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Enzyme-Regulated Microgel Collapse for Controlled Membrane Permeability Kazuyoshi Ogawa, Benlian Wang, and Etsuo Kokufuta* Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan Received February 13, 2001. In Final Form: May 8, 2001 This letter reports the preparation and the performance of a novel microgel system in which an enzyme (urease) is physically entrapped in the lightly cross-linked polymer network having thermosensitive and ionic moieties. The preparation can easily be performed by aqueous redox polymerization in the presence of both surfactant and enzyme. The microgel obtained undergoes a rapid change in diameter from 215 to 155 nm as an immobilized enzyme reaction sets in. When the gel particles are loaded into the pores of a cellulose membrane, we can enzymatically open the gate pore among the particles to allow the passage of a liquid through the membrane. These results strongly suggest that the enzyme immobilization method based on thermosensitive polyelectrolyte microgels has a large spectrum of technical applications.
Since the original use by Bernfeld and Wan1 of an acrylamide gel for the immobilization of enzymes through physical entrapment, a number of researchers have reported the preparation, characterization, and application of gel-entrapped enzymes. The technology for entrapping enzymes within gels is believed to be well established already. In addition, the nature of gel-entrapped enzymes can now be better understood. Our recent interest lies in the development of new concepts2 in the field of gelentrapped enzymes. Microgel particles consisting of cross-linked thermosensitive polymers, as well as their bulk gels, undergo an abrupt volume change in response to small changes of ambient temperature.3-7 Because the time taken for a gel to swell or shrink is proportional to the square of a linear dimension of the gel,8,9 the volume change for microgels is faster than that for the bulk gels with the same chemical structure. This has provided us a guiding principle in applications of thermosensitive microgels in both science and technology.10-13 At present, it is of importance to synthesize the microgels that swell or shrink in response to various kinds of stimuli. The attentions stated above have prompted us to study a novel microgel that undergoes a rapid size change in response to an immobilized enzyme reaction. The gel of this sort may be regarded as a “biochemo-mechanical system”14 capable of converting biochemical energy into mechanical work through the swelling and shrinking of the gel; therefore, an immobilized enzyme of this type is * To whom correspondence should be addressed. (1) Bernfeld, P.; Wan, J. Science 1963, 142, 678. (2) For examples, see: (a) Maugh, T. H. Science 1984, 223, 474. (b) Kokufuta, E. Prog. Polym. Sci. 1992, 16, 647. (3) Schild, H. G. Prog. Polym. Sci. 1992, 17, 163. From this review, we may learn a history of NIPA microgel studies. (4) Pelton, R. H.; Chibante, P. Colloids Surf. 1986, 20, 247. (5) Hirose, Y.; Amiya, T.; Hirokawa, Y.; Tanaka, T. Macromolecules 1987, 20, 1342. (6) Pelton, R. H.; Pelton, H. M.; Morphesis, A.; Rowell, R. L. Langmuir 1989, 5, 816. (7) Wu, C.; Zhou, S. Macromolecules 1997, 30, 574. (8) Tanaka, T.; Fillmore, D. J. J. Chem. Phys. 1979, 70, 1213. (9) Matsuo, E. S.; Tanaka, T. J. Chem. Phys. 1988, 89, 1695. (10) Kawaguchi, H.; Fujimoto, K.; Mizuhara, Y. Colloid Polym. Sci. 1992, 270, 55. (11) Matsumura, Y.; Hyodo, A.; Nose, T.; Ito, S.; Hirano, T.; Ohashi, S. J. Biomater. Sci., Polym. Ed. 1996, 7, 795. (12) Weissman, J. M.; Sunkara, H. B.; Tse, A. S.; Asher, R. A. Science 1996, 274, 959. (13) Suzuki, H.; Wang, B.; Yoshida, R.; Kokufuta, E. Langmuir 1999, 15, 4283.
distinguishable from those of the more usual type from the perspective of its utilization as a biocatalyst in a chemical conversion. Urease catalyzes the hydrolysis of urea into ammonia and carbon dioxide, which immediately react with water molecules to raise the pH of reaction system.15 When urease is immobilized in a polymer network having both thermosensitive and ionic moieties, this enzymatic pH change will alter the state of ionization of the network and thereby the volume-phase transition temperature of the gel.16 We used this principle and immobilized urease in the microgel network composed of N-isopropylacrylamide (NIPA, thermosensitive monomer) and N-vinylimidazole (VI, pH-sensitive ionic monomer). The enzyme reaction occurring within the gel phase eliminates the cationic charges of the network via the deprotonation of imidazole ions, so that the network will collapse to shrink the gel (see Figure 1). To prepare microgel particles with the immobilized urease, aqueous redox polymerization was performed in the presence of both enzyme and surfactant. Trimethylstearylammonium chloride (TMSAC) was used as the surfactant. Also used as a reactor was a 1 L conical flask equipped with a magnetic stirrer, a thermometer, a nitrogen gas inlet tube, and a reflux condenser with a gas outlet tube at the top. A pregel solution, which was prepared with thrice distilled oxygen-free water, has a composition (in w/v %) as follows: 1.11, NIPA; 0.103, VI; 0.015, N,N′-methylenebisacrylamide (cross-linker); 0.359, TMSAC; 0.026, urease (Jack bean source). The pregel solution (390 mL) was placed in the reactor and maintained at 45 °C with stirring (200 rpm). To remove oxygen well, nitrogen gas was continuously supplied above the surface of the pregel solution for 1 h before the reaction. The polymerization was initiated by adding 10 mL of aqueous O2-free solution of ammonium persulfate (0.8 w/w %), allowed to continue for 2 h, and terminated by blowing oxygen through a reactor. The resulting microgel suspension was allowed to stand overnight at 4 °C to precipitate (14) Kokufuta, E. Functional Immobilized Biocatalysts Prepared Using Stimulus-sensitive Polymer Gels. In The Polymeric Materials Encyclopedia - Synthesis, Properties and Applications; Salamone, J. C., Ed.; CRC Press: New York, 1996; Vol. 4, F-G, pp 2615-2621. (15) Reithel, F. J. Ureases. In The Enzymes, 3rd ed.; Boyer, P. D., Ed.; Academic Press: New York, London, 1971; Vol. 4, pp 1-21. (16) Kokufuta, E.; Zhang, Y.-Q.; Tanaka, T. J. Biomater. Sci., Polym. Ed. 1994, 6, 35.
10.1021/la0102354 CCC: $20.00 © 2001 American Chemical Society Published on Web 07/07/2001
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Figure 1. Schematic illustration for enzymatically induced volume collapse in a polyelectrolyte microgel system consisting of N-isopropylacrylamide, N-vinylimidazole, and immobilized urease. The enzyme reaction raises the pH of the gel phase, facilitating the deprotonation of imidazole ions even when the ambient pH is kept at a low level in the acidic range. This causes the collapse of the polymer network to shrink the gel, whereas the gel swells upon removal of the substrate from the outer medium.
Figure 2. Plots of normalized gel diameter (d/d0) against pH (a), temperature (b), and time (c). The normalization was performed with the equilibrium diameter (d0 ) 215 ( 3 nm) at 25 °C and at pH 4.0. The pH change at 25 °C as well as the temperature changes at pH 4.0 (open circles) and pH 6.5 (closed circles) are given by equilibrium diameters which were determined in different buffer solutions free from urea as the substrate: 1 mM acetate buffer (pH e 6.5) and 1 mM ammonium buffer (pH > 6.5). The timedependent changes of gel size initiated by addition of the substrate (10 mM urea) were studied in 1 mM acetate buffer (pH 4) at 25 °C using three different gel samples: closed circles, microgel particles with the immobilized active enzyme; open triangles, microgel particles with the enzyme which had been inactivated by heating the gel sample in 0.1 mM solution (pH 4) of acetic acid at 110 °C for 100 min; open circles, a cylindrical bulk gel (0.29 mm in diameter and 1 mm in length at pH 4) which was obtained from the same pregel solution containing the enzyme but not containing the surfactant. The time dependence of d/d0 for the microgel with the active enzyme is given by all the data obtained from seven measurements with different samples.
the surfactant, which was filtered out using a Gelman 5 µm filter. The suspension was then transferred to a dialyzing tube (Spectra/Por CE, MWCO 300 000) and dialyzed against a large volume of pure water. A combination of enzyme assay and total organic carbon analysis showed that the impurities (surfactants, free
enzymes, and unreacted monomers) remaining in the crude suspension were sufficiently eliminated. The purified suspension contained a total dry mass of 0.1 w/w % when evaporating it to dryness. About 1.6 w/w % of this dry mass corresponds to the overall amount of the immobilized enzyme. This value was estimated by mea-
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Figure 3. Enzymatically controlled permeation of liquid through a cellulose acetate syringe-filter whose pores are loaded with microgel particles with immobilized urease. +S denotes the substrate solution, i.e., 1 mM acetate buffer (pH 4) containing 10 mM urea, whereas -S is the substrate-free buffer. Pictures showing a swelling-shrinking change of microgel particles within the pore are synthesized with computer graphic software using a scanning electron micrograph of the filter membrane.
suring the concentration of proteins in the solution against which the microgel suspension had been dialyzed. The apparent Stokes diameter of microgel particles with the immobilized enzyme was determined by using a dynamic laser light scattering apparatus (Otsuka DLS7000) equipped with a 1.2 W argon ion laser (SpectraPhysics 2060-4S). First, we examined effects of pH and temperature on the particle size in the absence of urea (substrate) using acetate and ammonium buffers (1 mM each). As can be seen from Figure 2a,b, the gel shrinks with increasing pH and temperature. This indicates that our microgel possesses both amphipathic and electrolyte properties, which are due to the NIPA and VI moieties in the network, respectively. The time-dependent change in the gel diameter was studied in a substrate solution (10 mM urea, pH 4) with the two microgel samples, one of which had been heated so as to inactivate the enzyme immobilized. Also employed with the intention of studying the effect of gel size on shrinking kinetics was a cylindrical “bulk gel” obtained from the same pregel solution as used for the microgel preparation. From Figure 2c, it is clear that the microgel in which the enzyme was not inactivated shrinks in the presence of the substrate. It takes less than 10 min for the
microgel to reach a fully shrinking state (d/d0 ∼ 0.7) at a given temperature (25 °C), while the d/d0 for the bulk gel is 0.88 even at 1 h. This evidently demonstrates that our microgel particles undergo a rapid shrinking change due to the enzyme reaction. Then, the enzyme within the microgel serves as a size transformer in the presence of the substrate as a molecular stimulus. A membrane whose pores are loaded with the microgels can easily be prepared through the usual filtration method. We used a cellulose acetate syringe-filter (pore size ∼0.8 µm, Fuji film CALC80) fixed in a 25 mm plastic disk housing. To load the gel particles within the pores, the suspension was heated at 33 °C to shrink the gel (d ∼ 130 nm) and then pushed out from a 50 mL syringe to the filter attached to the top. The loading was stopped when it became difficult to press the plunger by hand. The filter with the loaded gel particles was away from the syringe and attached to a liquid flowmeter. The permeation of liquid through the filter membrane was studied at 25 °C using acetate buffer solutions (1 mM, pH 4) containing and free from 10 mM urea (see Figure 3). It was found that urea enhances the permeability as soon as it has soaked into the membrane. This enhanced permeability returned to the initial level when the substrate was swept
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with a stream of the urea-free solution. Such a permeation control can be repeated with a satisfactory reproducibility. No microgel was detected in the filtrate passed through the membrane within a lower detection limit (10-5 w/v %) of our light scattering measurements. These results strongly indicate that the microgel collapse is due only to the immobilized enzyme, because the free enzyme should be washed away during measurements. In conclusion, the present study demonstrates that the enzyme can be immobilized within the network of a microgel particle via a physical entrapping method; therefore, we may obtain a submicron-sized biochemomechanical gel that undergoes a rapid size change as the
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immobilized enzyme reaction sets in. Also demonstrated is the biochemical control of liquid permeation through the membrane whose pores were loaded with the biochemo-mechanical microgel particles. These results suggest that many of the stimulus-sensitive microgels are useful in the enzyme immobilization upon which unique applications of gel-entrapped enzymes would be developed. Acknowledgment. This work was supported in part by a Grant-in-Aid for Scientific Research to E.K. from the Ministry of Education, Japan (No. 08558092). LA0102354
