Immobilization of Laccase on Nanoporous Gold: Comparative Studies

Jan 21, 2009 - The present results indicated that the physical adsorption strategy was the best one for laccase immobilization on NPG. This was becaus...
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J. Phys. Chem. C 2009, 113, 2521–2525

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Immobilization of Laccase on Nanoporous Gold: Comparative Studies on the Immobilization Strategies and the Particle Size Effects Huajun Qiu,† Caixia Xu,† Xirong Huang,*,† Yi Ding,† Yinbo Qu,‡ and Peiji Gao‡ Key Laboratory of Colloid and Interface Chemistry of the Ministry of Education of China, Shandong UniVersity, Jinan 250100, P. R. China, and State Key Laboratory of Microbial Technology of China, Shandong UniVersity, Jinan, China ReceiVed: October 12, 2008; ReVised Manuscript ReceiVed: December 7, 2008

Nanoporous gold (NPG), prepared simply by dealloying Ag from Au/Ag alloy, was used in the present study as a carrier for laccase immobilization. Three immobilization strategies, i.e., physical adsorption, electrostatic attraction, and covalent coupling, were used to immobilize laccase on NPG. A detailed comparison among the three strategies was made in light of the loading, the specific activity, and the leakage of laccase. The present results indicated that the physical adsorption strategy was the best one for laccase immobilization on NPG. This was because of the potential covalent linkage between the nanoscale gold surface and the amino groups of the residue amino acids of laccase. The effects of the particle size of NPG on laccase loading and enzyme kinetics were also investigated. When the particle size of NPG got smaller, more laccase could access the inner pore and be immobilized. The kinetic study showed that the crushed NPG not only enhanced mass transfer of the substrate and its oxidation product but also favored the exposure of the active sites of the immobilized laccase to the substrate, i.e., the crushing facilitated the enhancement of the catalytic efficiency of laccase. Introduction Nanoporous gold (NPG) is a new kind of nanomaterial and has been the subject of much interest recently.1 NPG has the following properties: (1) it is a bulky material with a nanoscale microstructure, and therefore it has a large specific surface area and can be easily employed and recovered; (2) the preparation methodology is simple and reproducible; (3) the pore size is tunable in a wide range from a few nanometers to several microns, which facilitates a study of pore size-dependent properties; (4) prepared in concentrated nitric acid, its nanostructured surfaces are very clean and can be easily functionalized. These characteristics make this material an attractive candidate for a wide range of applications, e.g., as a catalyst for CO and glucose oxidation,2-4 methanol electro-oxidation,5 and reduction of oxygen and hydrogen peroxide.6 In addition, NPG is chemically and mechanically stable, conductive, and biocompatible. All these make NPG an attractive material as a support for the immobilization of enzymes7,8 and the construction of biosensors and bioreactors. As far as enzyme immobilization is concerned, there are three general strategies, i.e., physical adsorption, covalent coupling, and electrostatic attraction. Each strategy has its advantages and disadvantages. Which strategy is used usually depends on the properties of the carrier and enzyme of interest, the cost, and complexity of the procedure, etc.9 The use of porous materials as the carrier for enzyme immobilization has been widely investigated. A large number of porous materials such as polymer,10 inorganic sol-gel,11,12 and porous glass13 have been tried for this purpose. In recent years, porous silica has attracted more attention because of not * Corresponding author. Tel.: +86 531 88365433. Fax: +86 531 88365433. E-mail: [email protected]. † Key Laboratory of Colloid and Interface Chemistry of the Ministry of Education of China. ‡ State Key Laboratory of Microbial Technology of China.

only its large surface area but also its tunable pore sizes and good stability.14-17 Even so, NPG still has some advantages over porous silica. In addition to the easy preparation, recovery, and recycle of NPG, the most obvious difference is the excellent electric conductivity of NPG, which is very important for the construction of enzyme electrodes. Laccase is a copper-containing oxidase. It is able to catalyze a one-electron oxidation of various aromatic substrates with concomitant reduction of O2 to water.18-20 This free radical mechanism suggests that laccase has wide potential applications. In this paper, NPG was used as a carrier for laccase immobilization. The three enzyme immobilization strategies mentioned above were tried and compared to obtain a good result. The effect of the particle sizes of NPG on the properties of the immobilized laccase was also investigated. Experimental Section Chemicals. Laccase from Trametes Versicolor and its substrate 2,6-dimethoxyphenol (DMP) were purchased from Sigma. Au/Ag alloy (50:50, wt %) sheets with thickness of 25 µm were purchased from Changshu Noble Metal Co. Coomassie brilliant blue G-250 was a product of Sanland-chem International Inc. R-Lipoic acid, N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were obtained from Shanghai Yanchang Co. Other chemicals used were of analytical grade. A 50 mM phosphate-citric acid buffer solution and triply distilled water were used throughout the experiments. Preparation and Characterization of NPG. Dealloying of Au/Ag alloy was carried out in concentrated nitric acid for a certain period of time, which was similar to that reported in previous work.2,3,5,8 The microstructure of NPG was characterized with a JEOL JSM-6700F field emission scanning electron microscope, which was equipped with an Oxford INCA x-sight energy-dispersive X-ray spectrometer (EDS) for compositional analysis. The surface area of NPG was measured with Quad-

10.1021/jp8090304 CCC: $40.75  2009 American Chemical Society Published on Web 01/21/2009

2522 J. Phys. Chem. C, Vol. 113, No. 6, 2009 rasorb SI-MP (Quantachrome Instrument) using the BET method. Prior to N2 adsorption, all samples were degassed at 350 °C for 8 h. Immobilization of Laccase on NPG. Strategy 1 (covalent coupling): NPG (5 mg in fragments with a size of ∼0.5 mm) was incubated with 1 mL of R-lipoic acid solution (1.0 mM) for 24 h, and then it was rinsed five times with buffer solution (pH 6.8). The R-lipoic acid-modified NPG was reacted with a mixture of 75 mM EDC and 15 mM NHS solution for 10 h and rinsed again. After that, the NHS-terminated NPG was incubated at 4 °C with 200 µL of 7 mg mL-1 laccase solution (pH 6.8) for 24 h.21,22 Strategy 2 (electrostatic attraction): NPG (same as in strategy 1) was incubated with 1 mL of R-lipoic acid (1.0 mM) or methylene blue (1.0 mM) for 24 h, and then the lipoic acid or methylene blue-terminated NPG was incubated at 4 °C with 200 µL 7 mg mL-1 laccase solution for 24 h. Strategy 3 (physical adsorption): the bare NPG (same as in strategy 1) was incubated at 4 °C with 200 µL of 7 mg mL-1 laccase solution (pH 6.8) for 24 h. For every method, after 24 h incubation with laccase, the laccase-loaded NPG was rinsed five times to remove unbound laccase on the outer surface. The amount of the enzyme immobilized on NPG was calculated based on the difference between the amount of the protein added and that recovered in the supernatant and washing buffer. The protein concentration was determined by the Bradford method using bovine albumin as a standard. Unless otherwise specified, the experiment was performed at a room temperature of ∼25 °C. Activity of the Immobilized Laccase. At 30 °C, under stirring, the immobilized laccase prepared by one of the three strategies (on 5 mg NPG in fragment) was mixed with a mixture of 1 mL of buffer solution (pH 4.4, the optimum pH of the laccase) and 100 µL of 10 mM DMP. A plot of absorbance at 470 nm versus time was recorded on a Shimadzu UV-2550 spectrophotometer. The molar extinction coefficient of the oxidation product of DMP at 470 nm was 49.6 mM-1 cm-1. One unit of activity was defined as the amount of laccase required to have 1 µmol of DMP oxidized in 1 min. Leaching Test. The immobilized laccase (on 5 mg NPG in fragment) was mixed with 1 mL of buffer solution (pH 4.4) and incubated at 4 °C for 1 h, and then the NPG was removed. The remainder was mixed with 100 µL of 10 mM DMP at 30 °C, followed by recording the absorbance change at 470 nm with time on the spectrophotometer. This absorbance change was used to characterize the leached amount of laccase. Particle Size Effect of NPG. NPGs of three different particle sizes were used: sample 1 was a bulky piece (3 × 4 cm uncrushed plate); sample 2 was in small fragments with a size of ∼0.5 mm (obtained by crushing sample 1 with a pipet tip); sample 3 was fine grains of NPG at micron scale which was prepared by first crushing bulky NPG into fragments and then ultrasonicating them into fine grains. Enzyme Kinetics. The initial reaction velocities of the laccase catalyzed oxidation of DMP were first measured at different concentrations of DMP (the O2 in the reaction system was assumed to be saturated). Then a Lineweaver-Burk plot was made to obtain the apparent Michaelis-Menten constant of DMP (Km) and the apparent maximum velocity (Vmax). Vmax was proportional to the enzyme concentration, [E], as given in the following equation: Vmax ) kcat[E], where kcat was an apparent rate constant having a unit of reciprocal time (s-1). This kcat encompassed the chemical transformation events leading to product formation from the ternary enzyme conformation.23

Qiu et al. TABLE 1: The Amount of Laccase Loaded on NPG and the Specific Activity of the Immobilized Enzymea amount of laccase immobilized/ specific activity immobilization strategies (mg g-1) (U/mg of protein) covalent coupling electrostatic attraction physical adsorption a

16.0 8.2 15.5

0.83 0.8 0.81

Each data was an average of three replicate determinations.

Figure 1. SEM images of NPG with (A) and without (B) laccase.

Results and Discussion Selection of the Pore Size of NPG as a Carrier for Laccase Immobilization. The pore size of NPG was controllable by varying the etching time or initial alloy composition or by employing thermal annealing after dealloying. This feature facilitated our investigation on the effect of the pore sizes of NPG on the properties of the immobilized laccase. Our detailed study in this respect was reported elsewhere8 (using physical adsorption method). Our previous results demonstrated that NPG with a pore size of 40-50 nm was a good carrier for laccase immobilization because more laccase could be loaded on it while retaining good specific activity and stability. In present work, NPG with a pore size of 40-50 nm, obtained by dealloying Au/Ag alloy in concentrated HNO3 for ∼17 h, was thus chosen and used in the following experiments. Ag atoms in this sample were almost completely removed according to the EDS result. The Amount of Laccase Immobilized. Three commonly used strategies were tried. They were covalent coupling, electrostatic attraction, and physical adsorption. As shown in Table 1, the physical adsorption and the covalent coupling strategies resulted in an almost equal amount of laccase immobilized. The least amount of laccase was immobilized while using the electrostatic attraction strategy. As far as the physical adsorption strategy was concerned, it is well accepted that the interaction of nanoscale gold with NH2 was as strong as that with commonly used SH.24 The laccase from Trametes Versicolor has eight lysine residues. At least four lysine residues are on or near the surface of laccase (data from http:// www.ncbi.nlm.nih.gov/). On the concave surface of NPG, these surface lysine residues might contribute to the immobilization of laccase. In other words, some chemical adsorption occurred during the so-called physical adsorption process. Moreover, the amount of the protein in the leaching solution was below the detection limit of the Bradford method. So it could be concluded that the adsorption was a monolayer. Figure 1A was the SEM image of the laccase-loaded NPG using physical adsorption strategy. For comparison, the bare NPG was presented in Figure 1B. Because of laccase coverage (laccase was much less electron dense than the substrate gold), Figure 1A was relatively darker and more blurred than Figure 1B. For the covalent coupling strategy, its rationale was as follows (see Figure 2): First,

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Figure 2. Schematic illustrations of the immobilization of laccase on NPG and the molecular structure of methylene blue.

R-lipoic acid self-assembled monolayer was formed through Au-S bond; then the carboxylate groups were activated by NHS via esterification in the presence of the water-soluble EDC; finally, the terminal NHS groups were replaced by the amino groups on laccase surface, thereby resulting in a laccase monolayer on the surface of NPG. This explained why the physical adsorption and the covalent coupling strategies had almost the same amount of laccse immobilized on NPG. The size of laccase was about 6.5 nm × 5.5 nm × 4.5 nm.25 Its largest footprint on a surface was ca. 35.8 nm2. The surface area of NPG used in the experiments was ca. 14 m2 g-1 obtained through the N2 adsorption-desorption (BET) method. If laccases arranged themselves in an ideal monolayer, 1 g of NPG could load ca. 44 mg of laccase (the molecular weight of laccase was ca. 68 000 Da). This value was larger than the experimental value (∼16 mg g-1), but it was quite reasonable since the calculation assumed that there were no interactions between adsorption sites, no blockage, etc. It also indicated that not all the sites N2 adsorbed were suitable for laccase immobilization, i.e., the surface of NPG was not fully occupied by the enzyme. For the electrostatic attraction strategy, the surface charges of a carrier should be opposite to the net charges of an enzyme, which depended on the isoelectric point of the enzyme and the buffer pH used. The laccase used in the present work had an isoelectric point of 3.525 with the optimum pH at 4.4.8 It would be negatively charged when the buffer pH was higher than 3.5. So the lipoic acid or mercaptoacetic acid self-assembled monolayer on the NPG surface was not suitable for laccase immobilization by electrostatic attraction strategy because the COOH-functionalized surface was also negatively charged at a pH value higher than 3.5. As an alternative, a methylene blue (a sulfur-containing compound, its molecular structure was shown in Figure 2) self-assembled monolayer was chosen to make the surface of NPG positively charged over the pH range studied. Then, negatively charged laccase was obtained by adjusting the pH value of its buffer higher than 3.5. The amount of immobilized laccase increased with the increase of the buffer pH values (see Figure 3A) because of the increasing negative charge density of laccase. This trend was in agreement with the previous result observed under similar conditions.15 The instability of laccase at higher pH prevented the further study. The amount of immobilized laccase also depended on salt concentration. As shown in Figure 3B, the laccase loading decreased with the increase of the concentration of NaCl (from

Figure 3. The amounts of laccase immobilized via electrostatic attraction as a function of pH values of 50 mM buffer (A) or the concentrations of NaCl in 50 mM buffers (pH 6.8) (B).

0 to 1 M), i.e., addition of the inorganic salt NaCl was unfavorable for the laccase loading. Salt ions can partially screen the charges on laccase and on the surface of the methylene bluemodified NPG. This screening reduced the NPG-laccase electrostatic attraction, resulting in a decrease of the laccase loading on NPG. This screening also reduced both the intra- and the intermolecular electrostatic repulsions,26 which favored the immobilization of laccase. Therefore, the amount of laccase loaded on NPG in the presence of NaCl was governed by a new balance between the attraction and repulsion forces. At a given salt concentration, an electrostatic repulsion between

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Figure 4. Changes of the absorbance at 470 nm with time for the oxidation of DMP catalyzed by laccase from the leaching solution of the laccase-loaded NPG immobilized by (a) covalent coupling, (b) physical adsorption, and (c) electrostatic attraction strategies.

immobilized laccases made the dense packing of the enzyme impossible using the electrostatic attraction strategy. This might explain why the least amount of laccase was immobilized via electrostatic attraction. The Amount of Laccase Leached. The amout of laccase leached was below the detection limit of the Bradford method, so a more sensitive method was used, as mentioned in the Experimental Section. This method was based on the good catalytic effect of laccse on the oxidation of DMP. The more the leached laccase, the larger the initial velocity of the laccasecatalyzed oxidation of DMP. Figure 4 indicated that some laccase did remove from the NPG surface. The amount leached depended on the strategy used for the laccase immobilization. The electrostatic attraction resulted in the largest amount of leached laccase. The amounts of laccase leached from both physical adsorption and covalent coupling were relatively small and comparable, which should be due to the similar covalent linkage between the enzyme and the NPG surface as mentioned earlier. The small leakage might be caused by several free laccases which were physically entrapped in the inner pore of the NPG by its complex porous structure during the immobilization and gradually diffused out during the long time leaching process. The largest amount of leached laccase from electrostatic attraction could be ascribed to the charge screening effect of the salts in the buffer, which reduced the electrostatic interaction between the enzyme and the surface of NPG. As the concentration of the salts (ionic strength) in the buffer increased, more laccase was leached out for electrostatic attraction (data not shown). For covalent coupling and physical adsorption, the salt concentration had little effect by comparison. When a relative hydrophobic reagent polyethylene glycol (10% (w/v)) was added to the buffer (50 mM, pH 4.4), no extra laccase was leached for the three strategies. This result also indicated that there was little hydrophobic interaction during the immobilization.14 The Specific Activity of the Immobilized Laccase. As shown in Table 1, the three immobilization strategies resulted in an approximately equal laccase specific activity. For the physical adsorption and covalent coupling strategies, this conclusion seemed to be reasonable because the amino group of laccase used for linkage was theoretically the same for the two strategies. For the electrostatic attraction strategy, it was possible that the binding site of laccase used for Coulombic interaction was just near the amino group for the chemical linkage, thereby resulting in a similar effect. On the other hand, a coincidence could not be excluded.

Qiu et al.

Figure 5. Effect of the particle sizes of NPG on the amounts of the immobilized laccase.

Figure 6. Double reciprocal plot of the initial rate of the immobilized laccase catalyzed oxidation of DMP vs the concentration of DMP. The carrier for laccase immobilization was sample 1 (∆), sample 2 (0), and sample 3 (9)

The Particle Size Effect of NPG on the Laccase Loading and Kinetic Parameters. In this section, the physical adsorption strategy was selected because of its advantages (not only the reasons discussed above but also its simplicity and low cost) in laccase immobilization. The three different particle sizes of NPG were prepared as described in the Experimental Section. The particle size of NPG was in the following order: sample 1 > sample 2 > sample 3. The amounts of laccase immobilized on the three samples were correlated with the incubation time, which is shown in Figure 5. For the same time interval, the amount of laccase loaded on NPG increased with the decrease of the particle size of NPG. It is known that there was only a small increase in surface area by mechanical crushing of a bulky NPG into small pieces. Therefore, the large increase in the loading should be attributed to the increase of the accessibility of laccase to the inner pore. A similar conclusion could also be drawn from the enzyme kinetics. Figure 6 shows the Lineweaver-Burk plots of the immobilized laccase-catalyzed oxidation of DMP. The calculated kinetic parameters for the three samples on the basis of Figure 6 were listed in Table 2. After crushing, the Km value decreased a little, i.e., the affinity of the immobilized laccase to the substrate DMP increased a little. A similar result was also obtained when laccase was first immobilized on a bulky NPG and the laccase loaded NPG was then crushed for kinetic study (data not shown). This small increase in the affinity after crushing should be attri-

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TABLE 2: Kinetic Parameters of the Immobilized Laccase on Different Particle Sizes of NPG Km/mM

Vmax/ (mM min-1)

kcat/ min-1

Kcat/ Km /(mM-1 min-1)

0.25 0.19 0.17

0.32 1.02 1.19

720 900 950

2880 4737 5588

sample 1 sample 2 sample 3

buted to the decrease of the steric hindrance for the access of DMP to the active site of laccase (the steric hindrance resulted from the complex inner pore structure of NPG and the orientation of laccase on the surface). When the particle size of NPG decreased, the active site of more laccases might be exposed (i.e., the crushed NPG had less blocking effect on the active site of laccases). The mass transfer of the substrate DMP and its product within the pores was also increased after crushing. All these also resulted in a big kcat value for the two crushed samples. As compared with the free laccase (1257 min-1), the kcat value of the immobilized laccase was decreased by 25%. The reduction of active sites and/or the change of the active conformation of laccase after contact with the solid surface of NPG should be responsible for the decrease in kcat. Even so, the immobilized laccase on NPG still had some advantages over the free enzyme as demonstrated in our previous work,8 such as improved operation stability, thermal stability, and good reusability, etc. For the reusability of the carrier NPG, all these surface decorated NPG were immersed in concentrated HNO3 for ∼1 min and then washed to neutral pH for laccase immobilization again. The same amount of laccase could still be immobilized (for all the three strategies), indicating that the carrier could be completely refreshed by a simple concentrated HNO3 treatment. Conclusions The physical adsorption strategy was the simplest of the three strategies for laccase immobilization on NPG. As far as the amount, the specific activity, and the leakage of the immobilized enzyme were concerned, it was comparable to the covalent coupling strategy but superior to the electrostatic attraction strategy. Therefore, for NPG the physical adsorption strategy was the best one among the three strategies. When the particle size of NPG got smaller, more laccase could access the inner pore and be immobilized. The kinetic study showed that the crushed NPG not only enhanced mass transfer of the substrate and its oxidation product, but also favored for the exposure of the active sites of the immobilized laccase to the substrate, i.e., the crushing facilitated the enhancement of the catalytic

efficiency of laccase. Compared with silica, gold was more expensive; however, the experimental results showed that NPG could be completely refreshed by a simple concentrated HNO3 treatment. And because of its excellent electric conductivity, this material holds great promise in the field of biosensors. Acknowledgment. This work was financially supported by the Provincial Natural Science Foundation of Shandong, the National Natural Science Foundation of China (No.30570014), andNationalBasicResearchProgramofChina(No.2007CB936602). References and Notes (1) Erlebacher, J.; Aziz, M.; Karma, A. Nature 2001, 410, 450. (2) Xu, C. X.; Su, J. X.; Ding, Y. J. Am. Chem. Soc. 2007, 129, 42. (3) Yin, H. M.; Zhou, C. Q.; Xu, C. X.; Liu, P. P.; Xu, X. H.; Ding, Y. J. Phys. Chem. C 2008, 112, 9673. (4) Zielasek, V.; Jurgens, B.; Schulz, C.; Biener, J.; Biener, M. M.; Hamza, A. V.; Baumer, M. Angew. Chem., Int. Ed. 2006, 45, 8241. (5) Zhang, J. T.; Liu, P. P.; Ma, H. Y.; Ding, Y. J. Phys. Chem. C 2007, 111, 10382. (6) Zeis, R.; Lei, T.; Sieradzki, K.; Snyder, J.; Erlebacher, J. J. Catal. 2008, 253, 132. (7) Shulga, O. V.; Jefferson, K.; Khan, A. R.; D’souza, V. T.; Liu, J. Y.; Demchenko, A. V.; Stine, K. J. Chem. Mater. 2007, 19, 3902. (8) Qiu, H. J.; Xu, C. X.; Huang, X. R.; Ding, Y.; Qu, Y. B.; Gao, P. J. J. Phys. Chem. C 2008, 112, 14781. (9) Moelans, D.; Cool, P.; Baeyens, J.; Vansant, E. F. Catal. Commun. 2005, 6, 307. (10) Berlin, P.; Klemm, D.; Jung, A.; Liebegott, H.; Rieseler, R. Tiller. J. Cellulose 2003, 10, 343. (11) Park, C.; Clark, D. S. Biotechnol. Bioeng. 2002, 78, 229. (12) Lei, C.; Shin, Y.; Liu, J.; Ackerman, E. J. J. Am. Chem. Soc. 2002, 124, 11242. (13) Wang, P.; Dai, S.; Waezsada, S. D.; Tsao, A. Y.; Davison, B. H. Biotechnol. Bioeng. 2001, 74, 249. (14) Deere, J.; Magner, E.; Wall, J. G.; Hodnett, B. K. J. Phys. Chem. B 2002, 106, 7340. (15) Takahashi, H.; Li, B.; Sasaki, T.; Miyazaki, C.; Kajino, T.; Inagaki, S. Chem. Mater. 2000, 12, 3301. (16) DeLouise, L. A.; Miller, B. L. Anal. Chem. 2004, 76, 6915. (17) Hudson, S.; Magner, E.; Cooney, J.; Hodneet, B. K. J. Phys. Chem. B 2005, 109, 19496. (18) Xu, F. Biochemistry 1996, 35, 7608. (19) Thurston, C. F. Microbiology 1994, 140, 19. (20) Dodor, D. E.; Hwang, H. M.; Ekunwe, S. I. N. Enzyme Microbial Technol. 2004, 35, 210. (21) Wang, H.; Castner, D. G.; Ratner, B. D.; Jiang, S. Langmuir 2004, 20, 1877. (22) Li, D. X.; He, Q.; Cui, Y.; Duan, L.; Li, J. B. Biochem. Biophys. Res. Commun. 2007, 355, 488. (23) DeLouise, L. A.; Miller, B. L. Anal. Chem. 2005, 77, 1950. (24) Kumar, A.; Mandal, S.; Selvakannan, P. R.; Pasricha, R.; Mandale, A. B.; Sastry, M. Langmuir 2003, 19, 6277. (25) Piontek, K.; Antorini, M.; Choinowski, T. J. Biol. Chem. 2002, 277, 37663. (26) Wang, Y. J.; Angelatos, A. S.; Dunstan, D. E.; Caruso, F. Macromolecules 2007, 40, 7594.

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