pubs.acs.org/Langmuir © 2009 American Chemical Society
Surface Packing Characterization of Langmuir Monolayer-Anchored Enzyme Ravindrabharathi Narayanan,† Benjamin L. Stottrup,‡ and Ping Wang*,† †
Department of Bioproducts and Biosystems Engineering and Biotechnology Institute, The University of Minnesota, St. Paul, Minnesota 55108, and ‡Department of Physics, Augsburg College, Minneapolis, Minnesota 55454 Received March 26, 2009. Revised Manuscript Received July 14, 2009
We have synthesized a novel interface-anchoring alcohol dehydrogenase by covalent attachment of a hydrophobic polymer tail to the hydrophilic protein head. Analogous to a protein-based surfactant, this polymer-enzyme conjugate self-assembled at liquid-liquid or liquid-air interfaces to form a membrane similar to other surfactant monolayers. The packing and morphology of the interface-anchored enzymes play an important role in regulating the membrane behaviors including enzyme mobility and interfacial interactions of enzymes with reactant and product molecules. To characterize the surface assembly morphology of the interface-anchored enzymes, Langmuir film balance and fluorescence microscopy techniques were used. The Langmuir isotherm of the interface-anchored enzyme demonstrated a pronounced molecular rearrangement upon compression of the isotherm. This corresponded to changes in membrane morphology and state observed using fluorescence microscopy. The molecular diffusion within the novel interfaceanchored enzymes was further evaluated by using a fluorescence recovery after photobleaching technique. We report a diffusion coefficient of 6.7 10-10 cm2/s. The study represents the first in-depth analysis of surface packing and interfacial mobility of such interface-anchored enzymes.
Introduction All cellular processes depend on the discrete functional identities provided to the subcellular space by interfaces or membranes. In cellular membranes, lipid bilayers are stabilized through a clustering or assembling of the integral proteins.1 Among the various membrane-bound proteins, enzymes enable important biological reactions often associated with substrates and products diffusing across the membranes. Enzymes are macromolecules that contain both hydrophobic and hydrophilic groups. Interfacial enzymes such as lipases that are active near cellular membranes often contain large fractions of hydrophobic moieties on their surfaces and therefore have low solubility in water.2,3 Enzymes that do not possess such large hydrophobic moieties generally lack the capability of assembling and catalyzing reactions at interfaces.4,5 We have shown in previous studies that native watersoluble enzymes can be modified with hydrophobic polymers to achieve highly stable interfacial assembling.6-8 The enzyme molecules were conjugated with hydrophobic moieties like polystyrene and poly(lactic acid), leading to molecular structures similar to protein-based surfactants. In all the cases examined, the interface-assembled enzymes maintained very well their catalytic activities and afforded reaction rates that were up to 150 times higher than their native parent enzymes *Corresponding author: e-mail
[email protected]; Ph 612-624-3064. (1) Kaladhar, K.; Sharma, C. P. J. Biomed. Mater. Res., Part A 2006, 79A(1), 23–35. (2) Derewenda, U.; Brzozowski, A. M.; Lawson, D. M.; Derewenda, Z. S. Biochemistry 1992, 31(5), 1532–1541. (3) Louwrier, A.; Drtina, G. J.; Klibanov, A. M. Biotechnol. Bioeng. 1996, 50, 1–5. (4) Mori, T.; Fujita, S.; Okahata, Y. Carbohydr. Res. 1997, 298, 65–73. (5) Soler, G.; Bastida, A.; Blanco, R. M.; Fernandez-Lafuente, R.; Guisan, J. M. Biochim. Biophys. Acta 1997, 1339(1), 167–175. (6) Narayanan, R.; Zhu, G.; Wang, P. J. Biotechnol. 2007, 128(1), 86–92. (7) Zhu, G.; Wang, P. J. Biotechnol. 2005, 117(2), 137–213. (8) Zhu, G.; Wang, P. J. Am. Chem. Soc. 2004, 126(36), 11132–11133.
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exposed to the same reaction evnvironments.8 Moreover, interface-assembled enzymes demonstrated much improved stabilities with half-lifetimes several times longer than native enzymes.6,8 Such activity and stability observations are very much in contrast to traditional impression that water-soluble proteins undergo denaturation when exposed to liquid-liquid or air-liquid interfaces.4,5 Our further investigation suggested that the much reduced interfacial tension, as a result of the highly selective and concentrated assembling of the surfactantlike enzymes at interfaces, contributed significantly to the stabilization of the interface-anchored enzymes.9 In addition, it has been well-known that attachment of enzymes to other materials can substantially protect the enzymes from structural denaturation.10,11 On the contrast, native water-soluble enzymes generally achieve very low concentrations at interfaces and thus exposing their unprotected molecules to strong interfacial tension and shear stress. In addition to the well-demonstrated interfacial activity and stability of such polymer-conjugated enzymes, we also observed that the pattern and status of assemblies can significantly impact the performance of the enzymes at interfaces. Highly concentrated interfacial assemblies showed very little activity as a result of much limited mass transfer across such thick interfaces, while dilution of the interface can resume the activity of the enzyme due to the reduced mass transfer limitation of substrates.6 Such observations suggest that while interface-anchored enzymes maintain their activities, factors such as surface packing, assembly morphology, and interfacial mobility of the interface-anchored enzymes, mass transfer of substrates and products at the interface, and interfacial interactions among these molecules may all influence the efficiency of the interfacial biocatalysis. (9) Wang, L.; Zhu, G.; Wang, P.; Newby, B. Z. Biotechnol. Prog. 2005, 21(4), 1321–1328. (10) Wang, P. Curr. Opin. Biotechnol. 2006, 17, 574–579. (11) Kim, J.; Grate, J. W.; Wang, P. Chem. Eng. Sci. 2006, 61(3), 1017–1026.
Published on Web 08/13/2009
Langmuir 2009, 25(18), 10660–10665
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Figure 1. Interfacial assembly of PS-ADH conjugate. Left: Oregon green-labeled native ADH RS1 dissolved in water phase in a hexane-water biphasic system. Right: polymer-enzyme conjugate (dyed) at hexane-water interface.
In this work, to characterize the surface assembly morphology of the interface-anchored enzymes, we applied a Langmuir film balance along with a fluorescence microscope. The response of modified enzymes at the interface to expansion and compression of their dispersion area is an important characterization tool in assessing the phase behaviors of the enzyme monolayer. The resultant interfacial tension can be correlated to the adsorption/ desorption, aggregation/disaggregation, and probably intramolecular rearrangements of the interface-anchored enzymes. The 2-D interfacial molecular diffusion of the novel interface-anchored enzyme was further evaluated by using a fluorescence recovery after photobleaching (FRAP) technology.
Results and Discussion Interfacial Assembly of the Enzyme. A number of groups of enzymes are membrane bound, including phospholipases, peptidases, lactases, and glutaminases.12 Most of these enzymes contain hydrophobic side chains that interact with fatty acid groups of lipid membranes, thus anchoring the enzymes to the membranes. The most frequently studied interfacial enzymes are lipases, which act on the carboxyl ester bonds of triglycerides to liberate fatty acids and glycerol. Studies have shown that the active sites of lipases are covered by hydrophobic polypeptide segments, which will open upon binding to a lipid interface and therefore expose the active sites to their substrates, lipids.2,13 This mechanism enhances the hydrophobic interaction between lipases and lipid membrane, resulting in the interesting interfacial activation phenomenon.14-16 However, for enzymes with high hydrophilicity, it is relatively difficult for them to be adsorbed with a significant amount at oil-water or even Langmuir-Blodgett solid interfaces.17 On exposure to such interfaces, the enzyme molecules may undergo unfolding and result in rearrangement of the protein structure. This occurs because the enzyme experiences a thermodynamic environment at the interface that is different from their native bulk aqueous phase, which leverages effects of the various physical forces that determine its three-dimensional structure.18 One solution to avoid interfacial denaturation is to employ a modified Langmuir-Blodgett technique to prepare enzyme coatings on solid substrate supports.17 It was also reported that alkaline phophataseses, which tend to concentrate (12) Gennis, R. B. Biomembranes; Springer-Verlag: New York, 1989. (13) Brzozowski, A. M.; Derewenda, U.; Derewenda, Z. S.; Dodson, G. G.; Lawson, D. M.; Turkenburg, J. P.; Bjorkling, F.; Huge-Jensen, B.; Patkar, S. A.; Thim, L. Nature 1991, 351(6326), 491–494. (14) Louwrier, A.; Drtina, G. J.; Klibanov, A. M. Biotechnol. Bioeng. 1996, 50 (1), 1–5. (15) Dahim, M.; Brockman, H. Biochemistry 1998, 37(23), 8369–8377. (16) Brockman, H. L. Biochimie 2000, 82(11), 987–995. (17) Caseli, L.; Zaniquelli, M. E. D.; Furriel, R. P. M.; Leone, F. A. Colloids Surf., B 2002, 25, 119–128. (18) Izmailova, V. N.; Yampolskaya, G. P. Stud. Interface Sci. 1998, 7, 103–147.
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Figure 2. Effect of interfacial enzyme concentration on reduction of acetophenone.
Figure 3. Schematic of Langmuir film balance setup for surface pressure analysis and simultaneous optical observation.
at interfaces, could be active at the air-water interface over a broad range of pressures.19 As mentioned earlier, we have developed a method to conjugate enzymes with hydrophobic polymer molecules for highly efficient and stable interfacial catalysis, attributing to the effects of both material support and interfacial affinity assembling. Dehydrogenases like alcohol dehydrogenase from Rodococcus species (ADH RS1) along with cofactors catalyze a wide range of biosynthesis with high selectivity, with many promising synthetic processes involving hydrophobic chemicals.20,21 Biphasic reactions are generally desired for such biosynthesis. In this research, hydrophobic polystyrene (PS) was deliberately attached to the ADH RS1, via surface exposed lysine groups of the enzyme molecules,7,8 to enhance the surface hydrophobicity in order to allow the enzyme to assemble at oil-water interfaces. The protein content estimation by both Bradford analysis and reverse biuret method gave comparable results on the efficiency of enzyme modification, with an average modification yield as 53%. The resulting polystyrene-enzyme conjugate self-assembled at the oil-water interface as seen from Figure 1. Oregon green dye was used to label the enzymes. The green color observed at the oil-water interface for modified enzyme indicates a selective interfacial assembly, in contrast to native enzyme that remained in the bulk aqueous phase. To evaluate the activity of interface-anchored enzymes, organic synthesis of R-1-phenylethanol was examined as a model reaction. The interfacial enzyme was applied for reduction of acetophenone to R-1-phenylethanol in an organic phase of toluene. This reaction requires a cofactor NADH, which was applied in an aqueous phase of 7.5 pH phosphate buffer. The reaction was (19) Caseli, L.; Oliveria, R. G.; Masui, D. C.; Furriel, R. P. M.; Leone, F. A.; Maggio, B.; Elisabete, M.; Zaniquelli, M. E. D. Langmuir 2005, 21, 4090–4095. (20) Forde, J.; Oakey, L.; Jennings, L.; Mulcahy, P. Anal. Biochem. 2005, 338(1), 102–112. (21) Zhang, M.; Mullens, C.; Gorski, W. Anal. Chem. 2007, 79(6), 2446–2450.
DOI: 10.1021/la901076j
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Figure 4. Surface pressure isotherm of interface-anchored ADH RS1. On compression the monolayer behaves like (A) gel phase or liquid condensed phase; (B) conformational change or transition plateau, in this region the monolayer has a liquid phase and material can be seen to flow about the monolayer; and (C) dilute system. The trough area was controlled by placing Teflon bar of 75 cm2 in the middle.
carried out in a 50 mL reactor with magnetic stirring of the interface at 75 rpm. The rate of formation of phenylethanol was monitored using GC. An initial lag in the enzyme concentration-rate relationship was observed (Figure 2), resulting in a sigmoidal behavior. This indicates that the reaction kinetics of the interface-anchored enzyme does not follow the traditional Michaelis-Menten equation. The nontraditional behavior of interface-anchored enzymes was also observed in our similar work involving β-galactosidase which exhibited, on increasing interfacial enzyme concentration, a drop in reaction rate after reaching a maximum value.8 The underlying mechanism of interaction of interface-anchored enzymes and the reactants is not clear from these studies. It appeared that multiple layer assembly or aggregate formation of the enzymes at the interface that results in variation in mass transfer of substrate/product across the interface may hamper the reaction, as the activity of the enzyme can be resumed reversibly by diluting the interface to lower enzyme concentrations. A systematic study to elucidate the actual mechanism is needed to optimize the novel interfacial biocatalysis. Nevertheless, the PS-modified ADH RS1 maintained very good stability at the interface. Samples that were recovered from the interface and subsequently freeze-dried could be used repeatedly for several times without significant loss of its activity. Surface Pressure Analysis of Interface-Anchored Enzyme. A Langmuir film balance was used to characterize the surface assembly and morphology of the interface-anchored enzyme. A schematic of the Langmuir film balance setup is shown in Figure 3. When an amphiphile is spread on water at very low surface concentrations, the initial molecular organization is random. As the barrier is drawn across the trough, the surface area available to the amphiphiles is reduced and the molecules are compressed closer together. The surface pressure (π) will increase, provided the molecules remain on the surface and do not form bulk aggregates. As the pressure increases and the amphiphiles are compressed into a smaller area, the molecular organization increases as molecules make contact and begin to interact. Eventually, compression will result in a structural collapse as molecules are forced out of the two-dimensional plane. The Langmuir film balance study for observing the assembly behavior of modified enzymes clearly indicates a potential for monolayer 10662 DOI: 10.1021/la901076j
assembly of the enzymes at the interface (Figure 4). The isotherm indicates that for large surfaces the system behaves as a dilute monolayer, and there is little change in the surface tension of the subphase. In simpler Langmuir surfactant layers this region is often referred to as the “gas phase”. The plateau in the isotherm indicates the monolayer contains coexisting phases and is undergoing a transition between phases and that the monolayer can be further compressed without appreciably changing the pressure. The final transition of the observed monolayer looks typical of a condensed state as observed in fluorescence microscopy (Figure 4). The phase transition and surface packing of the interfaceanchored enzymes can be interpreted in terms of conformational change of the monolayer due to the work done during compression. The work done on the monolayer during compression is a result of contributions from relevant parameters like electrostatic energy, strain associated with stretching bonds, torsional potential, hydrogen bond formation, and van der Waals interaction terms.22-24 In the case of interface-anchored enzymes, the system is highly complex with two macromolecular moieties. This makes it difficult to quantify the individual parameters that contribute to the observed changes in the monolayer. However, the relatively flat plateau does indicate a discrete change in the monolayer behavior between two states. The surface pressure isotherm could be interpreted in terms of phase behavior, aggregation, and intramolecular rearrangement of the monolayer. A number of factors suggest the monolayer is undergoing a phase transition. The shape of the isotherm did not change on varying compression rates from 3 to 100 cm2/min. This implies thermal equilibrium of the monolayers upon compression and expansion. A further test of the monolayer’s stability was performed by holding the trough at constant area for over 20 min after a surface pressure of 11 mN/ m had been reached (above the plateau). In this test the surface pressure decreased by
