Biomacromolecules 2008, 9, 381–387
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Ionic Liquid-Based Preparation of Cellulose-Dendrimer Films as Solid Supports for Enzyme Immobilization Mozhgan Bagheri,† Héctor Rodríguez,‡ Richard P. Swatloski, Scott K. Spear, Daniel T. Daly, and Robin D. Rogers* Department of Chemistry, Center for Green Manufacturing, and Alabama Institute for Manufacturing Excellence, The University of Alabama, Tuscaloosa, Alabama 35487 Received September 13, 2007; Revised Manuscript Received October 21, 2007
Surface-active cellulose films for covalent attachment of bioactive moieties were achieved by codissolution of cellulose with polyamidoamine (PAMAM) dendrimers in an ionic liquid followed by regeneration of the composite as a film. Different generations of PAMAM were used for the formation of cellulose-dendrimer composites, as well as films with the dendrimer covalently bonded to the cellulose by means of the linker 1,3-phenylene diisocyanate. Surface characterization, thermal stability, and utility for immobilization of laccase were determined. The presence of the dendrimer amino groups was confirmed by detailed characterization of the films’ surfaces. These modified films exhibit acceptable thermal stability, comparable to that of other regenerated cellulose films, but the number of active functional groups on the surface is much smaller than the theoretical amount expected. Films made with 1,3-phenylene diisocyanate as linker for covalently bound cellulose and dendrimers exhibit a better performance for immobilization of laccase than those prepared by simple mixing of the cellulose and dendrimer. In general, a linear correspondence between the dendrimer generation within the films and the specific activity of immobilized laccase in such films was not observed.
1. Introduction Polymers from renewable resources have attracted much attention in many fields as biodegradable alternatives to products derived from the petrochemical industry. Cellulose is the most abundant natural polymer and it exhibits excellent biocompatibility and hydrophilicity. It is also nontoxic, biodegradable, and inexpensive, which makes it a useful support for enzyme immobilization.1 Different enzymes and cells have been attached in fibers, in membranes, and in gels of cellulose or cellulose derivatives through simple physical adsorption or weak ionic interaction between the enzyme and the surface of the support.2–6 However, in order to get a more stable, nonreversible binding of the biomolecules to the surface of cellulose, chemical activation and functionalization are preferred.7–9 Most of the methods for activation of cellulose membranes are based on the activation of the hydroxyl groups of cellulose. Dendrimers are characteristic synthetic macromolecules with highly branched structure and globular shape.10,11 They possess unique properties such as high density of active groups, good structural homogeneity, intense internal porosity, and good biocompatibility.12 Among the various types, polyamidoamine (PAMAM) dendrimers are of particular interest because of their globular structure, mimicking the three-dimensional structure of biomacromolecules, and their good biocompatibility.13 One of the successfully commercialized dendrimers is starburst polyamidoamine dendrimer developed by Tomalia, et al.14,15 Its surface functional groups (NH2, COOH, OH) proved to be useful in coupling molecules of interest (drugs, polyethylene glycol arms, etc.). PAMAM dendrimers of different generations are possible, including different number of amino * To whom correspondence may be addressed. † Current address: Department of Chemistry, College of Sciences, Shiraz University, Shiraz 71454, Iran. ‡ Permanent address: Department of Chemical Engineering, University of Santiago de Compostela, E-15782, Santiago de Compostela, Spain.
Scheme 1. Chemical Structure of PAMAM G2
groups, with each generation having double the number of groups as the previous one. Thus, generation 1 (G1) dendrimers have 8 amino groups, there are 16 in generation 2 (G2, Scheme 1), 32 in generation 3 (G3), and so on. Of particular interest are PAMAM dendrimers of generation 4 (G4) or lower, since they exhibit desirable biological properties such as nontoxicity and nonimmunogenicity for in vivo applications.16,17
10.1021/bm701023w CCC: $40.75 2008 American Chemical Society Published on Web 12/29/2007
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The high density of terminal amino groups in dendrimers provides a large number of reactive sites for cross-linkage when used as modifiers for polymers.18 Several researchers have added PAMAM dendrimers on polymer surfaces or into polymer blocks in order to achieve novel functional materials.19–24 Various strategies have been developed for attaching dendrimers to the surface of a support, mainly metal surfaces.25–31 Also recently, a new approach for biomacromolecular immobilization in solid supports through dendrimers has been demonstrated.13,32–35 Thin films and coatings of dendritic polymers offer interesting application possibilities, including uses as encapsulating coatings, redox ion channels, catalytic and biocompatible membranes, and adhesion promoters and inhibitors.36 Since the high density of terminal amino groups in PAMAM dendrimers provides a large number of reactive sites, this polyamine could be interesting as a modifier of cellulose surfaces. Through the formation of, for example, amide bonds between the amino groups of the dendrimer and carboxylic groups of the enzyme, solid supports based on cellulose functionalized with PAMAM can lead to high bioattachment performances. The role of the dendrimer here would not be limited to providing multiple covalent branching sites offering free amino groups for enzyme attachment, but it would also act as a spacer in the polymer matrix. There is limited literature available on dendrimerized polysaccharides. Some examples include the preparation of a chitosandendrimer hybrid37,38 and the preparation of a series of regioselectively dendrimerized cellulose derivatives by treatment of cellulose in an N,N-dimethylacetamide/LiCl solvent system with dendrons possessing isocyanate focal substituents.39 Here we describe the preparation and characterization of cellulose-dendrimer composites using the hydrophilic ionic liquid (IL) 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) to solubilize both cellulose6,40 and PAMAM followed by reconstitution into composite films. We have also studied the covalent attachment of the PAMAM dendrimer to the cellulose while codissolved in the IL using 1,3-phenylene diisocyanate as a linker. The efficiencies of these composite films were tested and compared under different treatment conditions using laccase from Rhus Vernificera (EC No. 3.1.1.3), a copper-containing redox enzyme produced by various classes of fungi and responsible for the degradation of polyphenolic compounds such as lignin,41 as a model enzyme.
2. Experimental Section 2.1. Materials. Starburst PAMAM dendrimers with ethylenediamine cores, of generations 1, 2, 3, and 4 (20 wt % in methanol solution for generations 1, 2, and 3 and 10 wt % for generation 4) were purchased from Aldrich and used directly without further purification. Microcrystalline cellulose (MCC), with degree of polymerization ∼200, and laccase from Rhus Vernificera (EC No. 3.1.1.3) were obtained from Sigma. Potassium phosphate and glutaraldehyde (Fisher Scientific) were used as received. All other reagents were supplied by Sigma-Aldrich and were of reagent grade. The IL [C4mim]Cl was synthesized in our laboratory by direct alkylation of 1-methylimidazole with chlorobutane, according to a previously published procedure.42 Its structure and purity were confirmed by 1H and 13C nuclear magnetic resonance spectroscopy. Additionally, it was characterized with Karl Fischer titration, which indicated a water content below 300 ppm. 2.2. Preparation of Films. Two strategies for the activation of cellulose surfaces for immobilizing biomacromolecules were studied: a straight blend of cellulose and PAMAM dendrimer; and bonding of
Bagheri et al. Table 1. Calculated Mass of Dendrimer (md), Number of Moles of Dendrimer (nd), and Number of Moles of Amino Groups (nNH2) Corresponding to the Addition of 0.3 mL of Methanol Commercial Solution of Dendrimer of Either Generation 1, 2, 3, or 4, in the Preparation of the Modified Cellulose Films, as Well as the Dendrimer Weight Percent of Its Mixture with 0.7 g of Cellulose generation md (×10-2 g) nd (×10-5) nNH2 (×10-4) 1 2 3 4
4.9 5.2 5.2 2.4
3.4 1.6 0.75 0.17
2.8 2.5 2.4 1.1
dendrimer, wt % 6.6 6.9 6.9 3.4
PAMAM dendrimer to the cellulose backbone using 1,3-phenylene diisocyanate as a linker. Details on the preparation of the different films are provided below. Regenerated Cellulose (CEL) films. MCC (0.7 g) was dissolved in [C4mim]Cl (10 g) using microwave pulse heating,40,43 taking care to avoid cellulose degradation. After complete dissolution, the 6.5 wt % solutions formed were cast as films on a glass plate using a coating rod (No. 60, purchased from R&D Specialties, Webster, NY). The films were reconstituted, and the IL solvent was leached using water. The films, dried in the atmosphere, had thicknesses of 0.1–0.2 mm. Cellulose-PAMAM Dendrimer (CEL-DEN) Films. MCC was dissolved in [C4mim]Cl as described above. After complete dissolution, 0.3 mL of PAMAM dendrimer of either generation 1, 2, 3, or 4 (yielding cellulose-dendrimer mixtures with less than 7 wt % of dendrimer on an ionic liquid-free basis, as indicated in Table 1) was added. Then, the mixture was homogenized manually while heated with microwave pulses. The process of casting the solution as a film and washing out the IL were analogous to that described above. Cellulose-Phenylene Diisocyanate-PAMAM Dendrimer (CEL-ISODEN) Films. To the homogeneous solution of MCC in [C4mim]Cl, prepared as previously indicated, 1,3-phenylene diisocyanate (0.05 g, 0.31 mmol) was added. The mixture was heated with microwave pulses while manually homogenized. Then, 0.3 mL of PAMAM dendrimer of either generation 1, 2, 3, or 4 (implying 0.034, 0.016, 0.007, and 0.002 mmol of dendrimer, respectively) was added to this mixture. Again, it was manually homogenized and cast as a film on a glass plate. The IL was finally removed by washing with water, and the film was allowed to dry. 2.3. Characterization of Films. Surface modification of the cellulose films was characterized by X-ray photoelectron spectroscopy (XPS). Measurements were carried out in a Kratos analytical analysis 165 multitechnique spectrophotometer using monochromatic Al KR X-rays (hν ) 1486.6 eV) as photon source, operated at 20 mA and 15 kV. Peak fit analyses of high-resolution spectra were performed after a five-point Savistsky-Golay smooth and a Shirley background correction. The amount of amino groups present on the composite surfaces was quantified by adsorption of bromophenol blue (BPB),44–46 since the anion of this colored substance will form ion pairs with protonated amino groups in the dendrimers. Films were immersed in 4 mL of buffered solution of BPB (1.22 × 10-5 M) at pH 7.5, for 12 h, followed by washing with 0.1 M phosphate buffer at pH 7. Absorbance measurements at 592 nm were determined using a Varian Cary 3C UV–visible spectrophotometer. The number of moles of amino groups was calculated according to the adsorbed BPB. Thermal stability of the modified cellulose films was characterized by thermogravimetric analysis (TGA), using a TA Instruments 2950 apparatus. Measurements were performed at a heating rate of 5 °C/ min from ambient temperature to 700 °C, under nitrogen atmosphere. 2.4. Enzyme Attachment to Modified Cellulose Films. In a first approach, modified cellulose films were placed directly in aqueous or buffered solutions of laccase (containing 5 mg of the enzyme, unless otherwise stated) and were incubated for 2 h at 30 °C for surface attachment. After the incubation period, films were washed with Triton
Cellulose Supports for Enzyme Immobilization Scheme 2. Laccase-Catalyzed Oxidation of SGZ, Used for Monitoring the Enzymatic Activity Assaya
a The reduced form (top) is the colorless substrate and the oxidized form (bottom) is the pink product.
X-100 (0.1%) followed by deionized water, to remove electrostatically surface-bonded enzymes. As an alternative treatment, the addition of a spacer between the support and the enzyme was considered. This strategy has been reported in the literature to improve the enzyme activity,47–50 since it provides a more flexible conformation, offering a greater freedom of movement, as well as minimizing unfavorable steric hindrance posed by solid supports.51 Glutaraldehyde was used as the first spacer by immersion of the modified films in a solution of 18 mL of 25 wt % glutaraldehyde and 23 mL of 0.1 M phosphate buffer (pH 7) and stirring for 12 h at room temperature. The use of 50 mL of cyanoborohydride coupling buffer (pH 7.5) together with the addition of the laccase, and allowing the reactions to proceed for 2 h at room temperature, resulted in the imine bonds being reduced and the enzyme attached.52 Finally, the films were washed with the phosphate buffer solution. In another method, diethyl succinate was chosen as the spacer. Here films were dried at ambient temperature and then were immersed in a solution of diethyl succinate and butyl alcohol at 60 °C for 24 h, followed by washing with copious amounts of deionized water. These films were then treated in a manner analogous to that described above. The incubation period for enzyme immobilization, in both cases, was analogous to that of the first approach described in this subsection. 2.5. Enzyme Assay by Oxidation of Syringaldazine. The specific activity of laccase immobilized in the functionalized cellulose films was monitored using the syringaldazine (SGZ) oxidation assay.42 This method is based on the laccase-catalyzed oxidation of SGZ, as shown in Scheme 2. The oxidized form has a characteristic pink color and a maximum absorbance at a wavelength of 555 nm in the visible region, whereas the substrate, or reduced form, is colorless and presents its maximum of absorbance at 371 nm. Thus, the reaction can be easily followed by UV/vis spectroscopy. Circular disks of each film (with a diameter of 1.6 cm and an area of 2.0 cm2) were cut and immersed in the enzymatic solution containing 2.5 mL of 100 mM phosphate buffer (pH 6.5) and 0.3 mL of 0.5 mM SGZ, in its reduced form. Incubation was carried out at 30 °C for 10 min or more, depending on the samples. Specific activities were calculated from the absorbance intensity measured at 530 nm, the excitation coefficient for oxidized SGZ (6.5 × 104 M-1 cm-1), and the film thickness. One unit of enzymatic activity was defined as the amount of enzyme required to oxidize 1 µmol of SGZ per minute at 30 °C. The amount of immobilized enzyme was determined from the activity measurements before and after soaking the membrane with the solution described, by means of an elemental mass balance. All absorbance measurements in this section were also carried out in the Varian instrument already described in section 2.3.
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3. Results and Discussion Characterization and enzymatic activity assays were carried out for four composites of cellulose-dendrimer (CEL-DEN) (each with a different generation of dendrimer), four covalently bonded films with 1,3-phenylene diisocyanate (CEL-ISO-DEN) (also, each with a different generation of dendrimer), and a cellulose-only film CEL, washed and unwashed. Moreover, four more films of the type CEL-DEN were prepared by adding glutaraldehyde to check the influence of its spacing function in the ability of the solid support for enzyme attachment. 3.1. Surface Characterization of Films. XPS Characterization. XPS N 1s spectra (Figure 1) clearly indicate surface modification of the cellulose films by the dendrimer was achieved. No N 1s peak was observed for washed CEL films (Figure 1a); however, if the IL is not thoroughly washed from the regenerated films, a peak is observed at ∼402 eV (Figure 1b) corresponding to nitrogen in the [C4mim]+ cation. The N 1s spectra of CEL-ISO-DEN films (Figure 1c, G2-modified; Figure 1d, G4-modified) reveal two components: one corresponding to amino groups of the dendrimers on the surface, at ∼400 eV, and another, at ∼402 eV, which might be attributed to residual [C4mim]+, to reaction of the dendrimer’s amino groups with CO2,21,53,54 or even to hydrogen bonding.55 The NH2 peak did increase with increasing generation of dendrimer. The C 1s spectra of a CEL film and CEL-DEN G2 are shown in Figure 2. The C 1s spectrum of the CEL film (Figure 2a) was fitted to three different carbon species: C-C at ∼285 eV, C-O at ∼287 eV, and the oxidized form COO at ∼289 eV.56–59 The C 1s spectrum of CEL-DEN G2 film (Figure 2b) shows a new component at ∼288 eV, related to the (CdO)N amide group in the dendrimer molecule.21,60 Colorimetric Characterization. The amino group is the most commonly used solid-supported functional group, and several colorimetric methods have been developed for its quantification.61–66 Nevertheless, according to our experience, the main problems associated with these tests are their limited sensitivity, the nonspecific adsorption of the chromophore on the support, and their incompatibility with polymeric supports when organic solvents are included in the protocol.61 Hence, pH dependence and charge of PAMAM dendrimers were used in this work as probes for the determination of amino groups in the support. The surface activation of the modified films was quantified by the formation of ion pairs with bromophenol blue (BPB), which allowed the photometric determination of amino groups. It has been shown that, in the amine-terminated PAMAM dendrimer, the protonation of the primary amino groups occurs between pH 7 and 10, whereas the protonation of the tertiary amino groups takes place between pH 3 and 7.67,68 Thus, pH 7.5 was selected for quantitative determination of amino groups at the surface of modified films. At this pH, BPB has negative charge and forms ion pairs with the positively charged primary amines, adsorbing on the films. After formation of ion pairs, the absorbance of the colored films was measured at a wavelength of 592 nm (Figure 3). The adsorption of BPB is larger for CEL-ISO-DEN films than for CEL-DEN films, and it also increases with increasing PAMAM generation, as expected. The quantitative results from the BPB test are summarized in Table 2. Although the amount of dendrimer used in the formation of CEL-DEN and CEL-ISO-DEN films was the same for each pair of the same generation, the calculated active moles of NH2 in the latter group were greater. This might be attributed to hydrogen bonding of amino groups to the cellulose backbone in CEL-DEN composites, leading to less primary amines
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Figure 1. XPS N 1s spectra of (a) regenerated cellulose, (b) unwashed regenerated cellulose, (c) film CEL-ISO-DEN G2, and (d) film CELISO-DEN G4.
Figure 3. Absorbance spectra, after adsorption of BPB at pH 7.5, of films CEL-DEN G1, G2, G3, and G4 (from bottom to top, solid lines) and CEL-ISO-DEN G1, G2, G3, and G4 (from bottom to top, dashed lines).
Figure 2. XPS C 1s spectra of (a) regenerated cellulose and (b) film CEL-DEN G2.
available and hence poorer surface activation. It is proposed that in the CEL-ISO-DEN films, the linker acts as spacer and
provides more flexibility to the dendrimer, thus permitting greater accessibility to the amino groups and making them more active. Nonetheless, for all films investigated, the low percentage of active NH2 groups on the surface suggests that most of the functional groups in the PAMAM dendrimer are consumed or encapsulated in the polymeric network. However, it must be said that the characterization method used has its drawbacks, and an accurate estimation of the number of active primary amines may be difficult due to steric hindrance. In absolute terms, it can be observed that the amount of calculated active moles of NH2 increases with increasing generation of PAMAM dendrimers as expected.
Cellulose Supports for Enzyme Immobilization
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Table 2. Calculated Moles of Active Surface Amino Groups (N) and the Corresponding Approximate Percent (%) Related to the Total Dendrimer Using the BPB Methoda modified films CEL-DEN G1 CEL-DEN G2 CEL-DEN G3 CEL-DEN G4 CEL-ISO-DEN CEL-ISO-DEN CEL-ISO-DEN CEL-ISO-DEN a
G1 G2 G3 G4
N (×10-9 mol)
%
1.0 1.1 1.4 1.4 3.3 4.8 6.1 7.5
0.02 0.02 0.03 0.04 0.06 0.10 0.14 0.19
Circular disks (d ) 1.6 cm, A ) 2.0 cm2) at pH 7.5.
Table 3. TGA Data Including First Onset Decomposition Temperature (Td1), Second Onset Decomposition Temperature (Td2), and the Residual wt % at 350 °C (R350) and at 500 °C (R500) material
Td1 (°C)
Td2 (°C)
R350
R500
MCC [C4mim]Cl PAMAM G2 CEL CEL-DEN G2 CEL-ISO-DEN G2
278 212 189 237 233 240
430
17 0 23 45 25 40
7 0 4 27 4 0
439 445 451 433
3.2. Thermal Stability. Thermal stability was investigated for the IL, MCC, PAMAM G2 dendrimer, regenerated cellulose, and composite films with dendrimer G2. Decomposition temperatures and residual weight percents, as reported by TGA analyses, are shown in Table 3. Cellulose is a polymer of moderate thermal stability, and rapid chemical decomposition occurs between 250 and 350 °C.69 Cellulose shows a first thermal degradation stage due to pyrolytic fragmentation and a second stage likely due to high cross-linking of the carbon skeleton.70 The original MCC is thermally stable up to 278 °C, whereas the regenerated cellulose, CEL film, showed onset of degradation at 237 °C. The modified films, with more than 90 wt % of cellulose in their composition, also show two stages of thermal degradation. They exhibit approximately the same thermal stability as the regenerated cellulose but a lower char yield at 500 °C. 3.3. Enzymatic Activity Assay. Laccase is a glycosylated protein with a typical molecular mass in the range 60–80 kDa, and 15–20 wt % of equal amounts of N- and O-linked carbohydrate. Since N-type glycosylation occurs exclusively on the nitrogen atoms of asparagine residues and O-glycosylation on the oxygen atoms of hydroxyls of serine and threonine residues, amide bonds can be formed between the primary amino groups of the dendrimer and the carboxylic residues of aspartic and glutamic acid. Thus, laccase activity was measured by the SGZ oxidation method, as indicated in section 2.5. Preliminary enzyme assays of CEL-DEN films made with all four generations (G1 to G4) of PAMAM dendrimer were carried out, looking for the best set of conditions. The effect of amount of enzyme present was investigated in the range 4–6 mg, for CEL-DEN G2 films. An increase in this initial amount led to an increase in the amount of immobilized protein, but simultaneously, a decrease in the specific activity of the immobilized enzyme was observed. These results might be ascribed to the fact that, with an increase in the amount of enzyme, it is likely immobilized through multiple interactions that could negatively affect its activity by altering its native conformation. Similar observations have already been reported by other researchers.47,71,72
Table 4. Specific Activity of Immobilized Laccase in CEL-DEN Films of Different Generations, with Initial Enzyme Dissolved in Either Deionized Water (A) or 0.01 M Phosphate Buffer at pH 6.5 (B) specific activity (µM/min/mg) generation
A
B
G1 G2 G3 G4
0.97 0.20 1.26 0.78
5.01 1.54 1.55 1.68
The effect of pH on the activity of the immobilized laccase was analyzed in the pH range 6–10 (0.01 M buffer). The results indicated that laccase immobilized on the film displayed lower efficiency in terms of specific activity with increasing pH. Hence, an initial amount of 5 mg of laccase and a pH of 6.5 were eventually selected as the conditions for carrying out the activity assays. The enzymatic activities of laccase, measured with the SGZ method, for incubated CEL-DEN composites made with different dendrimer generations are provided in Table 4. Two series of assays were carried out, with enzyme immobilized in the films from two different media: enzyme dissolved in pure water, and enzyme dissolved in a 0.01 M phosphate buffer solution at pH 6.5. Higher activities were obtained with the enzyme dissolved in the phosphate buffer. Other concentrations for the enzymatic solution (up to 0.1 M) were investigated, and the results corroborated increasing activity with increasing ionic strength. Under these conditions, the reaction with the substrate was faster, since the oxidized form of the substrate SGZ was visually observed after a short period of time (less than 10 min). But also intense leaching from the film was found with increasing ionic strength, especially for films made with G1 dendrimer. Since the films, after immobilization, were washed with Triton X-100 and deionized water to remove electrostatically surface-bonded enzyme without observation of any leaching, it can be assumed that the observed leaching during the activity assay is related to the substrate or its oxidized product coming out from the support. Some leaching is also detectable in the case of enzyme dissolved in deionized water, but of a much smaller magnitude. In both series of data in Table 4, no linear correlation between the dendrimer generation or the number of amino groups (see Table 1) and the activity of the immobilized enzyme was observed, the highest values being found for the lowest dendrimer generation. Decreases in the molecular mobility due to the multiple point attachment or the change of the microenvironment around the enzyme molecules can result in lowered activity, analogous to results found in the literature.48,49,51 Also, alterations in the expected enzyme activity can be explained by the existence of significant cross-linking among attached enzyme molecules, thus leading to conformational changes in the proteins and therefore decreasing the activity. To extend the molecular spacer and facilitate the attachment of laccase, the amino groups on the surface of CEL-DEN films were transformed into aldehyde groups by using glutaraldehyde. The so treated solid supports were tested for the specific activity of immobilized laccase, as well as the CEL-ISO-DEN films with different dendrimer generations. The effect of the addition of glutaraldehyde and of the covalent binding between cellulose and dendrimer through 1,3-phenylene diisocyanate within the film can be observed in Figure 4. The use of glutaraldehyde as additive negatively influenced the activation of the supports surfaces, lowering the specific
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Figure 4. Specific activity (as assayed by the syringaldazine method) of laccase attached to the different series of films, with different dendrimer generation: CEL-DEN films (black, left bars), CEL-DEN films with glutaraldehyde as an additive (light gray, central bars), and CEL-ISO-DEN films (dark gray, right bars).
activity of the enzyme. This may be related to alteration of the conformational structure and active center of the enzyme caused by the covalent binding. On comparison of the results for CEL-DEN and CEL-ISODEN films, the latter are found to exhibit higher specific activity. A plausible explanation is that surface activation is more effective in the CEL-ISO-DEN films as a result of the greater number of functional groups on the surface, as indicated by the spectroscopic studies described earlier. The evolution of laccase activity with dendrimer generation for the CEL-DEN films with glutaraldehyde and for the CELISO-DEN films follows an analogous trend to that observed in the CEL-DEN composites (Table 4). The previously given explanations for such behavior are also valid for any of the other series of films.
4. Conclusions Dendrimers are molecular structures that provide multiple branching sites with free amino groups for attachment of enzymes to solid supports. In this work, a simple method based on the ability of some ionic liquids (in this case, 1-butyl-3methylimidazolium chloride) to codissolve cellulose and other polymers has been presented for the modification of the surface of cellulose films with PAMAM dendrimers, either as composite materials or with covalent binding by means of a linker such as 1,3-phenylene diisocyanate. The presence in the modified films of the amino groups of the dendrimer molecules has been confirmed by detailed characterization of the films surface. In addition, these modified films exhibit acceptable thermal stability, comparable to that of regenerated cellulose-only films. The ability for enzyme immobilization on the modified cellulose films has been demonstrated using laccase as a model enzyme. It has been found that the series of films made with 1,3-phenylene diisocyanate as linker for covalently bonding cellulose and dendrimers exhibit a better performance for immobilization of the enzyme. Also, increase in the ionic
Bagheri et al.
strength of the medium for dissolution of enzyme has been shown to improve the specific activity of the attached laccase, although leaching may discourage the use of this option. Conversely, the addition of glutaraldehyde as additive for facilitating the attachment of the enzyme resulted in lower enzymatic activity, as calculated from the corresponding assays. In general, a linear correspondence between the dendrimer generations within the films and the specific activity of the immobilized laccase in such films was not observed. This seems to indicate the existence of intra- and intermolecular interactions within the films that may affect the number of available active groups for enzyme attachment. A deeper insight at a molecular level would be required for fully understanding the influence of the dendrimer generation on the relative amount of functional groups on the surface of the films prepared. Although there is much room for improvement of the performance of the materials presented in this work, the results are encouraging and suggest the possibility of using the described supports in interesting fields such as biosensors. Many other applications, for example, novel drug delivery systems, can also be envisioned for these biodegradable and biocompatible materials. Acknowledgment. The authors thank Dr. Earl Ada (The University of Alabama) for carrying out the XPS measurements, and Mr. Marcin Smiglak (The University of Alabama) for performing the TGA analyses. M.B. gratefully acknowledges the Iranian Ministry of Science, Research and Technology for financial support. H.R. is also grateful to the Ministerio de Educación y Ciencia (Spain) for the award of the FPI grant with reference BES-2004-5311 under project PPQ2003-01326.
References and Notes (1) Vigo, T. L. Polym. AdV. Technol. 1998, 9, 539–548. (2) Fishman, A.; Levy, I.; Cogan, U.; Shoseyov, O. J. Mol. Catal. B: Enzym. 2002, 18, 121–131. (3) Gupta, M. N. Eur. J. Biochem. 1992, 203, 25–32. (4) Tiller, J.; Berlin, P.; Klemm, D. Biotechnol. Appl. Biochem. 1999, 30, 155–162. (5) Roy, I.; Gupta, M. N. Process Biochem. 2003, 39, 325–332. (6) Turner, M. B.; Spear, S. K.; Holbrey, J. D.; Rogers, R. D. Biomacromolecules 2004, 5, 1379–1384. (7) Klemm, D.; Philipp, B.; Heinze, T.; Heinze, U. ComprehensiVe Cellulose Chemistry; Wiley-VCH: Chichester, 1998; Vol. 2. (8) Chesney, A.; Barnwell, P.; Stonehouse, D. F.; Steel, P. G. Green Chem. 2000, 2, 57–62. (9) Stollner, D.; Scheller, F. W.; Warsinke, A. Anal. Biochem. 2002, 304, 157–165. (10) Tomalia, D. A.; Naylor, A. M.; Goddard, W. A. Angew. Chem., Int. Ed. 1990, 29, 138–175. (11) Frechet, J. M. Science 1994, 263, 1710–1715. (12) Kojima, C.; Kono, K.; Maruyama, K.; Takagishi, T. Bioconjugate Chem. 2000, 11, 910–917. (13) Shen, L.; Hu, N. Biomacromolecules 2005, 6, 1475–1483. (14) Tomalia, D. A.; Baker, H.; Dewald, J.; Hall, M.; Kallos, G.; Martin, S.; Roeck, J.; Ryder, J.; Smith, P. Macromolecules 1986, 19, 2466– 2468. (15) Tomalia, D. A. Prog. Polym. Sci. 2005, 30, 294–324. (16) Roberts, J. C.; Bhalgat, M. K.; Zera, R. T. J. Biomed. Mater. Res. 1996, 30, 53–65. (17) Boas, U.; Christensen, J. B.; Heegaard, P. M. H. Dendrimers in Medicine and Biotechnology, RSC Publishing: Cambridge, 2006. (18) Xiao, Y.; Shao, L.; Chung, T.-S.; Schiraldi, D. A. Ind. Eng. Chem. Res. 2005, 44, 3059–3067. (19) Chung, T.-S.; Chng, M. L.; Pramoda, K. P.; Xiao, Y. Langmuir 2004, 20, 2966–2969. (20) Sui, G.; Micic, M.; Huo, G.; Leblanc, R. M. Langmuir 2000, 16, 7847– 7851. (21) Fail, C. A.; Evenson, S. A.; Ward, L. J.; Schofield, W. C. E.; Badyal, J. P. S. Langmuir 2002, 18, 264–268.
Cellulose Supports for Enzyme Immobilization (22) Zhao, M.; Liu, Y.; Crooks, R. M.; Bergbreiter, D. E. J. Am. Chem. Soc. 1999, 121, 923–930. (23) Mackay, M. E.; Hong, Y.; Jeong, M.; Hong, S.; Russell, T. P.; Hawker, C. J.; Vestberg, R.; Douglas, J. F. Langmuir 2002, 18, 1877–1882. (24) Cha, B. J.; Kang, Y. S.; Won, J. Macromolecules 2001, 34, 6631– 6636. (25) Fujiki, K.; Sakamoto, M.; Sato, T.; Tsubokawa, N. J. Macromol. Sci., Pure Appl. Chem. 2000, 37, 357–377. (26) Bar, G.; Rubin, S.; Cutts, R. W.; Taylor, T. N.; Zawodzinski, T. A. Langmuir 1996, 12, 1172–1179. (27) Tsukruk, V. V.; Rinderspacher, F.; Bliznyuk, V. N. Langmuir 1997, 13, 2171–2176. (28) Wells, M.; Crooks, R. M. J. Am. Chem. Soc. 1996, 118, 3988–3989. (29) Tokuhisa, H.; Zhao, M.; Baker, L. A.; Phan, V. T.; Dermody, D. L.; Garcia, M. E.; Peez, R. F.; Crooks, R. M.; Mayer, T. M. J. Am. Chem.Soc. 1998, 120, 4492–4501. (30) Li, J.; Piehler, L. T.; Qin, D.; Baker, J. R.; Tomalia, D. A.; Meier, D. J. Langmuir 2000, 16, 5613–5616. (31) Hierlemann, A.; Campbell, J. K.; Baker, L. A.; Crooks, R. M.; Ricco, A. J. J. Am. Chem. Soc. 1998, 120, 5323–5324. (32) Anzai, J.; Kobayashi, Y.; Nakamura, N.; Nishimura, M.; Hoshi, T. Langmuir 1999, 15, 221–226. (33) Fischer, M.; Vogtle, F. Angew. Chem., Int. Ed. 1999, 38, 885–905. (34) Yoon, H. C.; Hong, M.-Y.; Kim, H.-S. Anal. Biochem. 2000, 282, 121–128. (35) Yoon, H. C.; Hong, M.-Y.; Kim, H.-S. Langmuir 2001, 17, 1234– 1239. (36) Svenson, S. In Kirk-Othmer Encyclopedia of Chemical Technology, ed. Seidel, A., Ed.; John Wiley & Sons: Hoboken, NJ, 2007; Vol. 26, pp 786–812. (37) Sashiwa, H.; Shigemasa, Y.; Roy, R. Macromolecules 2001, 34, 3211– 3214. (38) Sashiwa, H.; Shigemasa, Y.; Roy, R. Carbohydr. Polym. 2002, 47, 191–199. (39) Hassan, M. L.; Moorefield, C. N.; Newkome, G. R. Macromol. Rapid Commun. 2004, 25, 1999–2002. (40) Swatloski, R. P.; Spear, S. K.; Holbrey, J. D.; Rogers, R. D. J. Am. Chem. Soc. 2002, 124, 4974–4975. (41) Harkin, J. M.; Obst, J. R. Science 1973, 180, 296–298. (42) Huddleston, J. G.; Visser, A. E.; Reichert, W. M.; Willauer, H. D.; Broker, G. A.; Rogers, R. D. Green Chem. 2001, 3, 156–164. (43) Swatloski, R. P.; Rogers, R. D.; Holbrey, J. D. US Patent 6824599, 2004. (44) Krchnak, V.; Vagner, J.; Lebl, M. Int. J. Pept. Protein Res. 1988, 32, 415–416. (45) Flegel, M.; Sheppard, R. C. J. Chem. Soc., Chem. Commun. 1990, 536–538. (46) Kay, C.; Lorthioir, O. E.; Parr, N. J.; Congreve, M.; McKeown, S. C.; Scicinski, J. J.; Ley, S. V. Biotechnol. Bioeng. 2000, 71, 110–118.
Biomacromolecules, Vol. 9, No. 1, 2008 387 (47) Yodoya, S.; Takagi, T.; Kurotani, M.; Hayashi, T.; Furuta, M.; Oka, M.; Hayashi, T. Eur. Polym. J. 2003, 39, 173–180. (48) Cao, L. Curr. Opin. Chem. Biol. 2005, 9, 217–226. (49) Nouaimi, M.; Moschel, K.; Bisswanger, H. Enzyme Microb. Technol. 2001, 29, 567–574. (50) Ho, L.-F.; Li, S.-Y.; Lin, S.-C.; Hsu, W.-H. Process Biochem. 2004, 39, 1573–1581. (51) Wang, Y.; Hsieh, Y.-L. J. Polym. Sci., Part A: Polym. Chem. 2004, 42, 4289–4299. (52) Turner, M. B.; Spear, S. K.; Holbrey, J. D.; Daly, D. T.; Rogers, R. D. Biomacromolecules 2005, 6, 2497–2502. (53) Yan, L.; Marzolin, C.; Terfort, A.; Whitesides, G. M. Langmuir 1997, 13, 6704–6712. (54) Sprik, M.; Delamarche, E.; Michel, B.; Roethlisberger, U.; Klein, M. L.; Wolf, J.; Ringsdorf, J. Langmuir 1994, 10, 4116–4130. (55) Kerber, S. J.; Bruckner, J. J.; Wozniak, K.; Seal, S.; Hardcastle, S.; Barr, T. L. J. Vac. Sci. Technol., A 1996, 14, 1314–1320. (56) Bartouilh de Taillac, L.; Porté-Durrieu, M. C.; Labrugère, C.; Bareille, R.; Amédée, J.; Baquey, C. Compos. Sci. Technol. 2004, 64, 827– 837. (57) Sigurdsson, S.; Shishoo, R. J. Appl. Polym. Sci. 1997, 66, 1591–1601. (58) France, R. M.; Short, R. D. Langmuir 1998, 14, 4827–4835. (59) Váquez, M. I.; Galán, P.; Casado, J.; Ariza, M. J.; Benavente, J. Appl. Surf. Sci. 2004, 238, 415–422. (60) Beamson, G.; Briggs, D. High resolution XPS of organic polymers; John Wiley: Chichester, 1992. (61) Hodges, R. S.; Merrifield, R. B. Anal. Biochem. 1975, 65, 241–272. (62) Cayot, P.; Tainturier, G. Anal. Biochem. 1997, 249, 184–200. (63) Abdellatef, H. E.; Khalil, H. M. J. Pharm. Biomed. Anal. 2003, 31, 209–214. (64) Kakabakos, S. E.; Tyllianakis, P. E.; Evangelatos, G. P.; Ithakissios, D. S. Biomaterials 1994, 15, 289–297. (65) Antoni, G.; Presentini, R.; Neri, P. Anal. Biochem. 1983, 129, 60–63. (66) Gaur, R. K.; Gupta, K. C. Anal. Biochem. 1989, 180, 253–258. (67) Niu, Y.; Sun, L.; Crooks, R. M. Macromolecules 2003, 36, 5725– 5731. (68) Cakara, D.; Kleimann, J.; Borkovec, M. Macromolecules 2003, 36, 4201–4207. (69) Klemm, D.; Heinze, T.; Philipp, B.; Wegenknecht, W. Acta Polym. 1997, 48, 277–297. (70) Klemm, D.; Philipp, B.; Heinze, T.; Wegenknecht, W. ComprehensiVe Cellulose Chemistry. Vol. 1: Fundamentals and Analytical methods; Wiley-VCH: Weinheim, 1998. (71) Ye, P.; Xu, Z.-K.; Che, A.-F.; Wu, J.; Seta, P. Biomaterials 2005, 26, 6394–6403. (72) Liu, X.; Guan, Y.; Shen, R.; Liu, H. J. Chromatogr., B 2005, 822, 91–97.
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