rather mild reaction conditions. Industry therefore strives to replace classical chemical production routes by ecologically responsible biotechnological alternatives.1−3 Especially the fine chemicals and pharmaceutical industry benefit from a high grade of enantiomeric purity achieved when enzymes are applied as catalysts in organic synthesis.1,4,5 The use of enzymes in industrial biotransformations still faces challenges that hamper their widespread applicability. One of these challenges arises from the chemical nature of synthetically interesting nonnatural substrates such as acrylates that are used in the preparation of acrylic esters.6 These non-natural substrates are often not soluble in water, and their molecular structure has a high degree of chirality. Enzymes, however, are very sensitive to the reaction conditions applied; they prefer buffered aqueous environments and often require an interface to become active. Different reaction media have been proposed to meet these challenges5 such as aqueous−organic solvent mixtures,7 pure organic solvents,8 ionic liquids,9 supercritical fluids,10 porous materials as carriers for immobilized enzymes,11 and water-inoil microemulsions.12,13 In the latter case, the enzyme is confined to nanometer-sized water droplets dispersed in a continuous oil phase that is stabilized by the addition of surfactants. Studies including lipases, esterases, and dehydrogenases showed that the catalytic activity of these enzymes in water-in-oil microemulsions strongly depends on the nature of the components, the overall amount of water, and the properties of the interfacial surfactant monolayer. In some © 2014 American Chemical Society

cases, one obtains an activity much higher than what can be observed in aqueous buffer solution, the so-called superactivity.13,14 Recently, bicontinuous microemulsions have also been identified as reaction medium for enzymatic catalysis.15−17 Bicontinuous microemulsions consist of interpenetrating nanometer-sized domains of water and oil that are separated by a spongelike surfactant monolayer.18−20 Compared to discrete droplets present in a water-in-oil microemulsion where the substrate diffuses freely in the continuous oil phase and the enzyme is trapped within the droplet, the spongelike structure of bicontinuous microemulsions allows us to confine the enzyme and substrate to the same structural motif. Similar diffusion rates in conjunction with the larger and, more importantly, connected interfacial monolayer might improve the enzyme−non-natural substrate contact and enhance the reaction kinetics and might even open up new reaction pathways. Microemulsions are complex multicomponent systems in which intermolecular interactions play a crucial role in the activity and stability of confined enzymes. For successful applications of microemulsions as reaction media, it is indispensable to understand the fundamental interactions between the enzyme and the bicontinuous microemulsion. To reach this goal, we follow a bottom-up approach. First, we study the changes in a well-defined simple microemulsion Received: October 31, 2013 Revised: February 24, 2014 Published: February 24, 2014 2993

dx.doi.org/10.1021/la4042088 | Langmuir 2014, 30, 2993−3000

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Surfactant mass fraction: msurfactant γA = msurfactant + m water

system that are induced by the introduction of lipase B from Candida antarctica (Cal B). As a lipase, Cal B has the ability to convert substrates at the oil−water interface. Cal B is a robust, well-investigated enzyme providing a broad substrate range, a high degree of enantioselectivity, and good stability against temperature changes and toward organic solvents.21 A small αhelix domain located close to the active center (which is, in contrast to other lipases, not covered by a lid domain) has been identified as a candidate for anchoring the lipase at the oil− water interface.22,23 In the following section, we focus on a bicontinuous microemulsion of composition H2O/NaCl−n-octane−pentaethylene glycol monodecylether (C10E5). To identify the location of the bicontinuous structure as a function of temperature and composition, the determination of respective phase diagrams is essential. Therefore, we systematically increased the concentration of Cal B in the microemulsion. The determination of the respective phase behavior, studies on the partitioning of Cal B between the interface and the water domains, and the self-diffusion of the microemulsion’s components provide unprecedented insight into the physicochemical properties of an enzyme in a bicontinuous microemulsion.

Concentration of lipase in the aqueous phase that is similar for all measurements: m CCal B = Cal B Vwater (5) Mass fraction of lipase in the surfactant mixture: mCal B δ= msurfactant + mCal B

2.A. Materials. Pentaethylene glycol monodecyl ether (denoted as C10E5, purity ≥97.0% GC) and n-octane (purity ≥99.5% GC) were purchased from Sigma, and wholly π-conjugated low-molecular-weight organogelator that displays triple-channel responses to fluoride ions was purchased from Aldrich (Germany). The aqueous solutions were prepared with doubly distilled water and sodium chloride (NaCl, purity 99.5%), which was purchased from Merck. Lipase B from Candida antartica (Cal B, lyophilized, recombinant, specific activity 5000 U g−1) was purchased from C-LEcta and purified according to the method in ref 24. 2.B. Phase Diagrams. The aqueous phase of the microemulsions was prepared from a stock solution consisting of water, 4 wt % NaCl, and 100 mg mL−1 Cal B. Dilutions were made using a solution of water and 4 wt % NaCl and were freshly prepared for each experiment. To prepare the microemulsion samples, all components were weighed into glass vials in the following order: surfactant, oil, and aqueous solution. The glass vials were sealed with glass stoppers and placed in water basins equipped with a thermostat (Thermo Scientific D30). A digital thermometer (Greisinger GMH 375) was placed close to the vial. Phase boundaries were measured by visual inspection with an accuracy of ±0.05 °C. To define the composition of the prepared microemulsions, we used the following parameters.18: Volume fraction of oil in the mixture of water and oil, which was kept constant at ϕ = 0.5 if not stated otherwise: Voil Voil + Vwater

Surfactant mass fraction γ in the total mixture: msurfactant γ= msurfactant + moil + m water

(6)

2.C. Partitioning of Cal B between the Microemulsion’s Interface and the Microemulsion’s Water Domain. Microemulsions were prepared in the three-phase region at γ = 0.05. Samples were placed in the water basin at their respective phaseinversion temperature. Once the samples reached their equilibrium state of being fully phase separated, the excess phase was carefully extracted using a syringe. The tip of the needle was cut flat to prevent contamination with the adjacent phase by disturbing the interface during the extraction procedure. The concentrations of Cal B in the excess water phase were measured by UV/vis spectroscopy. The measurements were performed on a Nanodrop 1300 microvolume spectrophotometer. Spectra were measured between 220 and 700 nm. The resulting absorptions at 280 nm correspond to the aromatic amino acids, mainly tryptophan. A calibration curve of increasing Cal B concentration in aqueous solution was used to identify the respective concentrations. 2.D. Pulsed Field Gradient NMR. Self-diffusion coefficients were measured by pulsed field gradient nuclear magnetic resonance (PFGNMR) using the stimulated spin echo sequence.25,26 Measurements were performed on a Bruker Avance III 400WB spectrometer operating at a 400.1 MHz 1H resonance. The magnet was equipped with a Bruker DIFF-30 gradient probe that delivers gradients of up to 11 T m−1 in the z direction. The measured echo was Fourier transformed, and the decay of selected peak intensities was fitted using the Stejskal−Tanner equation27

2. EXPERIMENTAL SECTION

ϕ=

(4)

⎛ ⎛ δ ⎞⎞ I = I0 exp⎜− γ 2δ 2g 2D⎜Δ − ⎟⎟ ⎝ ⎝ 3 ⎠⎠

(7)

where I0 is the intensity of a given peak without applied gradients, I is the intensity of a given peak with applied gradients, γ is the gyromagnetic ratio, Δ is the time interval between gradient pulses and thus the observation time, and δ and g are the duration and amplitude of the pulsed field gradients, respectively. The experiments were performed by recording NMR spectra as a function of g while δ and Δ were kept constant. The real temperature of the sample was determined via a calibration curve (i.e., via the known temperaturedependent peak shifts of pure ethylene glycol28,29). The temperature precision was around ±0.1 °C. Small volumes of the samples were placed in 4 mm NMR glass tubes to reduce convection that is caused by the unavoidable temperature gradient in the magnet. To prove that convection effects were not influencing the measurements, each measurement was repeated using different observation times, namely, Δ = 25 and 180 ms.30 We excluded the points that were measured at high temperatures because the difference between the self-diffusion coefficients, which were measured with different observation times, was greater than ±10% (which is the typical error for this experiment). The peaks in the 1H NMR spectra of the microemulsions are assigned as follows: the peak at 4.8 ppm originates from the O−H resonance of water, the peak at 3.7 ppm corresponds to the hydrogen atom of the oxyethylene headgroup of the surfactant, the intense peak at 1.3 ppm can be assigned to −CH2− groups of oil and surfactant, and the peak at 1.0 ppm results from the −CH3 groups of oil and surfactant. Unfortunately, it is not possible to measure the selfdiffusion coefficients of the enzyme because of its high molecular weight (33 273 Da22), which leads to slow tumbling and therefore a

(1)

(2)

Concentration of NaCl in the microemulsions, with respect to the composition of the aqueous phase: mNaCl ε= mNaCl + m water (3) A similar experimental procedure was followed to study the influence of Cal B on the miscibility gap H2O−C10E5 except that only surfactant and aqueous solution were weighed into the glass vials, respectively. We used the following definitions to describe the composition of the samples used to determine the miscibility gap H2O−C10E5: 2994


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short spin−spin relaxation time (T2). Short T2 values severely limit the power and flexibility of multipulse NMR experiments (i.e., the signalto-noise ratio decreases with decreasing T2).31 The measured self-diffusion of water in a microemulsion requires special consideration. The measured self-diffusion coefficient of water is a combination of the water molecules that diffuse freely and the ones that diffuse with the surfactant headgroup as a hydration shell.32 In the case of nonionic surfactant C10E5, 5 water molecules diffuse with each ethylene oxide group, hence 25 water molecules for each surfactant molecule.33 The fraction of water molecules diffusing with the surfactant headgroup is calculated according to the relationship p=

25nsurfactant n water

mass fractions, the bicontinuous microemulsion coexists with excess phases of oil and waterthe three-phase state (3) because the surfactant mass fraction is too low to solubilize the total volumes of water and oil. At higher surfactant mass fractions, a single-phase microemulsion region (1) is observed. The most important parameter needed to characterize a microemulsion is the X point, where the three-phase and the one-phase region meet. This point is characterized by the phase-inversion temperature denoted by T̃ , where the structure of the microemulsion is bicontinuous, and the least surfactant mass fraction needed to completely solubilize water and oil is denoted by γ̃. With the aim of performing enzymatic reactions under mild conditions, the X point was adjusted to 38 °C by adding 4 wt % NaCl to the water phase. NaCl is a lyotropic salt and a common parameter to tune the temperature dependence of the phase behavior in microemulsions. Cal B is water-soluble, which is why we can use aqueous solutions of Cal B as the water phase in microemulsions. The phase behavior of the microemulsion was characterized as a function of temperature and surfactant mass fraction using equal volumes of water and oil phases with increasing concentrations of Cal B in the water phase, namely, 1, 10, 50, and 100 mg mL−1. The resulting phase diagrams are presented in Figure 1. With increasing Cal B concentrations, we

(8)

where nsurfactant represents the moles of nonionic surfactant C10E5 and nwater represents the moles of water. Water self-diffusion coefficients were corrected according to Dm = (1 − p)Df + pD b

(9)

where Dm is the measured self-diffusion coefficient of water, Df is the self-diffusion coefficient of free water in the microemulsion, and Db refers to the self-diffusion coefficient of water that diffuses with the surfactant. Note that Db was assumed to be the self-diffusion coefficient of the nonionic surfactant according to the considerations above. The relative self-diffusion coefficients of water were calculated by dividing the self-diffusion coefficient of free water in the microemulsion by the self-diffusion coefficient of pure water. The relative self-diffusion coefficients of n-octane did not require any corrections, thus the relative self-diffusion coefficients were calculated by dividing the measured self-diffusion coefficient of n-octane by the self-diffusion coefficient of pure n-octane. The measured values of pure water and pure n-octane were in agreement with values that have already been published in the literature.34,35

3. RESULTS The first aim of the present study is to investigate the solubilization of an enzyme in a bicontinuous microemulsion that provides a large connected interface between interpenetrating aqueous and oil domains. Second, we were interested in the question of how the partitioning of the lipase between the interfacial layer and the water domain changes the properties of the respective microemulsion. Ultimately, we aim both to understand how enzymes catalyze the conversions of non-natural substrates in these artificial environments and to make use of this knowledge for applications in white biotechnology. For this puropose, we introduced lipase B from Candida antarctica into a bicontinuous microemulsion composed of H2O/NaCl−n-octane−pentaethylene glycol monodecylether (C10E5). Enzymatic catalysis in microemulsions involves the interplay of multiple parameters, biochemical as well as physicochemical. In this article, we restrict ourselves to the investigation of physicochemical parameters. 3.A. Phase Behavior of Cal B-Containing Microemulsions. The interaction of the three binary miscibility gaps (water−oil, water−nonionic surfactant, and oil−nonionic surfactant) leads to the appearance of a fish-shaped phase diagram for ternary nonionic systems.18 A ternary nonionic system runs through three different phase states as a function of temperature when the surfactant mass fraction γ is kept constant. At low temperatures, an oil-in-water microemulsion coexists with an excess oil phase in thermodynamic equilibrium denoted by 2. At high temperatures, a water-in-oil microemulsion coexists with an excess water phase denoted by 2̅. The transition between these two states takes place via a bicontinuously structured microemulsion. At low surfactant

Figure 1. Phase diagrams of the system H2O/NaCl/Cal B−n-octane− C10E5 as a function of temperature T and surfactant mass fraction γ at equal volumes of n-octane and the water phase for four different Cal B concentrations (no Cal B, white circle; 1 mg mL−1, gray triangle; 10 mg mL−1, black circle; 50 mg mL−1, white triangle; 100 mg mL−1, gray circle). The solid lines are guides for the eyes.

observe two effects, namely, that (1) T̃ shifts to lower temperatures and (2) γ̃ runs through a minimum between 1 and 50 mg mL−1. These effects will be explained in the following section. 3.B. Partitioning of Cal B between the Interface and the Water Domain. The shift of T̃ with increasing Cal B concentration can be explained by the influence of Cal B on the binary system water−nonionic surfactant. The phase behavior of a microemulsion is determined by the interaction of the three binary systems: water−oil, water−nonionic surfactant, and oil−nonionic surfactant. Because (a) Cal B is not soluble in 2995


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n-octane and (b) water and oil are not miscible over the relevant temperature and composition range, we consider only changes in the binary system H2O/NaCl−C10E5. Starting at a point on the upper miscibility gap at γA = 0.15, we added the same amount of Cal B to the water phase of system H2O/ NaCl−C10E5 as was added to the aqueous phase of the microemulsions. As is shown in Figure 2, the phase boundary systematically shifts to lower temperatures when the concentration of Cal B increases.

Table 1. Calculated Relative Mass of Lipase Located at the Interface of the Microemulsion H2O/NaCl−n-Octane− C10E5a C0/mg mL−1 1 5 10 50

m0

0.01 0.45 0.95 3.20

0.09 0.66 1.76 7.75

± ± ± ±

0.01 0.45 0.95 3.20

0.908 0.868 0.824 0.845

± ± ± ±

0.01 0.04 0.1 0.06

3.C. Diffusion in Cal B-Containing Microemulsions. The above-mentioned calculations show that Cal B is mainly located at the interfacial monolayer and may thus change its properties. To monitor the influence of adsorbed Cal B, we studied the microstructure of the respective microemulsions with PFG-NMR.36 PFG-NMR allows us to detect changes in the self-diffusion coefficients of water and oil that can be directly related to the type of microstructure present in the sample. At low temperatures, the self-diffusion coefficients of water are typically 2 orders of magnitude higher than those of oil (Dw ≫ Doil) because water is the continuous phase whereas oil is solubilized in microemulsion droplets. At high temperatures, one observes just the opposite (Dw ≪ Doil) because now the oil is the continuous phase and water is the dispersed phase. In the intermediate bicontinuous structure, both water and oil are continuous so that one observes Dw ≈ Doil. To compare the self-diffusion coefficients of water and oil in the microemulsion directly, one usually divides the experimental values by the selfdiffusion coefficients of the pure solvents. It thus holds for the relative self-diffusion coefficients, also called obstruction factors,36 D Drel = obs D0 (11) where Drel denotes the ratio between the self-diffusion coefficient of the solvent in the microemulsion (Dobs) and the self-diffusion coefficient of the pure solvent (D0). Geometrical obstruction effects and/or hydration effects caused by the enzyme may alter the self-diffusion coefficients of the solvents.37 Note that these effects depend mainly on the concentration of the enzyme present. Prior to measuring the microemulsion samples, we studied aqueous solutions containing 4 wt % NaCl and Cal B concentrations of 0, 1, 10, and 50 mg mL−1, respectively. The results presented in Table 2 show that the presence of Cal B does not alter the self-diffusion coefficient of water in the studied range of concentrations. In Figure 3, the relative self-diffusion coefficients of n-octane and water for microemulsions consisting of H2O/NaCl/Cal B− n-octane−C10E5 are presented as a function of temperature. The surfactant mass fractions were chosen close to the X point

C0 − Cw,mE C0

± ± ± ±

mi,mE/m0

Ci,mE is defined as mi,mE/Vwater. If we defined Ci,mE as mi,mE/VmE, where VmE is the volume of the microemulsion phase in the three-phase region, then the concentrations would be nearly twice as high.

To understand why γ̃ runs through a minimum, we studied the distribution of Cal B between the water domain and the interfacial monolayer of the microemulsion in the three-phase region. We prepared the respective three-phase state by decreasing γ to a value of γ = 0.05. The amount of Cal B in the excess water phase of the samples was determined via the absorption of aromatic amino acid residues at 280 nm using UV/vis spectroscopy as described in the Experimental Section. The relative amount located at the oil−water interface was then determined by applying the following mass balance, assuming that Cal B can be located only in (i) the excess water phase/the water domain of the microemulsion or (ii) adsorbed to the interfacial monolayer. =

0.91 4.56 8.24 42.2

Cw,mE/mg mL−1

a

Figure 2. Influence of Cal B on the phase boundary of the miscibility gap of the system H2O/NaCl−C10E5 as a function of temperature T and enzyme mass fraction in the water phase. The surfactant mass fraction was kept constant at γA = 0.15.

m i,mE

Ci,mE/mg mL−1

(10)

mi,mE is the mass of enzyme located at the interface, m0 is the mass of enzyme initially introduced into the microemulsion, C0 is the initial concentration of enzyme, and Cw,mE is the concentration of enzyme in the excess water phase. Table 1 shows the calculated values. The results show that the amount of Cal B in the excess water phase and at the interface increases with increasing initial concentration. The fact that the relative amount of Cal B at the interface remains almost unchanged, namely, between 80 and 90% for all samples, shows that the interface is not yet saturated with enzyme.

Table 2. Self-Diffusion Coefficients of Water, Dw, as a Function of the Concentration of Cal B, C0, at 33 °C C0/mg mL−1 0 1 10 50 2996

Dw/10−9 m2 s−1 2.74 2.82 2.73 2.54

± ± ± ±

0.02 0.06 0.04 0.01


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Figure 3. Relative self-diffusion coefficients of water (open symbols) and n-octane (filled symbols) in microemulsions that were prepared at equal volumes of n-octane and an aqueous solution consisting of 4 wt % NaCl and different Cal B concentrations, namely, no Cal B (top, left), 10 mg mL−1 (top, right), 50 mg mL−1 (bottom, left), and 100 mg mL−1 (bottom, right). The measurements were performed as a temperature cut through the one-phase region at a distance to the X point of Δγ = 0.005.

at γ = γ̃ + 0.005 to ensure that the structure of the microemulsion was indeed bicontinuous. At this surfactant mass fraction, the temperature range of the single-phase region was around 1 °C. The studied range of temperatures, as presented in Figure 3, is in most cases between 20 and 40 °C. This large range of temperature is required to study the transition from droplet to bicontinuous and back to droplet microemulsions. The self-diffusion coefficients of n-octane and water for all of the investigated samples present the same behavior. At low temperatures, the diffusion of water is fast whereas the diffusion of n-octane is slow. The opposite holds for high temperatures, whereas at intermediate temperatures the reduced self-diffusion coefficients are equal in magnitude, as was expected.36 Comparing the four plots in Figure 3, a distinct difference for the 100 mg mL−1 Cal B sample can be observed. For this lipase concentration, the relative self-diffusion coefficients of water at low temperatures are significantly lower than the values of the other three samples. A possible explanation could be that at these high enzyme concentrations a significant part of the water serves as a hydration shell for the enzyme and thus the average self-diffusion coefficient decreases. This explanation is supported by the values listed in Table 2. However, further studies are required to clarify this point. The crossover point of the relative self-diffusion coefficients of water and oil for a microemulsion was found to correlate with the phase inversion temperature T̃ . The theoretical relative self-diffusion coefficient at this point was found to be twothirds,32,38,39 reflecting an interfacial layer with a zero mean curvature (H = 0) as is the case for the bicontinuous

structure.40 Our results show that the crossover point indeed appears at T̃ . However, the relative self-diffusion coefficients differ from the theoretical value of two-thirds, and one obtains Drel = 0.5 (no Cal B), 0.5 (10 mg mL−1), 0.49 (50 mg mL−1), and 0.46 (100 mg mL−1). Such deviations have already been reported earlier and they were attributed to structural defects such as local tubular structures with nonzero mean interfacial curvatures.41−43 The self-diffusion coefficients of the nonionic surfactant C10E5 in the microemulsion samples at T̃ were found to be 0.12 × 10−9 m2 s−1 (0 mg mL−1), 0.13 × 10−9 m2 s−1 (10 mg mL−1), 0.11 × 10−9 m2 s−1 (50 mg mL−1), and 0.12 × 10−9 m2 s−1 (100 mg mL−1). Note that these values are a combination of the selfdiffusion coefficients of aggregated and free surfactant molecules and thus should be considered to be only estimates. Nevertheless, in comparing the self-diffusion coefficients obtained for the different Cal B concentrations one sees that the presence of Cal B does not influence the self-diffusion of C10E5 in the investigated microemulsion samples.

4. DISCUSSION Uppenberg et al. presented the amino acid sequence and the crystal structure of lipase B from Candida antarctica.22 They reported the dimensions of the lipase crystal to be 3 × 4 × 5 nm3. Trodler et al. modeled the structure and flexibility of Cal B in water and in different organic solvents using multiple molecular dynamics simulations.44 According to these simulations, the structure of Cal B is independent of the solvent. However, small deviations in the crystal structure were reported in the different solvents. These deviations were in the range of 2997


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δ = 0.0722). Unfortunately, the boost factor decreases rapidly with increasing Cal B mass fraction δ. Partitioning studies also show that the enzyme concentration in the excess water phase is increasing. At a concentration of 100 mg mL−1 (δ = 0.246), the efficiency of the microemulsion system is even lower than the efficiency of the original base microemulsion system (i.e., Cal B now even acts as an antiboosting biopolymer). Kabalnov et al. observed a similar effect when adding HMEHEC.48 The partitioning of HM-EHEC and the swelling of the microemulsion middle phase were found to run through a maximum with increasing polymer concentration. The authors discussed this observation in terms of a saturation of the surfactant monolayer with polymer and a counter osmotic pressure induced by the increasing polymer concentration in the excess water phase.

0.07 to 0.09 nm for polar solvents and in the range of 0.04 to 0.08 nm for nonpolar solvents. With regard to the flexibility of Cal B in the studied solvents, Trodler et al. showed that the core and the active site of the lipase have low flexibility whereas a higher flexibility of the residues located at the interface was found. On the basis of these calculations, one could argue that the structure of the lipase in bicontinuous microemulsions should be similar to its crystal structure. However, further experiments are required to clarify this assumption. The microstructure of microemulsion system D2O−noctane−C10E5 was well characterized by Sottmann et al.45 From small-angle neutron scattering experiments, the following parameters were determined: correlation length ξTS = 15 nm, domain size dTS = 30 nm, specific interfacial area S/V = 0.13 nm−1, and area per C10E5 molecule at the water−n-octane interface = 0.6 nm2. A comparison of the theoretical dimensions of Cal B (3 × 4 × 5 nm3) with the experimentally determined domain size of the bicontinuous microemulsion (15 nm) chosen in the present study reveals that the domain size of the microemulsion would be large enough to incorporate the enzyme. Note that enzymes such as lipase Cal B represent a class of biopolymers composed of amino acids as repeat units. The effect that the addition of polymers has on the properties of the respective microemulsions has been addressed very extensively in the literature. For example, the influence of the polymer’s molecular structure on the interfacial film properties and on the microstructure of the microemulsion has been studied.46−49 Jakobs et al. described the ability of a block copolymer to enhance of the efficiency of a microemulsion as efficiency boost factor f B.47 It holds that fB =

5. CONCLUSIONS We introduced lipase B from Candida antarctica (Cal B) into a bicontinuous microemulsion of composition H2O/NaCl−noctane−C10E5. The results obtained from phase diagrams, partitioning of the lipase between the microemulsions interface and the water domain, and pulsed-field-gradient NMR experiments lead to the following conclusions: (i) The addition of small amounts of Cal B resulted in an increase in the efficiency of the microemulsion. The shift was reversed at higher concentrations. (ii) The phase-inversion temperature of the microemulsion system systematically decreased with increasing Cal B concentration. The effect could be attributed to the shift in the miscibility gap in the binary H2O/NaCl− C10E5 system induced by the addition of Cal B. (iii) The partitioning of Cal B between the microemulsion phase and the water phase showed that with increasing Cal B concentration the concentration of both the enzyme in the water phase and at the interface increases. However, the ratio mi,mE/m0 was found to be nearly constant, which indicates that the interface is not saturated with enzyme under the chosen experimental conditions. (iv) The self-diffusion coefficients did not reveal any effect of Cal B on the bicontinuity of the microemulsion or on the self-diffusion behavior of the individual components.

γ0̃ − γ (1 ̃ − δ) γδ̃

(12)

where γ̃0 denotes the surfactant mass fraction at the X point of the microemulsion without polymer, γ̃ denotes the surfactant mass fraction at the X point of the microemulsion with polymer, and δ denotes the mass fraction of polymer in the surfactant/polymer mixture. We calculated Cal B boost factors using eq 12 for the concentrations studied. The values are presented in Table 3. Please note that the respective biopolymer mass fraction δ is also given (Experimental Section) and that γ̃0 = 0.145 was used for the calculations. The values suggest that at low δ Cal B acts as an efficiencyboosting biopolymer with a boost factor of 13.86, which is indeed comparable to a strong amphiphilic diblock copolymer of the PEP−PEO type (e.g., PEP5−PEO5; molecular mass = 10 650 g/mol; indices indicate equal molecular masses of the hydrophilic and the hydrophobic block in kg/mol; f B = 14.9 at

■

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Dr. Daniel Topgaard and Prof. Dr. Michael Hunger for help and valuable discussions concerning the PFGNMR measurements. Financial support was kindly granted by European COST action D43. We also thank M. Eng. Sven Richter for purifying the lipase.

Table 3. Boosting Factors for Microemulsions with Different Concentrations of Cal Ba C0/mg mL−1

γ̃

δ

fB

1 10 50 100

0.138 0.130 0.138 0.153

0.004 0.038 0.155 0.246

13.86 3.98 1.32 0.78

AUTHOR INFORMATION

Corresponding Author

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REFERENCES

(1) Farber, K. Biotransformations of non-natural compounds: state of the art and future development. Pure Appl. Chem. 1997, 69, 1613− 1632. (2) Wegman, M. A.; Janssen, M. H. A.; van Ranktwijk, F.; Sheldon, R. A. Towards biocatalytic synthesis of β-lactam antibiotics. Adv. Synth. Catal. 2001, 343, 559−576. (3) Martinez, C. A.; Hu, S.; Dumond, Y.; Tao, J.; Kelleher, P.; Tully, L. Development of a chemoenzymatic manufacturing process for pregabalin. Org. Process Res. Dev. 2008, 12, 392−398.

C0 is the concentration of Cal B in the initial aqueous solution, γ̃ is the surfactant mass fraction at the X point for each microemulsion, δ is the lipase mass fraction in the lipase/surfactant mixture, and f B is the boosting factor. a

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Physicochemical Aspects of Lipase B from Candida antarctica in

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