Enzyme Kinetics in Liquid Crystalline Mesophases - American ...

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Mar 25, 2015 - Mesophases can provide a perfect hosting environment for enzymes .... the fastest enzymatic reaction takes place within HII, followed by Ia3d ...

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Enzyme Kinetics in Liquid Crystalline Mesophases: Size Matters, But Also Topology Wenjie Sun, Jijo J. Vallooran, and Raffaele Mezzenga* ETH Zurich, Department of Health Sciences and Technology, Schmelzbergstrasse 9, CH-8092 Zurich, Switzerland S Supporting Information *

ABSTRACT: Lyotropic liquid crystalline systems (LLCs) are excellent immobilizing carriers for enzymes, due to their biocompatibility and well-defined pore nanostructure. Here we show that the liquid crystalline mesophase topology can greatly influence the enzymatic activity in a typical peroxidase (Horseradish peroxidase, HRP) enzymatic reaction. Enzyme kinetics was investigated in different LLC mesophases based on monolinolein, with varying symmetries and dimensions such as the 1D cylindrical inverse hexagonal phase (HII), the 2D planar lamellar phase (Lα), and two 3D bicontinuous cubic phases of double diamond (Pn3m) and gyroid (Ia3d) space groups. As expected, the mesophase with largest water channel size shows highest activity, regardless of the topology. Interestingly, however, when mesophases with different topologies have the same water channel size, then the topology plays the dominant role, and the enzyme showed the highest activity in the 3D tetra-fold connected Pn3m, followed by the Ia3d with trifold connectivity, and finally the 1D HII phase. This study demonstrates that the enzymatic activity in LLC mesophases depends on both the water channel size and the topology of the mesophase.

INTRODUCTION Lipid-based lyotropic liquid crystalline systems (LLCs), formed by the self-assembly of specific lipids, such as monoglyceride1−3 and phytantriol,4−6 have been attracting major attention due to their ability to be utilized in a wide range of applications, such as hosting matrices for material synthesis,7,8 protein crystallization,9−11 drug and nutrients delivery,12−16 and biomedical or environmental sensing.17−22 Mesophases with different characteristic topologies and symmetries, for instance, the 1D inverse hexagonal phase (HII), the 2D planar lamellar phase (Lα), and the 3D bicontinuous cubic phase (V2) can be obtained by manipulating the amphiphilic and aqueous composition of the system.3,4 The nanostructure formed depends on the geometries of the applied amphiphilic molecules and can be described by the critical packing parameter as reported by Israelachvili et al.23,24 or by more sophisticated energy minimization approaches.25,26 Small angle X-ray scattering (SAXS) was the main characterizing tool employed in these studies for the nanostructures and other structural parameters.10−22,27−29 Mesophases can provide a perfect hosting environment for enzymes due to their membrane-biomimetic structures; moreover, their intrinsic capacity to retain their structural features at thermodynamic equilibrium with a surrounding environment of excess water makes them ideal candidates for the design of reusable biosensors for biomedical and environmental detection. In meso enzyme-based biosensors have been widely investigated since the 1990s.17,18 A feat was achieved by © 2015 American Chemical Society

physically encapsulating enzymes within mesophases and following the evolution of the enzymatic reactions by electrochemical17−20 or optical21,22 methods. These highly sensitive analyte-specific biosensing systems can be used to detect numerous analytes, such as glucose (using glucose oxidase-GOx),17−19 toxic phenolic compounds, and hydrogen peroxide (using horseradish peroxidase-HRP) in biomedical and environmental fields.22,30−32 Conventional enzyme immobilization protocols include physical adsorption,33,34 covalent attachment,35,36 and encapsulation within inorganic37−41or organic matrices such as, hydrogels42−44 and mesophases.17−22 Although stable enzymematrix conjugates can be formed, the performance of the enzymes (i.e., activity and long-term stability) would be affected by the chemical or physical environment in which they are immobilized. The influence of the physical entrapment on the enzymes activity has been noticed since long time ago and pore-size dependences have been demonstrated in numerous mesoporous materials.38−41 Both size matching preference (enzymes show the highest activity and long-term stability when the pore size just exceed the molecular diameters of the enzymes)38−40 and monotonic increase of the enzyme activity with the increasing pore size,41 were observed. In our previous study,22 we investigated the water channel size dependence of Received: February 12, 2015 Revised: March 24, 2015 Published: March 25, 2015 4558

DOI: 10.1021/acs.langmuir.5b00579 Langmuir 2015, 31, 4558−4565



Figure 1. 1D SAXS spectra of scattered intensities vs scattering vector q showing Lα phase (red), bicontinuous cubic Ia3d (blue) and HII phase (green, respectively from front to back) obtained with M/SE (80:20) mixture, monoglyceride and M/TD (90:10) at the same 20% water, respectively. The phase symmetries (schematized in the right panel) were identified via the specific spacing of the reflection peaks following the labeled ratio and the water channel diameters d were calculated according to the equations described in the SI. SE), and the other with acetate buffer (pH = 4.65). The two halves were then mixed until the blend became totally homogeneous. To prepare mesophase samples with loaded enzyme and substrates for enzymatic studies, the desired amount of HRP solution (pH = 7) was loaded with monoglyceride (M), monoglyceride-sugar ester (M-SE) or monoglyceride-tetradecane (M-TD) mixtures in one syringe while the substrate ABTS and H2O2-containing acetate buffer (pH = 4.65) were loaded in the other syringe. In this way, the enzymatic reaction did not start before the mixing of the two syringes. Since the in meso enzymatic reaction typically presents an initial lag period, particular attention was given to finalize mixing well within the lag period, and to operate the mixing step within a constant time. Therefore, analysis of the lag period at different swollen conditions is simply affected by a constant offset. Small-Angle X-ray Scattering. The symmetries of the various mesophases were identified by Small-angle X-ray scattering (SAXS, Rigaku MicroMax-002+ microfocused X-ray source operating at 45 kV and 88 mA). The Ni-filtered Cu Kα radiation (λ = 1.5418 Å) was collimated by three pinholes of 0.4, 0.3, and 0.8 mm, respectively and the scattered intensity was collected by a 2D argon-filled Triton-200 Xray detector (20 cm diameter, 200 μm resolution) for over 30 min. The scattering vector is defined as q = 4 π sin(θ)/λ with a scattering angle 2θ and q was calibrated using silver behenate. The effective scattering vector ranged from 0.01 Å−1 to 0.44 Å−1. The viscous mesophase samples (HII, Pn3m, Ia3d) were placed on a Linkam hot stage sandwiched between two mica sheets spaced by a 1 mm O-ring. The more liquid-like fluid mesophase (Lα) was filled into 1.5 mm diameter quartz capillaries, and sealed with epoxy glue (UHU). For all SAXS measurements, the temperature was controlled at 37 °C. UV−vis Spectroscopy. The mesophase with loaded enzyme and substrate homogenized in the coupled Hamilton RN syringe setup was transferred to a demountable closed cuvette (Starna GmbH). The kinetics mode of the Cary 100 Bio UV−vis spectrophotometer was employed to record the whole progress curve of the enzymatic reaction of HRP at a fixed wavelength of 418 nm (maximum absorbance of the oxidized ABTS) at 37 °C. From the progress curves, the enzyme activity parameters and kinetics curves can be determined.

the in meso enzymatic activity of the model enzyme horseradish peroxidase HRP. To this end, the relatively small water channels of the standard double diamond bicontinuous cubic mesophase, Pn3m, formed by monolinolein, were enlarged to over twice its standard size by introducing a hydration-enhancing agent, sucrose stearate.45 It was found that the enzymatic activity was greatly increased with the swelling of the Pn3m water channels, asymptotically approaching its activity in bulk water. Although several studies have focused on the role of pore size on enzymatic activity, the influence of nanoconfinement geometries on enzymatic activity was not considered. Here we have characterized the peroxidase enzymatic activity in different liquid crystalline mesophases, ranging from the 1D cylindrical HII, to the 2D planar Lα, to the 3D V2. At the same water content, HII phase formed the largest pore size and in turn showed the highest enzymatic activity due to faster transport of the substrates within water channels, while the Lα phase with the lowest water layer domains showed the lowest enzymatic activity, as expected. Interestingly, when the enzymatic activities in mesophases with the same water pore sizes were compared, topology played the major role and 3D topological bicontinuous cubic phases showed a higher activity than the 1D topological HII phase. This work pushes forward our current understanding of the interplay between in meso enzyme activity and the physical nanoconfinment topologies and symmetries, which is crucial for unraveling enzyme kinetics in complex biological systems.


Materials and Sample Preparation. A commercial-grade monolinolein containing more than 90% monoglyceride (Dimodan U/J, Danisco, Denmark, Batch Number 015312) was used as received. Sucrose stearate S-1670 (SE), from Mitsubishi-Kagaku Foods Corporation, Japan, is a blended sugar ester with 75% of monoester and 25% of di-, tri-, and polyesters. Tetradecane (TD, 99.0%), Horseradish peroxidase (HRP, lyophilized, powder, ∼150 U/mg), 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, 98.0%), hydrogen peroxide (50% H2O2 in H2O), phosphate buffer (PBS, pH = 7), and acetate buffer (pH = 4.65), were purchased from Sigma-Aldrich. As described previously,22 a setup composed of two connected Hamilton RN syringes46 was used to homogenize mesophase samples within less than 3 min. Briefly, pure mesophases without enzyme and substrates inside were prepared by loading one syringe with lipid, either monolinolein (M) or a monolinolein-sugar ester mixture (M-

RESULTS AND DISCUSSION Equally Hydrated Mesophases with 1D, 2D, and 3D Topological Structures. The phase behavior of lipid−water systems can be adjusted by blending the lipid with other components (surfactant,47−49 cosurfactant,50 or oil51,52); accordingly, the mesophase compositions were tuned in order to obtain mesophases with different topological structures at the same hydration condition. The 1D HII, 2D Lα, and 3D Ia3d mesophases53 were established at the same water content (20%), when different lipid mixtures were applied: M/SE 4559

DOI: 10.1021/acs.langmuir.5b00579 Langmuir 2015, 31, 4558−4565



Figure 2. (a) Progress curves of HRP enzymatic reaction with 0.011 mg/mL HRP, 2 mM ABTS and 20 mM H2O2 within Lα phase (water layer thickness = 1.2 nm), bicontinuous cubic Ia3d (d = 2.6 nm), and HII phase (d = 3.4 nm) with the same water content 20%. (b) Relative activity and lag time comparison among HII, Ia3d, and Lα phases.

Figure 3. In meso enzymatic reaction kinetics curves of HRP within (a) hexagonal, (b) lamellar, and (c) cubic topological structures at 20 mM H2O2, 0.009, 0.011, and 0.008 mg/mL HRP, respectively.

hydration condition, 1D HII phase with the largest water channel of 3.4 nm, allows much easier access of the substrates to the enzymes, resulting in a shortest delay for the starting of the reaction. Because the enzyme diameter is of 6 nm, i.e., in all the cases larger than the size of the water domains, we expect that the diffusion-enhanced enzymatic behavior is reflected primarily on the diffusion of the substrate, by assuming the enzyme mostly immobilized within the mesophase. Furthermore, comprehensive enzymatic kinetics studies in these mesophases were performed. The obtained kinetics curves (Figure 3) show a behavior that is relatively complex and cannot be fitted by the common Michaelis−Menten equation:

(80:20) mixture, M/TD (90:10) mixture, and pure monolinolein, respectively. The azimuthally averaged 1D SAXS spectra are shown in Figure 1, and the water channel/domain sizes were calculated as previously described3 and as detailed in the Supporting Information (SI). Among these three identically hydrated mesophases with different topologies, the 1D HII phase has the largest water channel of diameter 3.4 nm while the 2D Lα phase has the smallest water domain of 1.2 nm. The enzymatic activity and kinetics studies in these three different topological structures were performed and are described in detail in the following section. Enzyme Activity in 1D HII, 2D Lα, and 3D Ia3d. In order to gain a better understanding of the interplay between hosting mesophase topologies and enzyme activities, enzyme reactions in the three identically hydrated mesophases with 1D, 2D, and 3D topologies were performed by maintaining identical final enzyme and enzymatic reactant concentrations, namely, 0.011 mg/mL HRP, 2 mM ABTS, and 20 mM H2O2. The progress of the enzymatic reactions, compared for the three mesophases in Figure 2a, indicates that the reaction rate gets steeper when going from Lα to Ia3d, to HII, indicating that the fastest enzymatic reaction takes place within HII, followed by Ia3d and then Lα. Further information was extracted from the reaction progress curves of the HRP reaction as follows. First, the calculated relative activities of HRP, with HII as reference, are 64% in Lα and 85% in Ia3d (Figure 2b). Second, the lag period time (the latency period with no significant activity before a detectable linear reaction regime starts), showed an opposite trend, with a shortest lag time found in HII ( Ia3d > HII (Figure 8 and SI Figure S1 at a different ABTS concentration). To our knowledge, this is the first report demonstrating that topological differences in structure can have such a significant influence on the enzyme activity. The difference in HRP activity can also be related to the differences in the diffusion rates of different topological structures. In SI Figure S2, the 4562

DOI: 10.1021/acs.langmuir.5b00579 Langmuir 2015, 31, 4558−4565



Figure 8. (a) Progress curves of the HRP enzymatic reaction with 0.009 mg/mL HRP, 7.8 mM ABTS and 20 mM H2O2 in Pn3m, Ia3d, and HII with the same water channel diameter of around 3.4 nm (b) Relative activity and lag time comparison among these phases.

has been reported by Lin et al.59,60 that the diffusional release rate was governed by the particle and the pore morphology. Therefore, water channel size is not the only factor that affects the performance of the enzymes in meso, and the topological connectivity of the nanoconfinement geometry also plays an important role.

S1) and the formed oxidized ABTS product versus the square root of time in three identically pore-sized mesophases with different symmetries: Pn3m, Ia3d, and HII (Figure S2). This material is available free of charge via the Internet at http:// pubs.acs.org.

CONCLUSIONS In this work, the influence of the physical nanoconfinement topologies of the LLC hosting matrix on the in meso enzyme activities was systematically investigated. In line with our previous study where the HRP reaction rate was faster in swollen bicontinuous cubic mesophases compared with the standard mesophase, water channel size dependence of the kinetics was observed for the enzymatic activity in mesophases of different geometries. At identical hydration conditions, the mesophase with the largest water channel diameter (the 1D cylindrical HII phase) exhibited the highest enzyme activity, followed by the second domain-larger 3D bicontinuous cubic Ia3d, and finally by the 2D planar lamellar phase, in which the water domains were the thinnest. Consistently, enzyme activities in bicontinuous cubic phases with 3D topological structures but different symmetries were higher for the cubic phase with larger channels. These results reveal that in identically hydrated mesophases with different space groups, the enzyme activity simply increases with increasing water channel size, irrespective of the hosting matrix topologies and symmetries. The greater water channel diameter provides a wider path for faster diffusion of substrate to the enzyme catalytic center and, in turn, results in higher enzymatic activity. However, when enzyme activities in differently hydrated mesophases of the same water channel sizes were compared, mesophase topology was found to be the determining factor in ruling kinetics, 3D tetra-fold connected cubic phases (Pn3m) showing increased activity over the 3D trifold connected cubic phases (Ia3d), and the 1D columnar mesophases (HII), respectively. This work provides the first experimental evidence that both the hydrophilic domain sizes and their topological connectivity control in meso enzymatic activity.


Corresponding Author

*E-Mail: raff[email protected] Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors gratefully acknowledge the China Scholarship Council and ETH Zurich for the financial support for this work. Dr. Wye-Khay Fong and Dr. Alexandru Zabara are kindly acknowledged for valuable discussions.


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* Supporting Information S

Details on calculations of the water channel diameters of mesophases, one more enzyme activity comparison within Pn3m, Ia3d and HII of the same water channel size (∼3.4 nm) at the identical enzymatic condition (ABTS = 6.8 mM) (Figure 4563

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DOI: 10.1021/acs.langmuir.5b00579 Langmuir 2015, 31, 4558−4565