Facile Synthesis and Novel Application of Zirconia Catalyzed and

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Facile Synthesis and Novel Application of Zirconia Catalyzed and Templated by Lysozyme Yanjun Jiang, Dong Yang, Lei Zhang, Yan Jiang, Yufei Zhang, Jian Li, and Zhongyi Jiang* Key Laboratory for Green Chemical Technology of Ministry of Education, School of Chemical Engineering and Technology, Tianjin UniVersity, Tianjin 300072, People’s Republic of China

In this study, lysozyme, which is a cationic antibacterial enzyme, was, for the first time, demonstrated to catalyze the hydrolysis/condensation of the precursor potassium hexafluorozirconate (K2ZrF6) and template the formation of zirconia particles at room temperature. The resulting zirconia particles were characterized using scanning electron microscopy (SEM), energy-dispersive X-ray spectrometry (EDX), thermogravimetric analysis (TGA), Fourier transform infrared (FT-IR) spectroscopy, and X-ray photoelectron spectroscopy (XPS). The prepared zirconia particles had an amorphous structure and exhibited a different morphology and surface roughness from those prepared via the conventional sol-gel approach. The catalytic and templating function of the lysozyme that was involved in the synthesis of zirconia particles was discussed, and a hydrolysis mechanism of K2ZrF6 was tentatively proposed. In addition, the zirconia was utilized as the carrier for the immobilization of yeast alcohol dehydrogenase (YADH), which displayed enhanced thermal and pH stability, compared to free YADH. Introduction Nature possesses an extraordinary and unique ability to produce biomaterials with astonishing properties, using organic templates to control the growth of the inorganic phase at ambient pressure and temperature and at a neutral pH value.1-7 A lucubrated example in biomaterials is biosilica, which can be produced by diatoms, radiolarians, and sponges, with an exquisite structure that exceeds human engineering capabilities.8-10 It has been observed that some biomolecules have important roles (such as catalyst and template) in the formation of biosilica.11-14 Silaffins were the first type of proteins that involved the formation of biosilica isolated from the diatoms. It was hypothesized that the self-assembly structure of polycationic silaffins provided a template for the polycondensation of silicic acid.1,15 Silicatein, which was isolated from siliceous spicules of the marine sponge Tethya aurantia, could catalyze both the hydrolysis and polycondensation of silica precursor.16,17 A peptide with 19 amino acid residues called R5 (H2NSSKKSGSYSGSKGSKRRIL-COOH) has been successfully used to precipitate silica and encapsulate enzymes into the biosilica.18,19 Thereafter, inspired by the biomineralization, some research groups investigated the biomimetic synthesis of silica in the presence of polyamines including poly(allylamine), poly(L-lysine) and poly(L-histidine).20-23 Currently, investigation of the mechanism underlying the biological formation of inorganic materials has developed as appealing domain in materials science, and a wide variety of inorganic materials (titanium dioxide, titanium phosphates, gallium oxohydride, gallium oxide, germania, etc.) have been successfully synthesized.24-30 Lysozyme (MW 16 kDa, 147 amino acid residues, pI = 10.5) is one of the most prominent members in the class of cationic enzymes, which can hydrolyze the glycosyl groups. In vivo lysozyme can catalyze the hydrolysis of specific peptidoglycan linkages in the cell wall of Gram-positive bacteria. Compared to the aforementioned biomolecules involved in biomineralization, lysozyme not only comprises several biomineralization* To whom correspondence should be addressed. Tel.: 86-22-2789 2143. Fax: 86-22-2789 2143. E-mail: [email protected].

mediating groups, such as nucleophilic and hydrogen-bonding acceptor groups, but also has some distinct advantages, such as high antimicrobial activity, easy availability, and low cost.31 Therefore, it is naturally conjectured that lysozyme should be an ideal candidate to catalyze and template the synthesis of inorganic oxides. Until now, lysozyme has been used to induce the formation of amorphous titania from potassium hexafluorotitanate or titanium(IV) bis(ammonium lactato) dihydroxide (Ti-BALDH).32 However, very few related reports that involve the synthesis of other inorganic oxides in the presence of lysozyme can be found, and the relevant mechanism is not clear yet.32-34 In this study, lysozyme was first used to catalyze the hydrolysis of K2ZrF6 and template the formation of zirconia under mild conditions. Moreover, the yeast alcohol dehydrogenase (YADH) was encapsulated in this type of zirconia, and its catalytic activity was primarily investigated. Zirconia is one of the ideal candidates for use as an immobilization carrier, because of its intrinsic physicochemical properties, such as good chemical and physical stability, high mechanical strength, large toughness, and strong acid and alkali resistance.35 However, the conventional approaches often require a long reaction time (the liquid-phase deposition method), high temperature (thermal synthesis method), high pressure (hydrothermal synthesis method), or high cost (rapid solid-state metathesis reaction).36-38 Hopefully, the biomimetic route illuminated in this study will help circumvent the aforementioned problems by enabling the synthesis process under mild conditions and at low cost. Experimental Section Materials. Lysozyme (EC 3.2.1.17), yeast alcohol dehydrogenase (YADH, EC1.1.1.1), and NADH (grade I, 98%) were purchased from Sigma. Potassium hexafluorozirconate (K2ZrF6) was obtained from Tianjin Reagent Chemicals Co. Ltd., China, and used as the zirconium precursor. All other chemicals were analytical grade. Synthesis of Zirconia. One milliliter (1 mL) of a 20 mg/mL native lysozyme solution was added into 9 mL of a 0.01 M K2ZrF6 solution, and the reaction mixture was incubated for 10

10.1021/ie071018m CCC: $40.75 © 2008 American Chemical Society Published on Web 02/20/2008

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min at room temperature. The precipitate was collected by centrifugation, washed three times with deionized water, and then dried under vacuum for 3 h. In the control experiment, a 0.01 M zirconium precursor solution was directly incubated for 24 h in the absence of lysozyme, and thermally denatured lysozyme in boiling water for 1 h was also used for zirconia synthesis. For comparison, alkali-catalyzed synthesis of zirconia particles was accomplished by adding 1 mL of 28% (w/w) ammonium hydroxide (NH4OH) into 9 mL of a 0.01 M zirconium oxychloride solution, and other conditions are kept the same as those in lysozyme-mediated synthesis. Encapsulation of YADH in Zirconia. One milliliter (1 mL) of a 20 mg/mL native lysozyme solution and 1 mL of a 1 mg/ mL phosphate-buffered saline solution (pH 7.0) of YADH were added into 8 mL of a 0.01 M K2ZrF6 solution. The reaction mixture was incubated for 10 min at room temperature, and then the precipitate was collected by centrifugation. YADHcatalyzed hydrogenation of formaldehyde to methanol coupling with the oxidation of NADH to NAD+ was used to evaluate the catalytic activity of free and encapsulated YADH. The activity of YADH was determined spectrophotometrically by directly measuring the absorbance of NADH at 340 nm, as was done in our previous report.39 The activity assays were performed over a temperature range of 20-60 °C and a pH range of 4.0-9.0. Characterization of Zirconia. Scanning electron microscopy (ESEM) (Philips XL30) was used to observe the morphology of resulting zirconia particles. Energy-dispersive X-ray spectrometry (EDX) equipment that was attached to the SEM system was used to analyze the elements that comprise the particles. Thermogravimetric analysis (TGA) of the prepared zirconia particles was performed in a Perkin-Elmer TG/DTA thermogravimetric analyzer by heating to 800 °C at a rate of 10 °C/ min under the air atmosphere. The powder X-ray diffraction (XRD) pattern was obtained with a Philips X’Pertpro diffractometer, using Co KR radiation with an accelerating voltage of 40 kV and a current of 40 mA to determine the identity of the samples. The infrared (IR) spectrum was obtained using a Nicolet-560 Fourier transform infrared (FT-IR) spectrometer. Thirty-two scans were accumulated with a resolution of 4 cm-1 for each spectrum. X-ray photoelectron spectroscopy (XPS) was used to characterize the binding energies and bonding states of Zr atoms in a Perkin-Elmer PHI 1600 ESCA system with a monochromatic Mg KR source and a charge neutralizer. Results and Discussion Morphology and Structure of Zirconia Prepared in the Presence of Lysozyme. In the absence of lysozyme, no precipitate was observed after the K2ZrF6 solution was incubated for 2 h. When the lysozyme solution (pH 7.0) was added into the K2ZrF6 solution, a white precipitate formed within several minutes, which was indicative of the catalytic function of lysozyme in the formation of these zirconium-containing materials. Figure 1a shows a typical SEM image of prepared zirconium-containing materials that were catalyzed by lysozyme. These particles exhibit an irregular form, ∼1 µm in size. They are much larger than those formed in the presence of cationic proteins secreted by the plant pathogenic fungus Fusarium oxysporum, which are quasi-spherical particles ∼7 nm in diameter.36 It is deduced that the high concentration of lysozyme can lead to the rapid hydrolysis of ZrF62-, and then the conglomeration of resultant zirconia nanoparticles occurs. The corresponding EDX spectrum (Figure 2), in conjunction with the SEM analysis, reveals that the atomic contents of zirconium,

Figure 1. Scanning electron microscopy (SEM) micrographs of zirconia particles prepared in the presence of (a) native lysozyme, (b) denatured lysozyme, and (c) ammonium hydroxide.

Figure 2. Representative energy-dispersive X-ray spectroscopy (EDX) spectrum of zirconia particles prepared in the presence of native lysozyme.

oxygen, and carbon elements in these particles are 4.1%, 21.0%, and 74.9%, respectively, which indicates that the particles are composed of zirconia and some organic compounds. The TGA curve of this composite is shown in Figure 3. There is an apparent weight loss (ca. 49.4%, by mass) from 250 °C to

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Figure 3. Thermogravimetric analysis (TGA) profile of zirconia particles prepared in the presence of native lysozyme.

Figure 4. Fourier transform infrared (FT-IR) spectra of (a) K2ZrF6 powder and (b) zirconia particles prepared in the presence of native lysozyme.

600 °C, which can be assigned to the combustion of some organic compounds. Since lysozyme is the only organic molecule in the reaction system, it can be deduced that the resulting zirconium-containing material is a composite of zirconia with lysozyme. This result suggests that, apart from the catalytic function, lysozyme may have some other roles (for example, as template) in the preparation of zirconia. To further investigate the composition and structure of these zirconia particles, XRD, FT-IR, and XPS analyses were conducted. Their XRD spectrum (data not shown) does not show any peak, which suggests that the zirconia may exist with amorphous strcture in these particles. However, the amorphous zirconium species crystallized to tetragonal zirconia, accompanying the complete lysozyme pyrolysis after calcination at 600 °C. Figure 4 presents the FT-IR spectra of pure K2ZrF6(Figure 4a) and as-prepared zirconia (Figure 4b). The FT-IR spectrum of zirconia particles shows the presence of a new resonance at 660 cm-1, which is absent in the spectrum of pure K2ZrF6. This band can be attributed to absorption of the ZrO-Zr, which indicates the formation of ZrO2 in the resulting materials. Meanwhile, two absorption bands centered at 1655 and 1544 cm-1 can be observed in Figure 4b, which are clearly missing in the FT-IR spectrum of pure K2ZrF6. These bands can be attributed to the amide I and II bands, respectively, which further indicated the presence of lysozyme in the resulting materials. Note that the characteristic peak of ZrF62- at 462 cm-1 still is present in Figure 4b, which indicates that there is some residual ZrF62- in the zirconia. XPS was conducted to investigate the surface elemental composition of the zirconia particles. As shown in Figure 5, in the XPS elemental survey spectrum of the zirconia sample, there is carbon (64.8 at. %), oxygen (19.5 at. %), zirconium (3.6 at. %), nitrogen (8.9 at. %), and F (3.2 at. %). The atomic ratio of Zr/F is ∼1:0.89, which indicates that K2ZrF6 may not

Figure 5. XPS survey spectrum of zirconia particles prepared in the presence of native lysozyme.

hydrolyze completely. The carbon, nitrogen, and some elemental oxygen should come from lysozyme that is occluded in the zirconia sample. To further investigate the states of these atoms, high-resolution XPS spectra of Zr, O, C, N, and F were recorded and are shown in Figure 6. From Figure 6a, one can find that the Zr3d range for the deposit is composed of two peaks, with binding energies of 182.8 and 185.0 eV, which can be assigned to Zr3d5/2 and Zr3d3/2, respectively. Note that the binding energy of Zr3d5/2 is higher than that in Zr metal (180.0 eV) and ZrOx (0 < x < 2, 181.4 eV), but similar to that in ZrO2 (182.9 eV).40 Therefore, the Zr atoms should present as the +4 covalent state, similar to that in ZrO2. Figure 6b illustrates the high-resolution scanning for O1s, which only contains a peak at 530.8 eV. It is well-known that the binding energy of O1s in the state of Zr-O-Zr is usually ∼531 eV in pure zirconia samples, and that in the state of -OH is more than 531 eV.40-42 This hints that ZrO2 is the main component in the zirconia sample, and the free Zr-OH groups are few. Figures 6c and 6d are the C1s and F1s XPS curves of the zirconia sample, respectively. The C1s XPS spectrum contains three peakssat 284.9, 286.7, and 288.0 eVsthat can be ascribed to the C-H, C-O, and CdO bonds, respectively, which result from lysozyme molecules in the sample. According to the previous report,43 the first peak at 284.9 eV also includes some devotion of adventitious hydrocarbon from the XPS instrument itself. There is only a peak at 399.8 eV in Figure 6d, which can be attributed to the N-H bonds of lysozyme. Figure 6e illustrates the highresolution XPS spectrum of F1s, which contains a single peak at 684.3 eV. This peak can be assigned to the F-Zr bonds of ZrF62-, further confirming that K2ZrF6 does not hydrolyze completely. It is inferred that the rapid hydrolysis velocity may be the main reason causing this fact. Function of Lysozyme in the Synthesis of Zirconia. It is reported that ZrF62- usually hydrolyzes according to the following reaction equilibrium:44

[ZrF6]2- + hH2O S [Zr(OH)hF6-h]2- + hHF If it is appropriate for our experiment, the pH value of the reaction solution will decrease along with the ZrF62- hydrolysis, because of the production of HF. To investigate the function of lysozyme in the formation of zirconia particles, the pH change of aqueous K2ZrF6 solution was monitored over time with and without lysozyme. As shown in Figure 7, the K2ZrF6 solution without lysozyme shows no appreciable change in pH value within 120 min. While in the presence of lysozyme, the pH value of the reaction mixture decreased from 4.64 to 3.54 within 120 min. This result suggests that lysozyme surely has the role of catalyst in the hydrolysis of ZrF62-. When the thermally

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Figure 7. pH value of aqueous K2ZrF6 solution changing with time (a) in the absence of lysozyme and (b) in the presence of native lysozyme.

Figure 6. High-resolution (a) Zr3d, (b) O1s, (c) C1s, (d) N1s, and (e) F1s XPS spectra of zirconia particles prepared in the presence of native lysozyme.

denatured lysozyme was added to the K2ZrF6 solution, the white zirconia precipitate also can form within several minutes. Furthermore, these particles (Figure 1b) exhibit smaller size than

those formed in the presence of native lysozyme, and the similar pH change of the reaction solution also can be observed. It is well-known that the primary catalytic activity of lysozyme is lytic action for hydrolyzing the peptidoglycan of bacterial cell walls. However, our result indicates that the catalytic activity of lysozyme in zirconia synthesis may come from some groups on its side chain, and it may be independent of the active site for lytic action and the three-dimensional integrity structure of lysozyme.32 When the lysozyme is thermally denatured, more catalytically active groups are exposed, which results in the formation of small zirconia particles. As a sharp contrast, the specific activity of silicatein was heavily dependent on the integrity of the protein’s native structure.24-26,45 Based on these results, a mechanism was proposed to tentatively elucidate the hydrolysis of K2ZrF6 catalyzed by lysozyme. Lysozyme includes not only several amino acid residues with -OH or -SH groups (such as serine, cysteamine, and lysine), but also many amino acid residues with -NH2 or -NH- groups (such as arginine and histidine). It is reported that these two types of amino acid residues are requisite functionalities of many biomineralization-mediated biomacromolecules. The nucleophilic attacking group, either -OH or -SH, has been proven to be essential for the hydrolysis of the precursor, whereas the hydrogen-bonding acceptor group, such as -NH2 or -NH-, would facilitate the hydrolysis reaction.46 As described in Scheme 1, it is assumed that hydrogen bonding between the hydroxymethyl and guanidyl group side chains of serine and arginine increases the nucleophilicity of the serine O atom, and, thus, facilitates the initial hydrolysis of ZrF62-. Subsequently, the forming Zr(OH)F52- complex would undergo an additional catalyst-mediated hydrolysis step with an equivalent of water to yield Zr(OH)2F42-. Ultimately, the condensation among the resulting hydroxyl groups led to the formation of zirconia. On the other hand, it is observed that lysozyme molecules are occluded in the zirconia particles, which indicates that lysozyme may serve other roles besides the catalytic function. To investigate its actual function, the zirconia particles also were prepared via the conventional sol-gel process, using alkali as a catalyst. As shown in Figure 1c, the alkali-catalyzed zirconia shows a much rougher surface and larger size, compared to the lysozyme-catalyzed sample. This phenomenon may be directly related to the nucleation mechanism concerning the growth of zirconia. There are apparently numerous nucleation sites, such as clusters of contiguous -OH side chains in lysozyme, which could feasibly condense the newly hydrolytic product from ZrF62-. By offering a favorable surface on which nuclei of zirconia can form, lysozyme may act initially as a template and

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Scheme 1. Proposed Hydrolysis Process of K2ZrF6 in the Presence of Lysozyme

Figure 8. Relative activities of free and immobilized YADH in zirconia particles (a) at different temperatures and (b) different pH values.

stabilize the small nuclei of zirconia to construct the surface with lower roughness.24 However, dynamic light scattering revealed that the average monomeric or oligomeric diameter of lysozyme dissolved in 50 mmol/L Tris-HCl buffer solution was only ∼3.5 nm, and the cluster diameter was ∼23.7 nm. This indicates that the original lysozyme templates were smaller than the resulting zirconia particles, which may be explained by the “dynamical template” concept, which is a synergistic growth mechanism that allows the production of superstructures that greatly exceed the size of the original template assemblies.47 YADH Encapsulation Using Lysozyme-Catalyzed Zirconia as the Matrix. Among the wide diversity of applications of encapsulation technologies, the immobilization of biological systems that include enzymes and cells in an inorganic matrix may be the most fascinating. Compared to the conventional acid/ base induced encapsulation technique, biomolecule-induced encapsulation shows some remarkable advantages, including the following: (i) it effectively protects the enzymes and cells from the attack of acid, alkali, or heat; (ii) it sufficiently preserves the native structures and activities; and (iii) it inherently ensures the mild conditions and low cost. To explore the potential application of biogenic zirconia, the yeast alcohol dehydrogenase (YADH, EC 1.1.1.1) from Saccharomyces cereVisiae, which is an important enzyme that can catalyze the conversion of formaldehyde to methanol with NADH as a co-enzyme,38 was immobilized in the lysozyme-

catalyzed zirconia. The enzyme activity measurements confirmed that 35% ((5.0) YADH was encapsulated in the zirconia matrix. The activities of encapsulated YADH at different temperatures and pH values are shown in Figure 8. From Figure 8a, one can observe that encapsulated YADH retains more than 60% of its maximum activity, even at 60 °C. Moreover, compared to free YADH, all the encapsulated YADH compounds show enhanced thermal stability, which can be attributed to the physical cage confinement of the matrix.48,49 The pH durability of YADH can be also significantly improved by entrapping in a zirconia matrix (see Figure 8b). At pH 4.0 and pH 9.0, more than 90% and 60% of its maximum activity are retained, respectively. To explain this phenomenon, it is supposed that the cages formed by the zirconia matrix offer a nature-like benign microenvironment for YADH molecules besides the cage confinement effect. The gap between the inner surface of the zirconia and the outer surface of YADH molecules is full of water molecules, which could sufficiently shunt the attack of acid or alkaline that surrounds the enzyme.49 Conclusions A novel and facile bio-inspired strategy for preparing zirconia under ambient conditions was successfully developed in this study. Lysozyme was proven to be an efficient catalyst for the hydrolysis of the zirconium precursor K2ZrF6 at room temperature. The proposed mechanism suggested that lysozyme hydrolyzed K2ZrF6 through its surface amino acid residues, rather than via the active sites for lytic action, which was quite different from that for silicatein. Meanwhile, lysozyme had a role as a template in the formation of zirconia, and the hydroxyl groups on the surface of lysozyme may direct the nucleation and growth of zirconia. In addition, yeast alcohol dehydrogenase (YADH) that was immobilized in this biogenic zirconia exhibited enhanced thermal and pH stability. Therefore, it is believed that this study may open a new route to design and

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produce numerous metal oxides for biological and technological applications. Acknowledgment Financial support from the National Natural Science Foundation of China (Grant No. 20576096), the Cross-Century Talent Raising Program of Ministry of Education of China, the program for Changjiang Scholars, Innovative Research Team in University (PCSIRT) and financial support from the Program of Introducing Talents of Discipline to Universities (No. B06006) are greatly appreciated. Literature Cited (1) Kro¨ger, N.; Deutzmann, R.; Sumper, M. Polycationic Peptides from Diatom Biosilica that Direct Silica Nanosphere Formation. Science 1999, 286, 1129. (2) Sundar, V. C.; Yablon, A. D.; Grazul, J. L.; Ilan, M.; Aizenberg, J. Fibre-optical Features of a Glass Sponge. Nature 2003, 424, 899. (3) Politi, Y.; Arad, T.; Klein, E.; Weiner, S.; Addadi, L. Sea Urchin Spine Calcite Forms via a Transient Amorphous Calcium Carbonate Phase. Science 2004, 306, 1161. (4) Aizenberg, J.; Sundar, V. C.; Yablon, A. D.; Weaver, J. C.; Chen, G. Biological Glass Fibers: Correlation between Optical and Structural Properties. Proc. Natl. Acad. Sci., U.S.A. 2004, 101, 3358. (5) Li, M.; Co¨lfen, H.; Mann, S. Morphological Control of BaSO4 Microstructures by Double Hydrophilic Block Copolymer Mixtures. J. Mater. Chem. 2004, 14, 2269. (6) Aizenberg, J.; Weaver, J. C.; Thanawala, M. S.; Sundar, V. C.; Morse, D. E.; Fratzl, P. Skeleton of Euplectella sp.: Structural Hierarchy from the Nanoscale to the Macroscale. Science 2005, 309, 275. (7) Mann, S.; Shenton, W.; Li, M.; Connolly, S.; Fitzmaurice, D. Biologically Programmed Nanoparticle Assembly. AdV. Mater. 2000, 12, 147. (8) Noll, F.; Sumper, M.; Hampp, N. Nanostructure of Diatom Silica Surfaces and of Biomimetic Analogues. Nano Lett. 2002, 2, 91. (9) Perry, C. C.; Keeling-Tucker, T. Biosilification: The Role of the Organic Matrix in Structure Control. J. Biol. Inorg. Chem. 2000, 5, 537. (10) Sumper, M.; Lehmann, G. Silica Pattern Formation in Diatoms: Species-Specific Polyamine Biosynthesis. ChemBioChem 2006, 7, 1419. (11) Li, M.; Schnablegger, H.; Mann, S. Coupled Synthesis and Selfassembly of Nanoparticles to Give Structures with Controlled Organization. Nature 1999, 402, 393. (12) Cha, J. N.; Stucky, G. D.; Morse, D. E.; Deming, T. J. Biomimetic Synthesis of Ordered Silica Structures Mediated by Block Copolypeptides. Nature 2000, 403, 289. (13) Sumper, M. Biomimetic Patterning of Silica by Long-chain Polyamines. Angew. Chem., Int. Ed. 2004, 43, 2251. (14) Tomczak, M. M.; Glawe, D. D.; Drummy, L. F.; Lawrence, C. G.; Stone, M. O.; Perry, D. J.; Pochan, C. C.; Deming, D. J.; Naik, R. R. Polypeptide-templated Synthesis of Hexagonal Silica Platelets. J. Am. Chem. Soc. 2005, 127, 12577. (15) Kro¨ger, N.; Lorenz, S.; Brunner, E.; Sumper, M. Self-assembly of Highly Phosphorylated Silaffins and Their Function in Biosilica Morphogenesis. Science 2002, 298, 584. (16) Cha, J. N.; Shimizu, K.; Zhou, Y.; Christiansen, S. C.; Chmelka, B. F.; Stucky, G. D.; Morse, D. E. Silicatein Filaments and Subunits from a Marine Sponge Direct the Polymerization of Silica and Silicones in vitro. Proc. Natl. Acad. Sci., U.S.A. 1999, 96, 361. (17) Zhou, Y.; Shimizu, K.; Cha, J. N.; Stucky, G. D.; Morse, D. E. Efficient. Catalysis of Polysiloxane Synthesis by Silicatein a Requires Specific Hydroxy and Imidazole Functionalities. Angew. Chem., Int. Ed. 1999, 38, 780. (18) Rodriguez, F.; Glawe, D. D.; Naik, R. R.; Hallinan, K. P.; Stone, M. O. Study of the Chemical and Physical Influences upon in vitro Peptidemediated Silica Formation. Biomacromolecules 2004, 5, 261. (19) Luckarift, H. R.; Spain, J. C.; Naik, R. R.; Stone, M. O. Enzyme Immobilization in a Biomimetic Silica Support. Nat. Biotechnol. 2004, 22, 211. (20) Jan, J. S.; Shantz, D. F. Biomimetic Silica Formation: Effect of Block Copolypeptide Chemistry and Solution Conditions on Silica Nanostructure. AdV. Mater. 2007, 19, 2951. (21) Brunner, E.; Lutz, K.; Sumper, M. Biomimetic Synthesis of Silica Nanospheres Depends on the Aggregation and Phase Separation of Polyamines in Aqueous Solution. Phys. Chem. Chem. Phys. 2004, 6, 854.

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ReceiVed for reView July 26, 2007 ReVised manuscript receiVed November 13, 2007 Accepted January 1, 2008 IE071018M