Laccase-Induced Grafting on Plasma-Pretreated Polypropylene

Biomacromolecules 2008, 9, 2735–2741

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Laccase-Induced Grafting on Plasma-Pretreated Polypropylene M. Schroeder,†,| E. Fatarella,‡ J. Kovacˇ,§ G. M. Guebitz,| and V. Kokol*,† Institute of Engineering Materials and Design, University of Maribor, Smetanova ul. 17, SI-2000 Maribor, Slovenia, Tecnotessile Societa` Nazionale di Ricerca Tecnologica, Via del Gelso 13, I-59100 Prato, Italy, Joeˇf Sˇtefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia, and Department of Environmental Biotechnology, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria Received April 24, 2008; Revised Manuscript Received July 18, 2008

A new environmentally friendly strategy for the sustainable functionalization of inert man-made polymer surfaces is mapped out for the first time using a combination of plasma pretreatment and enzymatic postgrafting. The efficiency of enzymatic covalent binding is investigated by grafting methacrylate monomers possessing different amino groups, primary, tertiary, and quaternary, onto a polypropylene surface using plasma pretreatment. Subsequent enzymatic grafting, using laccase and guaiacol sulfonic acid (GSA), is determined by surface analytical techniques, such as attenuated total reflectance Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. The grafting of GSA in the presence of a laccase is proven by a 10-fold increase in sulfur compared to the control. The covalent coupling between GSA and primary amine groups is determined by HPLC-MS using hexylamine as a model substrate. The advantage of technology is in the strong covalent binding of functional groups onto the synthetic polymer’s surface, which could then be suitably tailored by enzymes possessing substrate specificity and regional selectivity.

Introduction Polypropylene is a widely used material in textile and packaging industries due to its good mechanical properties, low production cost, and possibilities for recycling. The list is completed by its lightweight, chemical resistance, transparency, and suitable thermal properties. However, polypropylene is difficult to functionalize due to its chemical resistance. This study focused on the grafting of phenolic molecules on plasma-pretreated polypropylene using laccase enzymes. Laccases are copper-containing polyphenol oxidases that oxidize polyphenols, methoxy-substituted phenols, diamines, and a considerable range of other compounds using molecular oxygen as an electron acceptor.1 Several coupling reactions for organic synthesis have been reported using laccase-induced oxidation. These include the formation of carbon-oxygen bonds,2 carbon-carbon bonds, as described in the oxidative dimerization of salicylic esters3 or hydroxystilbenes,4 and a combination of oxidation followed by the Diels-Alder reaction.5,6 The following are also described: the formation of nitrogen-carbon bonds in the synthesis of 3-(3,4-dihydroxyphenyl)-propionic acid derivates,7 the amination of p-hydroquinones,8 the crosscoupling of 4-aminobenzoic acid with para-dihydroxylated compounds,9 derivatization of amino acids,10 and the synthesis of Tinuvin.11 To date, laccase-induced grafting has only been described for natural lignocellulose-based flax fibers containing phenols,12 lignin pulp,13 or surfaces with artificially added phenols such as dyed cellulose fibers.14 Polypropylene is limited by its hydrophobic and chemically inert surface, resulting in poor wettability and adhesion. Thus, polypropylene is inappropriate as such for enzymatic-induced coating. * To whom correspondence should be addressed. Tel.: +386 2 220 7896. Fax: +386 2 220 7990. E-mail: [email protected]. † University of Maribor. ‡ Tecnotessile Societa` Nazionale di Ricerca Tecnologica. § Joeˇf Sˇtefan Institute. | Graz University of Technology.

Plasma treatment is a commonly used technique for overcoming the drawbacks of synthetic inert polymers. First, the hydrophilicity can be increased,15,16 which is a major benefit for enzymatic applications in an aqueous environment. Several studies have described the improvement of simple adhesion between irradiated polypropylene and adhesive,17 wood fibers,18 or polyamide 6,19 respectively. Second, there are different strategies for introducing well-defined functionalities onto a polyolefin surface via irradiation. On the one hand, different groups such as epoxy, amine, carboxyl, hydroxyl, thiol, and aldehyde can be introduced by plasma treatment using nonpolymerizing gas such as oxygen, ammonia, or hydrogen sulfide.20-22 Such treatment induces activation by promoting the incorporation of different moieties of the process gas onto the material’s surface. On the other hand, monomers and their retention can be used, either indirectly by postgrafting23 or directly by simultaneous grafting24 using a polymerizing gas such as argon and helium, the macro-radicals generated onto the irradiated surface are able to initiate a free-radicals process, providing a homogeneous and specific density or distribution on the material’s surface. Thus, the manifold possibilities of introduced functional groups can widen the range of potential enzymes to induce postfunctional grafting or coating.25 In this study, nonwoven polypropylene fabrics were pretreated by argon plasma in the presence of different methacrylate monomers, to facilitate an enzyme-induced postgrafting of guaiacol sulfonic acid as a phenolic compound onto the surface. Monomers possessing different amino groups were used, primary, tertiary, and quaternary. The efficiency of the following enzymatic grafting on the surface was studied by ATR-FTIR and XPS.

Materials and Methods Chemicals and Enzyme. 2,2-Azinobis-(3-etylbenzothiazoline-6disulfonic acid (ABTS), 2-aminoethyl-methacrylate hydrochloride, 2-(N,N-dimethylamino)ethyl methylacrylate, 2-(methacryloyloxy)-ethyl-

10.1021/bm800450b CCC: $40.75  2008 American Chemical Society Published on Web 09/05/2008

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N,N,N-trimethylamonium chloride, guaiacol, and guaiacol sulfonic acid (4-hydroxy-3-methoxybenzenesulfonic acid potassium salt) were supplied by Sigma-Aldrich. The Coomassie Brilliant Blue reagent was purchased from BioRad. All other chemicals and solvents were of analytical grade. Cultivation of Trametes hirsuta (IMA2002) and purification of the laccase are described elsewhere.12 In brief, three steps purification was performed. After ultrafiltration with diatom earth, the laccase was further purified chromatographically using anion exchange and size exclusion techniques. Laccase Activity and Protein Assay. Laccase activity was elaborated on ABTS as substrate (436 ) 29300 M-1 cm-1). After the addition of 100 µL of enzyme to 1150 µL of citrate phosphate buffer (25 mM, pH 4.5) containing 250 µL of 5 mM ABTS, the formation of green radicals was followed at 436 nm using a Carry 50 UV-vis spectrophotometer. Thereby, one nkat corresponds to the generation of 1 nmol radicals per second. Plasma Treatment of Polypropylene Fabric. Nonwoven polypropylene fabric (melt blown nonwoven fabric, weight/area ) 45 g/m2) was immersed in an aqueous solution of 2-aminoethyl-methacrylate hydrochloride (20% (w/v)) and wrung at a pressure of 2 bar and velocity of 4 m min-1) by FL500/V foulard machine (Gavazzi s.r.l.). The samples were irradiated at different powers with argon plasma over an appropriate treatment time (180 s)26 and a radiofrequency of 13.56 kHz. The plasma reactor consisted of a cylindrically shaped AISI 316 stainless steel chamber (V ) 7850 cm3) able to work within a power range of 0-600 W was used, equipped with a high vacuum efficiency pump (18 m3/h) and four inlets for the introduction of the process gases. The distance between the electrodes (diameter of 40 cm) applied in the experimental setup was 80 mm and the samples were placed on a grid positioned in the middle of the two electrodes. Treated fabrics were washed with distilled water and dried in a ventilated oven at 70 °C for 1 h. The grafting of 2-(N,N-dimethylamino)ethyl methylacrylate and 2-(methacryloyloxy)-ethyl-N,N,N-trimethylamonium chloride was performed similarly in aqueous solutions, whereas 2-(tert-butylamino)ethyl-2-methylacrylate was dissolved in acetone (20% (w/v)) prior to plasma grafting. Enzymatic Grafting of Plasma-Pretreated Polypropylene Fabric. Plasma-pretreated polypropylene fabrics (2.5 × 2.5 cm) were incubated in 10 mL of citrate phosphate buffer (25 mM, pH 5.0) containing different amounts of guaiacol sulfonic acid (GSA; 0.4-2.0 mmol) with laccase (6 nkat) for 2 h at 50 °C. After the desired incubation time, the reaction was stopped by adding 1 mL of 1 M sodium fluoride, the fabrics were then washed under tap water, rinsed three times with distilled water, and dried in a desiccator for 72 h. Different samples were prepared as control, one with nonpretreated fabric incubated with enzyme and gauaiacol sulfonic acid and another with pretreated fabrics immersed only in GSA. These series were complemented by grafting using polyphenols. Therefore, GSA was incubated with laccase under the same conditions as above, followed by adding the fabric to the reaction mixture. To mask the remaining radicals on the polyphenol, amino ethane was added before incubation of the fabric. ATR-FTIR Measurements. Monomer plasma grafting, as well as enzymatic postgrafting, was investigated by means of infrared spectroscopy. ATR FTIR spectra of plasma grafted monomers were recorded at room temperature using a Perkin-Elmer spectrometer with an ATR zinc selenide cell. The integrated carbonyl stretching adsorptions from the normalized spectra were considered for a semiquantitative evaluation of the methacrylic monomers grafting efficiency. An average of 16 scans was applied using a resolution of 4 cm-1. ATR FTIR spectra of the enzymatically coated fabrics were obtained by recording 16 scans over a range of 4000 cm-1 to 650 cm-1 with a resolution of 4 cm-1 and intervals of 1 cm-1, using a Perkin-Elmer apparatus (Spectraflash 600 plus, Swiss) equipped with diamond crystal (Specac) ATR device. XPS Analysis. The XPS analysis, also known as electron spectroscopy for chemical analysis (ESCA), was carried out on a PHI-TFA

Schroeder et al. Table 1. Grafting Efficiency of Primary Amine Monomer on Polypropylene Fabric Using Different Discharge Powera

a

power (W)

mmol amine/g fabric

100 200 300 400

16.8 52.3 159.2 84.6

180 s.

XPS spectrophotometer produced by Physical Electronic Inc. The untreated and differently treated polypropylene fabrics were fixed by metallic plate with a hole in the metallic sample’s holder. Thereby, three different sample locations were analyzed from two experimental series. The analyzed area was 0.4 mm in diameter, and the analyzed depth was about 3-5 nm. The sample’s surfaces were excited by X-ray radiation from a monochromatic Al source at photon energy of 1486.6 eV. Wide-scan spectra were acquired at pass energy of 187 eV for identification and quantification of elements on the surfaces of the fabrics. The atomic concentrations of surface region were calculated using relative sensitivity factors provided by the spectrometer’s manufacturer. High-energy resolution spectra of C 1s, O 1s, and S 2p were acquired with energy resolution of about 0.6 eV with analyzer pass energy of 29 eV, to reveal binding energies of XPS peaks associated with different chemical states of elements. A low-energy electron gun-neutralizer was used due to sample charging. The accuracy of binding energies was about (0.4 eV. Three places on all samples were analyzed for sample homogeneity. The results for the composition between different places scatter was about 5%, which reflects a fairly homogeneous surface composition. Enzymatic Coupling of Guaiacol Sulfonic Acid and Hexylamine. To 40 mL of citrate phosphate buffer (25 mM, pH 5.0) containing 1.3 mL (1 g; 10 mmol) of hexylamine and immobilized laccase and 10 mL of a buffered (phosphate buffer, 25 mM, pH 5.0) solution of guaiacol sulfonic acid was added, giving a final concentration of 1.0 mmol. The reaction mixture was incubated for 2 h at 50 °C. After the desired incubation time samples were taken for the detection of coupling products by HPLC-MS. The MS analysis was done on an Agilent MSD (Waldbronn, Germany) with direct injection using the following parameters: drying gas temperature, 350 °C; drying gas pressure, 40 psi; drying gas flow (nitrogen), 10 L min-1; capillary voltage, 3500 V; fragmentor voltage, 70 V; mass range, 70-400 m/z. The data were analyzed using the software Chemstation Rev. A 10.01 (Agilent, Waldbronn, Germany). For measurement in positive mode 0.01% acetic acid was added. To check for fragments formed during the massmetry, the fragmentor voltage was set from 25 to 150 V.

Results Plasma Treatment of the Polypropylene. The binding of 2-aminoethyl-methylacrylate (AEMA) hydrochloride on the surface of the polypropylene were proved by the stretching band of carboxyl group (νs OsCdO) at 1730 cm-1 as well as peaks between 1650 cm-1 and 1490 cm-1, which can be associated with N-H deformation. The peaks at 1450 cm-1 and 1380 cm-1 relate to CH3 and CH2 deformations resulting from the polypropylene backbone and are therefore shown in all samples. Those peaks between 2950 and 2800 cm-1, corresponding to various aliphatic CsH stretching modes, were used for normalization. Polypropylene treated with argon plasma only was also investigated to prevent any contribution from any secondary reaction on the investigated signal. As shown in Table 1, the best yield for the grafting of AEMA in 180 s was obtained at a discharge power of 300 W, indicating that the generation of macro-radicals onto the polypropylene surface is optimal under those conditions. Grafting yields of

Laccase-Induced Grafting on Polypropylene Table 2. Grafting Efficiency of Different Amine Monomers on Polypropylene Fabric Using a Discharge Power of 300 Wa

a

monomer

mmol amine/g fabric

primary amine tertiary amine quaternary amine

159.2 134.5 150.7

180 s.


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Table 3. Absorbance at Different Wave Numbers of FTIR Spectra of Differently Plasma-Pretreated Polypropylene (PP)a wave numbers sample

1250 cm-1

1104 cm-1

1049 cm-1

PP untreated; control sample PP grafted with primary amino groups PP grafted with tertiary amino groups PP grafted with quaternary amino groups

0.14

0.14

0.14

0.96

1.19

1.25

0.21

0.20

0.21

0.17

0.16

0.17

a After enzymatic coating with guaiacol sulfonic acid normalized in the area between 2950 and 2800 cm-1.

Table 4. Ratio of Different Atoms from XPS Spectra of Differently Treated Polypropylene ratio

Figure 1. Correlation map for polypropylene fabric grafted with primary amine (AEMA) at a power of 300 W (180 s), with the spectra recorded for the treated sample within the spectral range of 1800-1500 cm-1. The map showed that the spectra of the amino monomer are homogeneously distributed onto the polypropylene surface.

2-aminoethyl-methacrylate hydrochloride (primary amine), 2-(N,Ndimethylamino)ethyl methylacrylate (tertiary amine), and 2-(methacryloyloxy)-ethyl-N,N,N-trimethylamonium chloride (quaternary amine) performed at 300 W and summarized in Table 2 support this observation. Investigation of the carboxyl stretching band’s adsorption ratio at 1720 cm-1 (attributed to AEMA) and the bending of the CsH group at 1440 cm-1 (attributed to polypropylene) confirmed the homogeneity in monomer distribution (Figure 1). Enzymatic Grafting of Plasma-Pretreated Polypropylene. Plasma-pretreated polypropylene fabrics were treated with guaiacol sulfonic acid (GSA) in the presence of a laccase. As control, no plasma-pretreated polypropylene, immersed with GSA and enzyme, were taken. The effects of different amine monomers grafted onto the polypropylene surfaces regarding further GSA laccase coating were studied to elaborate the nature and mechanism of the enzymatically induced coating. As shown in Table 3, the use of a primary amine monomer resulted in the most successful grafting. The reddish coloration of the treated surfaces indicated that GSA was coupled onto the plasmapretreated polypropylene only in the presence of laccase. The color formation was also observed for the enzymatic polymerization of GSA. These results are confirmed also by XPS analyses (Table 4). The addition of ethane amine to the reaction mixture resulted in amplification of the color, and no grafting effect was exhibited on the polypropylene. ATR-FTIR Analysis of Plasma-Treated and PostGrafted Polypropylene. As is evident from Figure 2 and from the results in Table 3, new peaks appeared in the IR spectra after enzymatic treatment. The band at 1250 and 1228 cm-1

sample

O/C

N/C

S/C

PP untreated (blank) PP plasma pretreated, immersed with GSA (control sample) PP plasma pretreated, 100 mg GSA and laccase PP plasma pretreated, 250 mg GSA and laccase 250 mg GSA and laccase without plasma pretreatment (control sample) reaction mixture of GSA and laccase reaction mixture of GSA and laccase, stopped with ethanamine

0.030 0.153

0.005 0.052

0.001

0.306

0.089

0.010

0.311

0.108

0.013

0.090

0.027

0.001

0.283

0.113

0.008

0.255

0.130

0.003

may be attributed to the grafting of guaiacyl and syringyl units.27,28 The area between 2950 and 2800 cm-1, corresponding to various aliphatic C-H stretching modes, was used for normalization. The peak at 1049 cm-1, characteristic for the stretching band of aromatic sulfates,29 showed increased absorbance only on the plasma-pretreated and -postgrafted polypropylene in the presence of the GSA and laccase. The guaiacyl peak at 1250 cm-1 was also high for all plasma-treated samples. Furthermore, the coated samples also showed a broad peak at 1639 cm-1, which can be related to the quinone. XPS Analysis of Plasma-Treated and Post-Grafted Polypropylene. Figure 3 shows the XPS wide-scan spectra obtained on the untreated polypropylene fabric, on the fabric treated by 250 mg GSA without plasma pretreatment, and on the sample pretreated with plasma and then treated in 250 mg GSA and laccase. In these spectra, four characteristic peaks for oxygen (O 1s; 533 eV), carbon (C 1s; 286 eV), and nitrogen (N 1s; 401 eV) were identified, whereas sulfur (S 2p; 168 eV) is hardly seen on this scale due to low concentration. The ratios of atomic concentrations relating to carbon were calculated for oxygen, nitrogen, and sulfur, and the results are summarized in Table 4. The highest ratio for S/C concentration was found for the sample pretreated by plasma and then treated with 250 mg GSA and laccase. The XPS method is not only able to identify and quantify the presence of elements on the surface but can also identify chemical bonding of elements on the surface by deconvolution of elemental XPS spectra into corresponding peaks. Figure 4 shows high-energy resolution C 1s spectra of untreated polypropylene fabrics, fabrics pretreated by plasma

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Figure 2. FTIR spectra of (a) untreated polypropylene fabric, (b) polypropylene pretreated with plasma, and (c) polypropylene pretreated with plasma and incubated in 50 mg GSA with 50 µL laccase.

Figure 3. XPS wide-range spectra obtained on untreated polypropylene fabrics (a), on fabrics treated with 100 mg GSA in the presence of laccase without plasma pretreatment (b), and on fabrics pretreated with plasma and further treated with 100 mg GSA in the presence of laccase (c).

and fabrics pretreated by plasma and then treated with GSA in the presence of laccase. These spectra were deconvoluted into subpeaks, and according to the literature, the following peak assignment was carried out.30-32 For all cases, the C 1s spectrum from untreated fabric shows one strong peak at a binding energy of 285.0 eV assigned to CsC and CsH bonds and actually expected for the bonding of polypropylene backbone’s carbon atoms. All spectra also contained a peak at 286.5 eV associated with CsO bonds and a peak at 288.0 eV associated with CdO or OsCsO bonds. C 1s spectra from the plasma-pretreated fabrics, as well as from the plasma/GSA-treated fabrics (Figure 4b,c), showed an additional peak at 289.0 eV associated with OsCdO group. For the fabrics pretreated by plasma and then treated with GSA, the S 2p spectrum is presented in Figure 5, showing

that sulfur was present on the surface. The curve-fitting analysis of the S 2p spectrum showed that sulfur atoms are bonded with two chemically different bonds. A minor double peak at 163.7 eV is associated with S2- oxidation state, whereas the major double peak at binding energy of 168 eV is associated with S3+ and S4+ oxidation states. Enzymatic Coupling of Guaiacol Sulfonic Acid and Hexylamine. Chromatographic purification of the reaction mixture was performed to conclusively identify the products. An adequate resolution was achieved. Using mass spectroscopy in negative ion mode, two peaks at different retention times were identified (m/z 203.7 [M - H]-) and (m/z 301.7 [M H]-), respectively. They can be clearly associated to the initial GSA and the hexylamine conjugate.

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Figure 4. XPS high-energy resolution spectrum C 1s obtained on (a) untreated polypropylene fabrics, (b) on plasma-pretreated fabrics, and (c) on fabrics that were pretreated with plasma and then incubated with 250 mg GSA in the presence of 50 µL laccase. Different peaks associated with different chemical states of carbon atoms are shown.

Discussion Plasma Pretreatment. Polypropylene fabric was pretreated with argon plasma to facilitate an enzymatically induced coating of polyolefine’s inert surface. Thereby, the inertness and chemical simplicity of polypropylene may allow a closer look at those mechanisms and conditions for the tailored enzymatic coupling and functionalization of surfaces. Plasma treatments can be performed under a wide-range of conditions and various parameters. The impact of the applied plasma on the grafting yield was elaborated during the first stage. The stretching band of the carboxyl group (νs OsCdO) at 1730 cm-1 is due to the

plasma grafting of the methacrylate monomer. Corresponding to various aliphatic CsH stretching modes, peaks between 2950 and 2800 cm-1 were used for normalization. Macro-radicals generated by the active species in the plasma chamber are induced, as previously investigated,33 by increasing the applied power enhancement. As was also demonstrated, optimum power has to be verified because secondary reactions, such as recombination or deactivation, are promoted by increasing the amount of radicals. Accordingly, to enhance the deposition of functional monomers onto the polypropylene surface, the experiments were investigated within the range of 100-400 W.

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Figure 5. XPS high-energy resolution spectrum S 2p obtained on fabrics pretreated with plasma and further incubated with 100 mg GSA in the presence of 50 µL laccase. Different peaks associated with different chemical states of sulfur atoms are shown.

The effectiveness of plasma treatment on the grafting of functional molecules was characterized by means of ATR-FTIR spectroscopy, as reported elsewhere.26 It was found that the most suitable power for ensuring maximum grafting yield is 300 W (Table 1). The observed trend shows that, by increasing the applied power, the activation reaction is promoted until etching and radical recombination reactions. A chemical imaging analysis was carried out to determine the homogeneity of the treatment; the distribution of a relevant signal all-over a portion of the sample (0.2 × 0.2 cm2) was investigated for the sample treated under optimized conditions. The correlation map with the spectra recorded for the treated sample (Figure 1) showed that the amine was distributed evenly over the surface. Different amino groups, primary, tertiary, and quaternary, were investigated to provide an insight into the chemistry of the following linking reaction. As reported in literature, in a polymerization reaction the reactivity of the methacrylate monomers is unaffected by the polarity of the molecules but, in the case of n-alkyl acrylates and methacrylates, as the length of the n-alkyl group increases, the grafting percentage and homopolymer content decreases; this was attributed to their increase in steric hindrance. In our case, the steric hindrance increased by increasing the substituents in the amino groups, so a decrease of the grafting yield was expected from primary to quaternary amine. The experimental data, recorded at optimum power for promoting polypropylene activation, confirmed that the grafting efficiency is unaffected by the polarity of different monomers (Table 2) and that negligible effects in the free radical grafting process are induced by steric hindrance, probably because the amino substituent is too far from the methacrylic reactive group. Enzyme-Induced Grafting of Guaiacol Sulfonic Acid. Polypropylene fabrics, differently treated as well as untreated, exhibit a simple XPS survey spectra containing four characteristic peaks for oxygen (O 1s; 533 eV), carbon (C 1s; 286 eV), nitrogen (N 1s; 401 ev), and sulfur (S 2p; 168 eV). Ratios relating to the carbon were calculated for oxygen, nitrogen, and sulfur and are summarized in Table 4. Certain statements can be made based on these results. First, the amount of sulfur is related to the presence of GSA. Second, the amount of grafted GSA onto the polymer surface increased when increasing concentration was applied during the enzymatic process. Third,

both enzyme and pregrafted amino groups are essential for a successful grafting. As indicated in Figure 4a, the XPS C 1 spectrum from the untreated fabrics shows, in addition to a single hydrocarbon peak, a small amount of oxygen-related carbon bonds (CsO and CdO), which can be attributed to contamination of the fabric’s surface during handling of the samples under atmospheric pressure. The relative weight of the CsO groups increased after plasma treatment. The main change in the chemical states of carbon after plasma treatment was the appearance of carboxylic groups on the surface (Figure 4b). We relate this group with the grafted methylacrylate in correlation with previous studies.34 The surfaces of polypropylene fabrics after plasma pretreatment, followed by treatment with 250 mg GSA in the presence of laccase (Figure 4c), showed significant increases of CsO, CdO, OsCsO, and OsCdO bonds with respect to the peak relating to native CsC/CsH bonds. This demonstrates the efficiency of grafting and strong oxidation at the surface. We should note that the increases of some of these peaks can also be attributed to CsN. Besides the chemical states of carbon at the surface, the data on composition (O/C ratio in Table 4) also show strong oxidation. Further evidence of successful grafting is given by the XPS S 2p spectrum (Figure 5) from the surfaces of polypropylene fabrics after plasma pretreatment, followed by treatment with 250 mg GSA in the presence of laccase. It shows the S2-, S3+, and S4+ oxidation states. While S2- probably originates from impurities (e.g., adsorbed enzyme), S3+ and S4+ are exclusively related to the grafted sulfonic acid. Because a major part of the sulfur is contributed by the S3+ and S4+, these data significantly confirm that the amount of sulfur corresponds to a GSA coating onto polypropylene fabrics. These results were confirmed by ATR-FTIR analysis relating to the peak at 1049 cm-1, characteristic for the stretching band of aromatic sulfates.29 This peak at 1049 cm-1, as well as the guaiacyl peak at 1250 cm-1,27 were used to evaluate the impacts of the different amino groups on the coating (Table 3). The study exhibited clearly, that only the primary amino groups acted as reactive partners. Thus, the possibility of a simple adsorption, as well as an ionic interaction, can be ruled out by the lack of grafting on quaternary and tertiary amino groups, respectively. The formation of nitrogen-carbon bonds was described for the laccase-catalyzed synthesis of 3-(3,4-dihydroxyphenyl)-

Laccase-Induced Grafting on Polypropylene

propionic acid derivates,7 for the amination of p-hydroquinones,8 for the cross-coupling of 4-aminobenzoic acid with paradihydroxylated compounds9 and for the synthesis of Tinuvin.11 During this study, cross-coupling appeared to proceed via quinone substructures. A broad peak at 1639 cm-1 in the FTIR spectra was assigned to quinones. We recently hypothesized that a laccase-induced cross-linkage of benzotriazol with phenol occurred via nucleophilic attack of the nitrogen at the quinone substructures.11 Coupling of primary amine would follow this reaction mechanism and validates the results from the FTIR analysis. Besides the described coupling reaction, laccase-induced polymerization occurs in parallel. These homomolecular couplings have been reported for organic synthesis, as well as coating using laccase-induced oxidation.12 However, there are different possibilities for laccase-induced grafting. Besides the amination described above, the grafting of polyphenols also occurs in parallel.

Conclusions The present work describes a combination of two sensitive procedures using a combination of plasma pretreatment and enzymatic postgrafting for generating a novel functionalization of inert polymer surfaces under mild conditions without damages and extensive use of chemicals over a wide range. The efficiency of enzymatic grafting, using laccase and guaiacol sulfonic acid (GSA), on methacrylate monomer plasma-pretreated polypropylene surface is investigated and determined by ATR-FTIR spectroscopy and XPS. The grafting of GSA with amino monomer is determined by HPLC-MS and using hexylamine as a model substrate. The major advances of the combination procedure is due to the fact that the enzymatic reaction is not restricted to polymers already containing functional groups, such as natural (lignin, cellulose) or synthetic (polyvinyl alcohol), but can also be extended to inert polymers such as polyolefines. Acknowledgment. This research was supported by a Marie Curie Transfer of Knowledge Fellowship of the EC sixth FP under Contract No. MTKD-CT-2005-029540, by Slovenian Research Agency (J2-7018-0795), and Slovenia-Italy (C5) scientific and technological collaboration programme.

References and Notes (1) Thurston, C. Microbiology 1994, 140, 19–26. (2) Manda, K.; Go¨rdes, D.; Mikolasch, A.; Hammer, E.; Schmidt, E.; Thurow, K.; Schauer, F. Appl. Microbiol. Biotechnol. 2007, 76, 407– 416. (3) Ciecholewski, S.; Hammer, E.; Manda, K.; Bose, G.; Nguyen, V. T. H.; Langer, P.; Schauer, F. Tetrahedron 2005, 61, 4615–4619. (4) Ponzoni, C.; Beneventi, E.; Cramarossa, M. R.; Raimondi, S.; Trevisi, G.; Pagnoni, U. M.; Riva, S.; Forti, L. AdV. Synth. Catal. 2007, 34989, 1497–1506. (5) Hajdok, S.; Leutbecher, H.; Greiner, G.; Conrad, J.; Beifuss, U. Tetrahedron Lett. 2007, 4829, 5073–5076.


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(6) Witayakran, S.; Ragauskas, A. J. Green Chem. 2007, 9, 475–480. (7) Mikolasch, A.; Hammer, E.; Jonas, U.; Popowski, K.; Stielow, A.; Schauer, F. Tetrahedron 2002, 58, 7589–7593. (8) Niedermeyer, T. H. J.; Mikolasch, A.; Lalk, M. J. Org. Chem. 2005, 70, 2002–2008. (9) Manda, K.; Hammer, E.; Mikolasch, A.; Niedermeyer, T.; Dec, J.; Jones, A. D.; Benesi, A. J.; Schauer, F.; Bollag, J. M. J. Mol. Catal. B: Enzym. 2005, 41, 86–92. (10) Manda, K.; Hammer, E.; Mikolasch, A.; Go¨rdes, D.; Thurow, K.; Schauer, F. Amino Acids 2006, 31, 409–419. (11) Schroeder, M.; Pereira, L.; Rodriguez Couto, S.; Erlacher, A.; Schoening, K.-U.; Cavaco-Paulo, A.; Guebitz, G. M. Enzyme Microb. Technol. 2007, 40, 1748–1752. (12) Schroeder, M.; Aichernig, N.; Guebitz, G. M.; Kokol, V. Biotechnol. J. 2007, 2, 334–341. (13) Lund, M.; Ragauskas, A. J. Appl. Microbiol. Biotechnol. 2001, 55, 699–703. (14) Kim, S. Y.; Zille, A.; Murkovic, M.; Guebitz, G. M.; Cavaco-Paulo, A. Enzyme Microb. Technol. 2007, 40, 1782–1787. (15) Wei, Q.; Li, Q.; Wang, X.; Huang, F.; Gao, W. Polym. Test. 2006, 25, 717–722. (16) Lai, J.; Sunderland, B.; Xue, J.; Yan, S.; Zhao, W.; Folkard, M.; Micheal, B. D.; Wang, Y. Appl. Surf. Sci. 2006, 252, 3375–3379. (17) Muehlhan, C.; Weidner, S.; Friedrich, J.; Nowack, H. Surf. Coat. Technol. 1999, 116-119, 783–787. (18) Yuan, X.; Jayaraman, K.; Bhattacharyya, D. Composites, Part A 2004, 35, 1363–1374. (19) Seo, Y.; Ninh, T. H. Polymer 2004, 45, 5387–5392. (20) Oehr, C.; Mu¨ller, M.; Elkin, B.; Hegemann, D.; Vohrer, U. Surf. Coat. Technol. 1999, 116-119, 25–35. (21) Kurdi, J.; Ardelean, H.; Marcus, P.; Jonnard, P.; Arefi-Khonsari, F. Appl. Surf. Sci. 2002, 189, 119–128. (22) Gendry, M.; Durand, J.; Cot, L. Thin Solid Films 1987, 149, 313– 324. (23) Cernakova, L.; Kovacik, M.; Zahoranova, A.; Cernak, M.; Mazur, M. Plasma Chem. Plasma Process. 2005, 25, 427–437. (24) Badyal, J. P.; Ward, L.; York, D. W.; Brooker, A. T.; Summers, S.; Crowther, J. M.; Roberts, N. P. S.; Yates, A. T.; Saswati, D. A method for providing laundry-durable finish for textile by plasma-induced graft polymerisation WO 2005028741, 2005. (25) Fatarella, E.; Nesti, S.; Kokol, V.; Schroeder, M.; Cortez, J. Functionalised polymer materials and production method thereof MI2007A001611, 2007. (26) Castelvetro, V.; Fatarella, E.; Corsi, L.; Giaiacopi, S.; Ciardelli, G. Plasma Process. Polym. 2006, 3, 48–57. (27) Lawther, J. M.; Sun, R. Ind. Crop. Prod. 1996, 5, 87–95. (28) Sun, R. C.; Tomkinson, J.; Wang, S. Q.; Zhu, W. Polym. Degrad. Stab. 2000, 67, 101–109. (29) Taddei, P.; Arosio, C.; Monti, P.; Tsukada, M.; Arai, T.; Freddi, G. Biomacromolecules 2007, 84, 1200–1208. (30) Moulder, J. F.; Stickle, W. F.; Sobol, P. E.; Bomben, K. D. Handbook of X-Ray Photoelectron Spectroscopy; Physical Electronics Inc.: Eden Prairie, Minnesota, 1995. (31) Briggs, D. Surface analysis of polymers by XPS and static SIMS; Cambridge University Press: New York, 1998. (32) Beamson, G.; Briggs, D. High Resolution XPS of Organic PolymerssThe Scienta ESCA300 Database; J.Wiley & Sons: Chichester, 1992. (33) Andreozzi, L.; Castelvetro, V.; Ciardelli, G.; Corsi, L.; Faetti, M.; Fatarella, E.; Zulli, F. J. Colloid Interface Sci. 2005, 289, 455–465. (34) Kwon, O. J.; Myung, S. W.; Lee, C. S.; Choi, H. S. J. Colloid Interface Sci. 2006, 295, 409–416.

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