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Surface modification of poly(ethylene terephthalate) (PET) fibers by a cutinase from Fusarium oxysporum
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PRBI-10502; No. of Pages 8
Process Biochemistry xxx (2015) xxx–xxx
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Surface modification of poly(ethylene terephthalate) (PET) fibers by a cutinase from Fusarium oxysporum Maria Kanelli a , Sozon Vasilakos b , Efstratios Nikolaivits a , Spyridon Ladas c , Paul Christakopoulos d , Evangelos Topakas a,∗ a Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 5 Iroon Polytechniou Str., Zografou Campus, Athens 15780, Greece b MIRTEC, Materials Industrial Research & Technology Center S.A, Athens 17342, Greece c Surface Science Laboratory, Department of Chemical Engineering, University of Patras, 26504 Rion, Patras, Greece d Biochemical and Chemical Process Engineering, Division of Sustainable Process Engineering, Department of Civil, Environmental and Natural Resources Engineering, Luleå University of Technology, SE-97187 Luleå, Sweden
a r t i c l e
i n f o
Article history: Received 5 June 2015 Received in revised form 27 August 2015 Accepted 30 August 2015 Available online xxx Keywords: Enzymatic modification Poly(ethylene)terephthalate (PET) fabric Cutinase FT-IR ATR XPS Dyeing
a b s t r a c t Synthetic polyester fabrics occupy a great part of the textile industry production satisfying variable ordinary needs. Nonetheless, their high hydrophobicity constitutes an important weakness that impedes process manufacture, as well as permeability and evaporation of sweat when used in clothing industry. The enzymatic treatment of these materials is a modern and eco-friendly procedure that aims at the increase of the hydrophilicity through superficial modification. In this study, the enzymatic surface hydrolysis of poly(ethylene terephthalate) (PET) fabric is succeeded using a recombinant cutinase from Fusarium oxysporum. The effect of various parameters is studied for the enzymatic modification of PET, such as temperature, pH, enzyme loading and reaction time. The optimal parameters are found to be 40 ◦ C, pH 8, and 1.92 mg enzyme loading per gram of fabric. The controlled enzymatic hydrolysis of PET textile is further confirmed and characterized using various spectroscopic and analytical methods, including Fourier Transform Infrared (FT-IR) in the Attenuated Total Reflectance mode (ATR) and X-ray photoelectron spectroscopy (XPS). Tensile test and dyeability analyses were also employed achieving a K/S increase up to 150%, confirming the successful surface modification without degrading the quality of the starting material. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction In modern times, technology and science have met advances that have encouraged fundamental changes and improvements in several industrial sectors, such as textile industry. Synthetic fabrics prove to be useful in many applications because of their advantages as far as their physical properties are concerned, presenting strength and elasticity [1]. In addition, synthetic textiles present less wrinkles, fast drying properties and can be produced massively in a short period of time with lower cost as opposed to natural textiles. Their main drawback is the low hydrophilicity that impedes the process of finishing and dyeing, as well as the permeability and evaporation of sweat [2–5] and stain resistance [6]. Towards this
∗ Corresponding author. Fax: +30 210 7723163. E-mail addresses: [email protected], [email protected] (E. Topakas).
direction, enzymatic treatment consists an innovative biotechnological and eco-friendly approach to increase fabrics’ hydrophilicity and improve their properties, as opposed to chemical treatment, which has higher manufacturing cost and energy consumption and leads to strength loss and degradation of fabric bulk properties [7–12]. Enzymes are macromolecular compounds that present selectivity and cause superficial modification of the fibers, as they cannot diffuse further into the fibers’ bulk part because of their size [13–15]. Modification of poly(ethylene terephthalate) (PET) fibers aims at the surface hydrolysis of the ester bonds releasing carboxyland hydroxyl- end groups [12,16] and as a result terephthalic ions in the reaction supernatant. According to literature, enzymes suitable for polyester modification are esterases, lipases and cutinases [16–18]. Cutinases are serine esterases that belong to the ␣/-hydrolase fold family. Their role in nature is the hydrolysis of ester bonds in cutin, a wax biopolyester found in plant cuticle of aerial surfaces of plants [19]. Furthermore, cutinases have the ability to hydrolyze
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the ester bonds of synthetic polyesters and this ability makes them suitable for many industrial applications such as the surface modification of PET [17,20]. In literature, commonly used hydrolases for PET surface modification are cutinases from Fusarium solani sp. pisi [7–9,21]. However, it has previously been reported that a cutinase from Fusarium oxysporum proved to be more efficient as far as PET fabric modification is concerned, presenting higher activity in comparison to a cutinase from F. solani sp. pisi. [22]. In addition, cutinase from F. oxysporum is considered as highly organic solvent-tolerant, indicating its potential in biotechnological applications including the essential area of Biocatalysis [20]. In the present work, the ability of a F. oxysporum cutinase (FoCut5a) for the surface hydrolysis of PET fibers was investigated. The potential of this enzyme in PET modification was proved by its capability to hydrolyze two PET model substrates, bis(benzoyloxyethyl) terephthalate (3PET) and commercial bis(2hydroxyethyl) terephthalate (BHET) [23]. Various parameters were studied aiming at the efficient surface modification of PET textile, such as the effect of temperature, pH, incubation time and enzyme loading. The extent of the enzymatic treatment was monitored by the quantification of terephthalic acid (TPA) and its derivatives (TPA equivalents) released in reaction supernatant. In addition, the modification of the polymeric material was further justified through the application of different analytical techniques, including FTIR-ATR, SEM, XPS, DSC, TGA, as well as through dyeability tests. 2. Material and methods 2.1. Materials, chemicals and enzymes Commercial PET woven fabric with tricot knit was kindly supplied by Colora S.A (Thessaloniki, Greece) with density 126 columns in−1 and 58 rows in−1 , weight 52.90 g m−2 and thickness 42 m. The polyester fabric was washed with detergent Felosan NFG from CHT Bezema (Tübingen–Germany) for the removal of paraffins used during weaving. Recombinant cutinase from F. oxysporum was heterologously expressed in Escherichia coli BL21 (DE3) and purified by immobilized metal affinity chromatography, as described previously [23]. TPA and BHET were supplied by Merck (Whitehouse Station, New Jersey) and Sigma (St. Louis, MO), respectively. Trifluoroacetic acid and acetonitrile were of HPLC grade (Sigma, St. Louis, MO). The reactive dyes used were Novacron Deep Cherry S–D, Novacron Yellow S-3R (Everberg, Belgium) and Jakazol Black 133% (Huntsman, Gujarat, India). All other chemicals were of analytical grade.
spectrophotometrically at 241 nm in a BOECO S-20 Spectrophotometer (Hamburg, Germany). Possible hydrolysis products were TPA, BHET and mono(2-hydroxyethyl) terephthalate (MHET). A calibration curve of TPA was used for the calculation of the total amount of products released since all three products have the same molar absorption due to their carbonyl groups [8]. Samples of 0.6 mL of the supernatant were taken and the enzyme was precipitated with the addition of 1:1 methanol prior to centrifugation. Subsequently, in 1 mL of the supernatant sulfuric acid was added (18 mM) in order to convert TPA and any of its derivative ions into their neutral counterparts. Control samples without the addition of the enzyme or without the addition of fabric were measured as well. The reaction supernatants from the hydrolysis at optimal conditions, were concentrated and analyzed by HPLC, using a SHIMADZU LC-20AD pump equipped with a Jasco UV-975 detector recording at 241 nm. The reversed phase column Eurospher-100 C18 from KNAUER (Berlin) was maintained at room temperature. A linear gradient method, involving 1% trifluoroacetic acid solution and acetonitrile as eluents, at a flow rate of 0.8 mL/min, was applied, as previously described [8].
2.4. Dyeing and color measurements Polyester fabrics were dyed in 390 cm3 glass tubes in a laboratory scale dyeing Ahiba Texomat machine (Datacolor, Lawrenceville, NJ, USA). According to dyeing procedure, 55 g L−1 of Na2 SO4 and 2% w/w of each dye were dissolved and introduced in the glass tubes where fabrics were also introduced. The dye bath was heated up to 60 ◦ C and after 20 min, 5 g L−1 Na2 CO3 was added. Finally, 0.4 g L−1 of NaOH were incorporated 30 min before the end of the dyeing procedure. For the fabric neutralization to pH 6 a small amount of HCOOH was added after removing the fabrics from each dye bath, and then, a wash procedure followed at 95 ◦ C with soap (Cibapon R) for 1–2 h, in order to remove the non-reacted dye. Color changes (E) were evaluated with a color tristimulus colorimeter (Data Color International, Spectraflash SF450) (Lawrenceville, NJ, USA). The spectrophotometer was calibrated according to the manufacturer’s instructions, using the supplied black and white calibration standards. The color alterations were calculated using the CIE L*a*b* System, established by the “Commission Internationale de l’Eclairage-CIE” according to ASTM D 2244-68. For each sample, four repeated measurements were taken to determine the color coordinates L, a, b. “L” indicates the brightness, “a” describes the red-green content and “b” the yellow-blue content. Color change can be calculated using Eq. (1):
2.2. Enzymatic treatment of polyester fabric Polyester fabrics were cut into pieces of 0.5 g and were incubated in glass vessels with bath ratio of textile mass over buffer mass 1:50 under stirring (170 rpm) for 24 h. For the enzymatic treatment, different parameters were studied including the effect of temperature (25–50 ◦ C), pH (phosphate buffer 100 mM pH range 5.8–8.0, glycine-NaOH buffer 100 mM pH range 9.0–10.0), incubation time (0–24 h) and enzyme loading (0.096–3.840 mg g−1 of fabric). After enzymatic treatment, the fabrics were washed with 2 g L−1 Na2 CO3 at 60 ◦ C for 1 h and afterwards double-washed with deionized water for 1 h, as previously described [13]. All experiments were performed in duplicate. 2.3. Monitoring of enzymatic modification For the determination of the polyester hydrolysis degree, TPA or its derivatives released in samples’ supernatants were quantified
E =
(L2 − L1 )2 + (a2 − a1 )2 + (b2 − b1 )
2
(1)
Studies have concluded that the E = 1.0 is a color difference that can be perceived by 50% [24] by the human eye, while E = 2.0 is a color difference that can be seen at 100% [25]. In order to determine the dye absorption, the color strength (K/S) of the dyed samples was measured at the wavelength of minimum reflection by using Eq. (2): K (1 − R)2 = S 2R
(2)
where, R refers to the reflection value of the sample. Practically, the E value is used when it is desired to compare two shades. The color strength, i.e. the ratio K/S, is the parameter that is used to evaluate the dyeing process and dyeing capacity.
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2.5. Tensile test Tensile tests were carried out in an SDL Atlas tensile test machine (Rock Hill, SC, USA) operating at grip separation speed of 50 mm min−1 . All measurements were run at 25 ◦ C with specimen dimensions 3 cm width × 10 cm length. 2.6. Spectrometric and physicochemical analysis of modified fabrics The chemical structure of the enzymatically treated PET was examined by Fourier Transform-Infrared Spectroscopy Attenuated Total Reflectance (FT-IR ATR) analysis using a Bruker Tensor 27 FTIR Spectrometer, equipped with a diamond of simple reflection with penetration depth ∼2 m. The changes on the surface of the modified textiles were spotted with Scanning Electron Microscopy (SEM) using FEI Quanta 200 (Hillsboro, Oregon, USA) equipped with a solid state and a large field detector. The textile surface was first coated with a thin layer of gold (∼23 nm) by Physical Vapor Deposition method (PVD) using the SC7620 Sputter Coater (Laughton, England). For the X-ray Photoelectron Spectroscopy (XPS) analysis of control and modified fabric two square specimens (equal to or larger than ∼12 « 12 mm2 ), were loaded in the introduction chamber of a Leybold-Specs MAX200 (Cologne, Germany) system and were fastened by metallic screws on the MAX200 Al plate sample holder. The XPS measurements took place at room temperature and ∼10−8 mbar pressure, using non-monochromatic MgK␣ X-rays (photon energy 1253.6 eV) and a Hemispherical Electron Energy Analyser (SPECS EA200) with Multi-Channel Detection, properly calibrated according to ISO15472 and ISO24237. The analyser operated under conditions optimized for better signal intensity (constant pass energy of 100 eV, maximum lens aperture). The analysed sample area was a 4 × 7 mm2 rectangle near the center of each specimen. Estimated C and O signal contributions from the holder surface and the contamination layer (∼0.8 nm) due to exposure to the atmosphere were subtracted-off from the measured area. The differential scanning calorimetry (DSC) measurements were conducted in a Mettler DSC 1 STARe System. A heating-coolingheating cycle was applied from 20 to 350 ◦ C, under nitrogen flow (10 mL min−1 ), with heating and cooling rate at 10 ◦ C min−1 for determining first melting point, crystallization point and second melting point. Thermogravimetric analysis (TGA) was performed in a Mettler TGA/DSC 1 thermobalance from 30 to 600 ◦ C with heating rate at 10 ◦ C min−1 under nitrogen flow (10 mL min−1 ) for detecting degradation temperature at maximum weight loss. 3. Results and discussion
Fig. 1. Effect of temperature on PET surface hydrolysis by FoCut5a cutinase. The enzymatic modification was conducted at pH 7 for 24 h and 0.94 mg gfabric −1 enzyme loading.
Fig. 2. Effect of pH on PET surface hydrolysis by FoCut5a cutinase. The enzymatic modification was performed at 40 ◦ C, for 24 h and 0.94 mg gfabric −1 enzyme loading.
37 ◦ C has been chosen by other scientific groups as well to test the activity of other enzymes on PET fabrics or films. For example, a temperature of 37 ◦ C was selected for the enzymatic modification of polyester fibers with a cutinase from F. solani pisi [7] and 40 ◦ C for PET membrane hydrolysis using TEXAZYM EM commercial enzyme [26].
3.1. Optimization of the surface modification process by FoCut5a 3.1.1. Effect of temperature The enzymatic hydrolysis of the polyester fabric leads to the exposure of carboxyl- and hydroxyl- end groups on PET surface, while TPA or TPA equivalents, were released in the reaction supernatant. The effect of temperature was investigated when PET fabric was treated with 0.94 mg cutinase g−1 of fabric in phosphate buffer pH 7, for 24 h in the range of 25–50 ◦ C. Maximum release of TPA or its derivatives (29.2 M) was observed at 40 ◦ C, while over 45 ◦ C a small decrease of enzymatic modification occurred (Fig. 1) due to low enzyme thermostability [23]. The present results are in accordance to the modification of semi-crystalline PET fibers where a TPA equivalent concentration of ∼34 M was reached at 37 ◦ C using a commercial lipase from Thermomyces lanuginosus with 1.13 g g−1 of fabric enzyme loading [10]. The temperature of 40 ◦ C or this of
3.1.2. Effect of the pH In order to study the effect of initial pH in the enzymatic modification of PET fibers, the reactions were carried out in the range of pH 5.8–10.0 using potassium phosphate and glycine-NaOH buffers, 0.94 mg g−1 of fabric enzyme loading, at 40 ◦ C, for 24 h. Maximum TPA equivalents release (30 M) was found at pH 8 that was close to TPA release found at pH 7, however higher pH values decreased enzymatic activity (Fig. 2). In addition, higher pH values could increase the chemical hydrolysis of PET fibers in depth, resulting in a serious decrease of the final’s product quality, as in alkali treatment [8]. For PET films or fabrics modification with cutinases, pH 8 is commonly applied according to literature [8–10,26], as in the case of a cutinase from Thermomonospora fusca that was used as a biocatalyst for the surface hydrolysis of PET fabrics at 50 ◦ C [18].
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Fig. 3. Effect of enzyme loading on PET surface hydrolysis by FoCut5a cutinase. The enzymatic modification was conducted at 40 ◦ C, pH 8, for 24 h.
Fig. 5. HPLC chromatogram of PET hydrolysis reaction supernatant when treated at 40 ◦ C, pH 8 and enzyme loading 1.92 mg gfabric −1 . The control and the hydrolyzed sample are depicted with a dashed and a continuous line, respectively. Table 1 Results of dyeing trials. L, a, and b are the color coordinates of dyed samples, K/S is the color strength and E is the color difference between dyed samples. The enzymatic modification was carried out at optimal conditions (40 ◦ C, pH 8, enzyme loading 1.92 mg gfabric −1 , 3 h). D65/10
L
a
b
K/S
E
Controla Modifieda Controlb Modifiedb Controlc Modifiedc
82.39 81.17 82.01 74.81 85.65 83.49
1.81 1.73 9.80 18.09 2.92 3.79
−8.90 −10.15 −7.75 −5.94 −1.62 9.41
0.15 0.17 0.18 0.45 0.10 0.24
1.75
a b c
11.13 11.27
Dyed with Black Jakazol 133%. Dyed with Deep Cherry S–D. Dyed with Novacron Yellow S-3R.
after the first 3 h. The polyester fabric surface kept being hydrolyzed with a slow rate and a plateau was reached after 2 h and maintained. Fig. 4. Time course of PET surface hydrolysis by FoCut5a cutinase. The enzymatic modification was conducted at 40 ◦ C, pH 8 and enzyme loading 1.92 mg gfabric −1 .
3.1.3. Effect of enzyme loading and time course For a sustainable textile modification process, the amount of the enzyme used is a crucial parameter due to the high cost of the biocatalyst. The effect of enzyme loading was studied in the range of 0.096–3.840 mg g−1 of fabric. As it is obvious in Fig. 3, the enzyme loading does not lead to a significant further increase of TPA equivalents over the concentration of 1.92 mg g−1 of fabric, with maximum TPA equivalents release ∼35 M. In literature, the hydrolysis activity of another cutinase from T. fusca has been studied, at 60 ◦ C, pH 7 with a TPA equivalent release of about 12 mM for amorphous and 200 M for semi-crystalline PET fibers with 11.30 mg g−1 of fabric enzyme loading [10]. From the reaction time course study (Fig. 4), it is shown that the hydrolysis ceases after the first 2–3 h possibly due to the gradual protein deactivation at 40 ◦ C, a result drawn from the enzyme’s thermostability analysis [23]. Our results are in accordance with Silva et al. [27] who used a wild type and a genetically modified T. fusca cutinase with which the TPA equivalents release was ceased the first 3–5 h, under the conditions of 60 ◦ C, pH 7.5 and various enzyme concentrations. The aforementioned theory of protein deactivation is supported by the fact that a second addition of enzyme at the third hour of hydrolysis reaction leads to increased product release of about 26%. Indicatively, a test took place at the optimum conditions of temperature and pH in which a second equal amount of enzyme was added
3.2. Identification of hydrolysis products As mentioned previously, the enzymatic hydrolysis of PET fabrics results in TPA and its equivalents release through ester bond cleavages. After studying the optimal conditions for the enzymatic treatment, HPLC analysis of the reaction medium was employed in order to identify the hydrolysis products. The standards used were commercially available, with retention times of 11.3 and 13.1 min for TPA and BHET, respectively. The enzymatic treatment of PET with FoCut5a clearly released TPA, while BHET was not detected (Fig. 5). On the other hand, various unidentified peaks were spotted with one among them at 12.3 min, possibly corresponding to MHET, as was previously described in literature following the same HPLC analysis methodology [8]. The release of only TPA and MHET has previously been reported when PET was hydrolyzed using recombinant cutinases from Thermobifida cellulosilytica and T. fusca [28]. 3.3. Analysis of enzymatically modified PET fibers 3.3.1. Dyeing of the enzymatically modified fabric As it can be seen from the E values reported in Table 1 for treated and untreated fabrics, there is a significant color variation after enzymatic modification in all cases of examined dyes. The increased K/S values of enzymatically treated samples is related to the presence of more OH end groups after enzymatic hydrolysis of the polymer, which results in a significant increase of dye reacting
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Fig. 6. Dyeing effects of unmodified (lower) and enzymatically modified at optimal conditions (upper) PET fabrics using the following dyes: Jakazol Black 133% (a), Novacron Deep Cherry S-D (b) and Novacron Yellow S-3R (c).
Table 2 Dye concentrations in each dyeing bath before and after dyeing of the PET fabrics. The enzymatic treatment was performed at optimal conditions (40 ◦ C, pH 8, enzyme loading 1.92 mg gfabric −1 , 3 h). Dye bath
Before (mg/mL)
After (mg/mL)
%
Controla Modifieda Controlb Modifiedb Controlc Modifiedc
3.786 3.737 3.169 3.321 2.749 2.747
3.462 2.788 1.653 1.689 1.867 1.693
−8.57 −25.41 −47.84 −49.15 −32.09 −38.36
a b c
Dyed with Black Jakazol 133%. Dyed with Deep Cherry S–D. Dyed with Novacron Yellow S-3R.
with the fabric. The K/S values for Black Jakazol 133%, Deep Cherry and Novacron Yellow dyes were 0.02, 0.27 and 0.14, respectively (minus the K/S value of the unmodified fabric). In literature, the K/S values found for enzymatically modified PET fabrics under the conditions of 60 ◦ C, pH 7.5 and enzyme loading 12.5 mg g−1 of fabric, dyed with Reactive Black 5 were 0.11 for modification with a recombinant T. fusca cutinase (Tfu 0883) and 0.26 for T. fusca cutinase after a double mutation (Q 132A/T101A) [27]. The K/S values using a T. fusca cutinase and a lipase from T. lanuginosus were 0.32 and 0.12, for PET fabrics modified at pH 7, 60 and 37 ◦ C and enzyme loading 0.2 and 20 g L−1 respectively, using Astrazone Blue BG dye [10]. Moreover, for enzymatic treatment at 35 ◦ C and pH 7.2 with hydrolases from T. fusca and F. solani f. sp. pisi at 8 and 16 U, respectively, the K/S values were 0.07 and 0.45 respectively after dyeing with Reactive Red 2 [9]. In Fig. 6 the dyed modified fabrics are presented using three dyes in comparison to each control, respectively. Dye concentrations in each dyeing bath before and after dyeing are reported in Table 2. The reported values indicate increased dye absorption by the enzymatically treated fabrics in all cases. The increase of dye absorption is marginal in the case of Deep Cherry dye and higher in the cases of Black Jakazol and Novacron Yellow dyes. Despite the fact that the enzymatically treated PET fabric appears to absorb much more Black Jakazol dye from the dyeing bath compared to the untreated fabric, these samples exhibit very low E. This contradiction is found because during the dyeing procedure excess dye molecules “anchored” on the enzymatically treated fabric without, however, adequately bonding to it. The “anchored” dye molecules which had not reacted with the PET fabric were removed during the washing followed by acidification process. 3.3.2. Mechanical measurements The mechanical properties such as tensile strength of PET fabrics are of great importance and they define the product’s applications [18]. Therefore the enzymatic treatment should not cause any debasement that would render the final product unsuitable to use. According to Table 3, there is no obvious effect on the tensile prop-
Table 3 Results from tensile test on untreated and treated at optimal conditions (40 ◦ C, pH 8, enzyme loading 1.92 mg gfabric −1 , 3 h) PET fabrics.
Control Modified
Load at peak (kgf)
Elongation at break (mm)
Strain at peak (%)
21.52 ± 1.10 20.26 ± 0.70
17.52 ± 1.60 17.43 ± 1.10
34.32 ± 1.65 33.88 ± 2.16
erties of the treated fabrics. This result is desirable and expected, as it has previously been reported that no significant decrease of tensile strength on PET fabrics is presented after enzymatic treatment with a commercial lipase (EC 3.1.1.3) from Candida antarctica and a commercial cutinase (EC 3.1.1.74) [3]. Furthermore, no significant differences on burst strength measurements were reported between untreated and enzymatically treated samples of PET fabrics using a cutinase as biocatalyst [15]. 3.3.3. FT-IR ATR analysis Aiming at the spectroscopic identification of the enzyme mediated modification of PET surface, FT-IR ATR analysis was carried out, as shown in Fig. 7. The ester bond of PET consists a strong band at 1710–1750 cm−1 , which corresponds to the carbonyl bond C O due to stretch vibrations and at 1210–1320 cm−1 , which corresponds to the C O bond due to stretch vibrations as well. As it can be discerned from Fig. 7, the intensity of the C O and C O bands at 1718 cm−1 and 1250 cm−1 for the enzymatically modified fabric is decreased most possibly because of the breakage of the ester groups as a result of the polyester hydrolysis. The FT-IR ATR analysis of amorphous and crystalline oriented PET membranes also presented an undoubted decrease of the bands at 1720 and 1260–1246 cm−1 , after modification with the TEXAZYM EM commercial enzyme formulation [26]. This fact is proved by Du et al. where a software was employed to determine the second derivative spectrum of a PET sample hydrothermally degraded, indicating that the band from 1640 to 1780 cm−1 consisted of overlapped peaks that belonged either to main chain ester bonds or end carboxyl groups. During degradation the ester bonds decreased with a parallel increase of the end groups, causing a small fall in the overall band after 30 days of the sample hydrolysis [29]. 3.3.4. SEM analysis The enzymatic surface modification has the advantage of retaining the surface characteristics and preserving the textile properties, as the enzyme does not interfere with the bulk mass of the fibers. On the contrary, an alkali treatment of a polyester fabric concludes to higher hydrophilicity but also influences the fabric’s strength. As shown in the SEM images of untreated and enzymatically treated PET fibers respectively, the enzymatic treatment is visualized by the presence of small etches and cracks of the treated fibers compared to the untreated ones, nevertheless no significant changes were detected (Fig. 8). However, this is not the case when the polyester surface is treated with NaOH as indicated in literature [10] leading
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Fig. 7. FT-IR ATR spectrum of untreated (black line) and enzymatically modified at optimal conditions PET fabric (gray line).
Fig. 8. Surface SEM depictions of untreated (a) and enzymatically modified at optimal conditions (b) PET fibers.
to clear fiber deterioration. Nevertheless, there is no homogeneity between the treated fibers as the hydrolysis signs appear more vivid in some parts of the fibers and not at all in others. 3.3.5. XPS analysis Besides the characteristic XPS peaks of O and C, the PET samples, mainly the modified one, exhibited a small unexpected N content, probably due to superficial residual enzyme. The shape of the predominant C peak region in all specimens suggested the presence of several chemical states for C1s, corresponding to the different distinct functional groups in the polymer chain. In order to extricate the contributions of various states, a peak fitting was applied according to relevant information from literature [10] for
the binding energies (BEs) and using the same peak-width for all components (Fig. S1). The effect of the modification was indicated by the changes in the relative intensities of the peaks, which yielded the relative abundance of specific C1s components, after subtracting holder and contamination contributions, as presented in Fig. 9. It is clear that the enzymatic modification resulted in a small relative increase of the hydrolyzed groups (12 and 5% for C O C, C OH and C O + HO C O groups, respectively) over the aliphatic/aromatic groups (C C) that decreased about 7%. The same pattern has been reported before, after XPS analysis of PET hydrolysed with a lipase from T. lanuginosus and a cutinase from T. fusca [21].
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Acknowledgements The financial support of general Secretariat of Research and Technology (GSRT) of Greece-ESPA 2007-2013 is gratefully acknowledged. Furthermore, Onassis Institute should be gratefully thanked for the support provided to Maria Kanelli throughout this work. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.procbio.2015.08. 013. References
Fig. 9. Relative abundance (%) of different C1s components from XPS analysis of control (black) and modified at optimal conditions (gray) PET fabric (C C ∼ 285 eV; C O C, C OH ∼ 286 eV; C O ∼ 289 eV; OH C O ∼ 291 eV).
3.3.6. DSC-TGA analysis In order to find any change of the thermal properties of the treated fabrics, DSC and TGA analyses were carried out. The heating–cooling–heating cycle applied during DSC analysis allowed the detection of the first melting point (256 ◦ C), the crystallization point (211 ◦ C) and the second melting point (254 ◦ C) of the polyester textile. No remarkable differences in the thermal properties were observed between the untreated and treated polyester fabrics. From TGA analysis the thermal degradation of the fabric is presented with maximum weight loss at 426 ◦ C for the control and at 424 ◦ C for the enzymatically modified fabric. As it can be discerned, there are no significant changes in the thermal properties of the PET fabrics. That is positive since the product’s degradation is not desired, nevertheless small variations exist and are indicative of the enzymatic modification. 4. Conclusions In the present investigation, the potential of a F. oxysporum cutinase for the enzymatic surface modification of PET textiles was studied, aiming at a sustainable and controlled functionalization of the synthetic product. The enzymatic process is proved to be environmentally friendly due to the mild conditions followed (40 ◦ C, pH 8), without affecting the mechanical and thermal properties of the PET fabric, as proved by tensile tests and DSC-TGA analyses, respectively. The subtle superficial changes were confirmed utilizing a variety of analytical techniques, including FT-IR ATR analysis, XPS, SEM, as well as through dyeability tests using reactive dyes. Both FT-IR ATR and XPS analyses justified the presence of free hydroxyl and carboxyl groups on the outermost layers of the PET polymer, which was further confirmed by the reaction of these free groups with different reactive dyes. The dyeing ability of the PET fabrics improved significantly with an increase in K/S values of 150% and 140% after enzymatic treatment with Deep Cherry and Novacron Yellow dyes, respectively. Therefore, it can be concluded that the enzymatic treatment using the F. oxysporum cutinase, is capable of functionalizing textile surface made of PET, without compromising the polymer bulk properties, such as strength. This asset will be further exploited for the modification of other polyesters with interesting properties, such as biobased polyhydroxyalkanoates or polylactic acid, which are biodegradable and can be applied in numerous fields including biomedical, pharmaceutical and packaging industries.
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Surface modification of poly(ethylene terephthalate) (PET) fibers by a cutinase from Fusarium oxysporum Maria Kanelli a , Sozon Vasilakos b , Efstratios Nikolaivits a , Spyridon Ladas c , Paul Christakopoulos d , Evangelos Topakas a,∗ a Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 5 Iroon Polytechniou Str., Zografou Campus, Athens 15780, Greece b MIRTEC, Materials Industrial Research & Technology Center S.A, Athens 17342, Greece c Surface Science Laboratory, Department of Chemical Engineering, University of Patras, 26504 Rion, Patras, Greece d Biochemical and Chemical Process Engineering, Division of Sustainable Process Engineering, Department of Civil, Environmental and Natural Resources Engineering, Luleå University of Technology, SE-97187 Luleå, Sweden
a r t i c l e
i n f o
Article history: Received 5 June 2015 Received in revised form 27 August 2015 Accepted 30 August 2015 Available online xxx Keywords: Enzymatic modification Poly(ethylene)terephthalate (PET) fabric Cutinase FT-IR ATR XPS Dyeing
a b s t r a c t Synthetic polyester fabrics occupy a great part of the textile industry production satisfying variable ordinary needs. Nonetheless, their high hydrophobicity constitutes an important weakness that impedes process manufacture, as well as permeability and evaporation of sweat when used in clothing industry. The enzymatic treatment of these materials is a modern and eco-friendly procedure that aims at the increase of the hydrophilicity through superficial modification. In this study, the enzymatic surface hydrolysis of poly(ethylene terephthalate) (PET) fabric is succeeded using a recombinant cutinase from Fusarium oxysporum. The effect of various parameters is studied for the enzymatic modification of PET, such as temperature, pH, enzyme loading and reaction time. The optimal parameters are found to be 40 ◦ C, pH 8, and 1.92 mg enzyme loading per gram of fabric. The controlled enzymatic hydrolysis of PET textile is further confirmed and characterized using various spectroscopic and analytical methods, including Fourier Transform Infrared (FT-IR) in the Attenuated Total Reflectance mode (ATR) and X-ray photoelectron spectroscopy (XPS). Tensile test and dyeability analyses were also employed achieving a K/S increase up to 150%, confirming the successful surface modification without degrading the quality of the starting material. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction In modern times, technology and science have met advances that have encouraged fundamental changes and improvements in several industrial sectors, such as textile industry. Synthetic fabrics prove to be useful in many applications because of their advantages as far as their physical properties are concerned, presenting strength and elasticity [1]. In addition, synthetic textiles present less wrinkles, fast drying properties and can be produced massively in a short period of time with lower cost as opposed to natural textiles. Their main drawback is the low hydrophilicity that impedes the process of finishing and dyeing, as well as the permeability and evaporation of sweat [2–5] and stain resistance [6]. Towards this
∗ Corresponding author. Fax: +30 210 7723163. E-mail addresses: [email protected], [email protected] (E. Topakas).
direction, enzymatic treatment consists an innovative biotechnological and eco-friendly approach to increase fabrics’ hydrophilicity and improve their properties, as opposed to chemical treatment, which has higher manufacturing cost and energy consumption and leads to strength loss and degradation of fabric bulk properties [7–12]. Enzymes are macromolecular compounds that present selectivity and cause superficial modification of the fibers, as they cannot diffuse further into the fibers’ bulk part because of their size [13–15]. Modification of poly(ethylene terephthalate) (PET) fibers aims at the surface hydrolysis of the ester bonds releasing carboxyland hydroxyl- end groups [12,16] and as a result terephthalic ions in the reaction supernatant. According to literature, enzymes suitable for polyester modification are esterases, lipases and cutinases [16–18]. Cutinases are serine esterases that belong to the ␣/-hydrolase fold family. Their role in nature is the hydrolysis of ester bonds in cutin, a wax biopolyester found in plant cuticle of aerial surfaces of plants [19]. Furthermore, cutinases have the ability to hydrolyze
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the ester bonds of synthetic polyesters and this ability makes them suitable for many industrial applications such as the surface modification of PET [17,20]. In literature, commonly used hydrolases for PET surface modification are cutinases from Fusarium solani sp. pisi [7–9,21]. However, it has previously been reported that a cutinase from Fusarium oxysporum proved to be more efficient as far as PET fabric modification is concerned, presenting higher activity in comparison to a cutinase from F. solani sp. pisi. [22]. In addition, cutinase from F. oxysporum is considered as highly organic solvent-tolerant, indicating its potential in biotechnological applications including the essential area of Biocatalysis [20]. In the present work, the ability of a F. oxysporum cutinase (FoCut5a) for the surface hydrolysis of PET fibers was investigated. The potential of this enzyme in PET modification was proved by its capability to hydrolyze two PET model substrates, bis(benzoyloxyethyl) terephthalate (3PET) and commercial bis(2hydroxyethyl) terephthalate (BHET) [23]. Various parameters were studied aiming at the efficient surface modification of PET textile, such as the effect of temperature, pH, incubation time and enzyme loading. The extent of the enzymatic treatment was monitored by the quantification of terephthalic acid (TPA) and its derivatives (TPA equivalents) released in reaction supernatant. In addition, the modification of the polymeric material was further justified through the application of different analytical techniques, including FTIR-ATR, SEM, XPS, DSC, TGA, as well as through dyeability tests. 2. Material and methods 2.1. Materials, chemicals and enzymes Commercial PET woven fabric with tricot knit was kindly supplied by Colora S.A (Thessaloniki, Greece) with density 126 columns in−1 and 58 rows in−1 , weight 52.90 g m−2 and thickness 42 m. The polyester fabric was washed with detergent Felosan NFG from CHT Bezema (Tübingen–Germany) for the removal of paraffins used during weaving. Recombinant cutinase from F. oxysporum was heterologously expressed in Escherichia coli BL21 (DE3) and purified by immobilized metal affinity chromatography, as described previously [23]. TPA and BHET were supplied by Merck (Whitehouse Station, New Jersey) and Sigma (St. Louis, MO), respectively. Trifluoroacetic acid and acetonitrile were of HPLC grade (Sigma, St. Louis, MO). The reactive dyes used were Novacron Deep Cherry S–D, Novacron Yellow S-3R (Everberg, Belgium) and Jakazol Black 133% (Huntsman, Gujarat, India). All other chemicals were of analytical grade.
spectrophotometrically at 241 nm in a BOECO S-20 Spectrophotometer (Hamburg, Germany). Possible hydrolysis products were TPA, BHET and mono(2-hydroxyethyl) terephthalate (MHET). A calibration curve of TPA was used for the calculation of the total amount of products released since all three products have the same molar absorption due to their carbonyl groups [8]. Samples of 0.6 mL of the supernatant were taken and the enzyme was precipitated with the addition of 1:1 methanol prior to centrifugation. Subsequently, in 1 mL of the supernatant sulfuric acid was added (18 mM) in order to convert TPA and any of its derivative ions into their neutral counterparts. Control samples without the addition of the enzyme or without the addition of fabric were measured as well. The reaction supernatants from the hydrolysis at optimal conditions, were concentrated and analyzed by HPLC, using a SHIMADZU LC-20AD pump equipped with a Jasco UV-975 detector recording at 241 nm. The reversed phase column Eurospher-100 C18 from KNAUER (Berlin) was maintained at room temperature. A linear gradient method, involving 1% trifluoroacetic acid solution and acetonitrile as eluents, at a flow rate of 0.8 mL/min, was applied, as previously described [8].
2.4. Dyeing and color measurements Polyester fabrics were dyed in 390 cm3 glass tubes in a laboratory scale dyeing Ahiba Texomat machine (Datacolor, Lawrenceville, NJ, USA). According to dyeing procedure, 55 g L−1 of Na2 SO4 and 2% w/w of each dye were dissolved and introduced in the glass tubes where fabrics were also introduced. The dye bath was heated up to 60 ◦ C and after 20 min, 5 g L−1 Na2 CO3 was added. Finally, 0.4 g L−1 of NaOH were incorporated 30 min before the end of the dyeing procedure. For the fabric neutralization to pH 6 a small amount of HCOOH was added after removing the fabrics from each dye bath, and then, a wash procedure followed at 95 ◦ C with soap (Cibapon R) for 1–2 h, in order to remove the non-reacted dye. Color changes (E) were evaluated with a color tristimulus colorimeter (Data Color International, Spectraflash SF450) (Lawrenceville, NJ, USA). The spectrophotometer was calibrated according to the manufacturer’s instructions, using the supplied black and white calibration standards. The color alterations were calculated using the CIE L*a*b* System, established by the “Commission Internationale de l’Eclairage-CIE” according to ASTM D 2244-68. For each sample, four repeated measurements were taken to determine the color coordinates L, a, b. “L” indicates the brightness, “a” describes the red-green content and “b” the yellow-blue content. Color change can be calculated using Eq. (1):
2.2. Enzymatic treatment of polyester fabric Polyester fabrics were cut into pieces of 0.5 g and were incubated in glass vessels with bath ratio of textile mass over buffer mass 1:50 under stirring (170 rpm) for 24 h. For the enzymatic treatment, different parameters were studied including the effect of temperature (25–50 ◦ C), pH (phosphate buffer 100 mM pH range 5.8–8.0, glycine-NaOH buffer 100 mM pH range 9.0–10.0), incubation time (0–24 h) and enzyme loading (0.096–3.840 mg g−1 of fabric). After enzymatic treatment, the fabrics were washed with 2 g L−1 Na2 CO3 at 60 ◦ C for 1 h and afterwards double-washed with deionized water for 1 h, as previously described [13]. All experiments were performed in duplicate. 2.3. Monitoring of enzymatic modification For the determination of the polyester hydrolysis degree, TPA or its derivatives released in samples’ supernatants were quantified
E =
(L2 − L1 )2 + (a2 − a1 )2 + (b2 − b1 )
2
(1)
Studies have concluded that the E = 1.0 is a color difference that can be perceived by 50% [24] by the human eye, while E = 2.0 is a color difference that can be seen at 100% [25]. In order to determine the dye absorption, the color strength (K/S) of the dyed samples was measured at the wavelength of minimum reflection by using Eq. (2): K (1 − R)2 = S 2R
(2)
where, R refers to the reflection value of the sample. Practically, the E value is used when it is desired to compare two shades. The color strength, i.e. the ratio K/S, is the parameter that is used to evaluate the dyeing process and dyeing capacity.
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2.5. Tensile test Tensile tests were carried out in an SDL Atlas tensile test machine (Rock Hill, SC, USA) operating at grip separation speed of 50 mm min−1 . All measurements were run at 25 ◦ C with specimen dimensions 3 cm width × 10 cm length. 2.6. Spectrometric and physicochemical analysis of modified fabrics The chemical structure of the enzymatically treated PET was examined by Fourier Transform-Infrared Spectroscopy Attenuated Total Reflectance (FT-IR ATR) analysis using a Bruker Tensor 27 FTIR Spectrometer, equipped with a diamond of simple reflection with penetration depth ∼2 m. The changes on the surface of the modified textiles were spotted with Scanning Electron Microscopy (SEM) using FEI Quanta 200 (Hillsboro, Oregon, USA) equipped with a solid state and a large field detector. The textile surface was first coated with a thin layer of gold (∼23 nm) by Physical Vapor Deposition method (PVD) using the SC7620 Sputter Coater (Laughton, England). For the X-ray Photoelectron Spectroscopy (XPS) analysis of control and modified fabric two square specimens (equal to or larger than ∼12 « 12 mm2 ), were loaded in the introduction chamber of a Leybold-Specs MAX200 (Cologne, Germany) system and were fastened by metallic screws on the MAX200 Al plate sample holder. The XPS measurements took place at room temperature and ∼10−8 mbar pressure, using non-monochromatic MgK␣ X-rays (photon energy 1253.6 eV) and a Hemispherical Electron Energy Analyser (SPECS EA200) with Multi-Channel Detection, properly calibrated according to ISO15472 and ISO24237. The analyser operated under conditions optimized for better signal intensity (constant pass energy of 100 eV, maximum lens aperture). The analysed sample area was a 4 × 7 mm2 rectangle near the center of each specimen. Estimated C and O signal contributions from the holder surface and the contamination layer (∼0.8 nm) due to exposure to the atmosphere were subtracted-off from the measured area. The differential scanning calorimetry (DSC) measurements were conducted in a Mettler DSC 1 STARe System. A heating-coolingheating cycle was applied from 20 to 350 ◦ C, under nitrogen flow (10 mL min−1 ), with heating and cooling rate at 10 ◦ C min−1 for determining first melting point, crystallization point and second melting point. Thermogravimetric analysis (TGA) was performed in a Mettler TGA/DSC 1 thermobalance from 30 to 600 ◦ C with heating rate at 10 ◦ C min−1 under nitrogen flow (10 mL min−1 ) for detecting degradation temperature at maximum weight loss. 3. Results and discussion
Fig. 1. Effect of temperature on PET surface hydrolysis by FoCut5a cutinase. The enzymatic modification was conducted at pH 7 for 24 h and 0.94 mg gfabric −1 enzyme loading.
Fig. 2. Effect of pH on PET surface hydrolysis by FoCut5a cutinase. The enzymatic modification was performed at 40 ◦ C, for 24 h and 0.94 mg gfabric −1 enzyme loading.
37 ◦ C has been chosen by other scientific groups as well to test the activity of other enzymes on PET fabrics or films. For example, a temperature of 37 ◦ C was selected for the enzymatic modification of polyester fibers with a cutinase from F. solani pisi [7] and 40 ◦ C for PET membrane hydrolysis using TEXAZYM EM commercial enzyme [26].
3.1. Optimization of the surface modification process by FoCut5a 3.1.1. Effect of temperature The enzymatic hydrolysis of the polyester fabric leads to the exposure of carboxyl- and hydroxyl- end groups on PET surface, while TPA or TPA equivalents, were released in the reaction supernatant. The effect of temperature was investigated when PET fabric was treated with 0.94 mg cutinase g−1 of fabric in phosphate buffer pH 7, for 24 h in the range of 25–50 ◦ C. Maximum release of TPA or its derivatives (29.2 M) was observed at 40 ◦ C, while over 45 ◦ C a small decrease of enzymatic modification occurred (Fig. 1) due to low enzyme thermostability [23]. The present results are in accordance to the modification of semi-crystalline PET fibers where a TPA equivalent concentration of ∼34 M was reached at 37 ◦ C using a commercial lipase from Thermomyces lanuginosus with 1.13 g g−1 of fabric enzyme loading [10]. The temperature of 40 ◦ C or this of
3.1.2. Effect of the pH In order to study the effect of initial pH in the enzymatic modification of PET fibers, the reactions were carried out in the range of pH 5.8–10.0 using potassium phosphate and glycine-NaOH buffers, 0.94 mg g−1 of fabric enzyme loading, at 40 ◦ C, for 24 h. Maximum TPA equivalents release (30 M) was found at pH 8 that was close to TPA release found at pH 7, however higher pH values decreased enzymatic activity (Fig. 2). In addition, higher pH values could increase the chemical hydrolysis of PET fibers in depth, resulting in a serious decrease of the final’s product quality, as in alkali treatment [8]. For PET films or fabrics modification with cutinases, pH 8 is commonly applied according to literature [8–10,26], as in the case of a cutinase from Thermomonospora fusca that was used as a biocatalyst for the surface hydrolysis of PET fabrics at 50 ◦ C [18].
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Fig. 3. Effect of enzyme loading on PET surface hydrolysis by FoCut5a cutinase. The enzymatic modification was conducted at 40 ◦ C, pH 8, for 24 h.
Fig. 5. HPLC chromatogram of PET hydrolysis reaction supernatant when treated at 40 ◦ C, pH 8 and enzyme loading 1.92 mg gfabric −1 . The control and the hydrolyzed sample are depicted with a dashed and a continuous line, respectively. Table 1 Results of dyeing trials. L, a, and b are the color coordinates of dyed samples, K/S is the color strength and E is the color difference between dyed samples. The enzymatic modification was carried out at optimal conditions (40 ◦ C, pH 8, enzyme loading 1.92 mg gfabric −1 , 3 h). D65/10
L
a
b
K/S
E
Controla Modifieda Controlb Modifiedb Controlc Modifiedc
82.39 81.17 82.01 74.81 85.65 83.49
1.81 1.73 9.80 18.09 2.92 3.79
−8.90 −10.15 −7.75 −5.94 −1.62 9.41
0.15 0.17 0.18 0.45 0.10 0.24
1.75
a b c
11.13 11.27
Dyed with Black Jakazol 133%. Dyed with Deep Cherry S–D. Dyed with Novacron Yellow S-3R.
after the first 3 h. The polyester fabric surface kept being hydrolyzed with a slow rate and a plateau was reached after 2 h and maintained. Fig. 4. Time course of PET surface hydrolysis by FoCut5a cutinase. The enzymatic modification was conducted at 40 ◦ C, pH 8 and enzyme loading 1.92 mg gfabric −1 .
3.1.3. Effect of enzyme loading and time course For a sustainable textile modification process, the amount of the enzyme used is a crucial parameter due to the high cost of the biocatalyst. The effect of enzyme loading was studied in the range of 0.096–3.840 mg g−1 of fabric. As it is obvious in Fig. 3, the enzyme loading does not lead to a significant further increase of TPA equivalents over the concentration of 1.92 mg g−1 of fabric, with maximum TPA equivalents release ∼35 M. In literature, the hydrolysis activity of another cutinase from T. fusca has been studied, at 60 ◦ C, pH 7 with a TPA equivalent release of about 12 mM for amorphous and 200 M for semi-crystalline PET fibers with 11.30 mg g−1 of fabric enzyme loading [10]. From the reaction time course study (Fig. 4), it is shown that the hydrolysis ceases after the first 2–3 h possibly due to the gradual protein deactivation at 40 ◦ C, a result drawn from the enzyme’s thermostability analysis [23]. Our results are in accordance with Silva et al. [27] who used a wild type and a genetically modified T. fusca cutinase with which the TPA equivalents release was ceased the first 3–5 h, under the conditions of 60 ◦ C, pH 7.5 and various enzyme concentrations. The aforementioned theory of protein deactivation is supported by the fact that a second addition of enzyme at the third hour of hydrolysis reaction leads to increased product release of about 26%. Indicatively, a test took place at the optimum conditions of temperature and pH in which a second equal amount of enzyme was added
3.2. Identification of hydrolysis products As mentioned previously, the enzymatic hydrolysis of PET fabrics results in TPA and its equivalents release through ester bond cleavages. After studying the optimal conditions for the enzymatic treatment, HPLC analysis of the reaction medium was employed in order to identify the hydrolysis products. The standards used were commercially available, with retention times of 11.3 and 13.1 min for TPA and BHET, respectively. The enzymatic treatment of PET with FoCut5a clearly released TPA, while BHET was not detected (Fig. 5). On the other hand, various unidentified peaks were spotted with one among them at 12.3 min, possibly corresponding to MHET, as was previously described in literature following the same HPLC analysis methodology [8]. The release of only TPA and MHET has previously been reported when PET was hydrolyzed using recombinant cutinases from Thermobifida cellulosilytica and T. fusca [28]. 3.3. Analysis of enzymatically modified PET fibers 3.3.1. Dyeing of the enzymatically modified fabric As it can be seen from the E values reported in Table 1 for treated and untreated fabrics, there is a significant color variation after enzymatic modification in all cases of examined dyes. The increased K/S values of enzymatically treated samples is related to the presence of more OH end groups after enzymatic hydrolysis of the polymer, which results in a significant increase of dye reacting
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Fig. 6. Dyeing effects of unmodified (lower) and enzymatically modified at optimal conditions (upper) PET fabrics using the following dyes: Jakazol Black 133% (a), Novacron Deep Cherry S-D (b) and Novacron Yellow S-3R (c).
Table 2 Dye concentrations in each dyeing bath before and after dyeing of the PET fabrics. The enzymatic treatment was performed at optimal conditions (40 ◦ C, pH 8, enzyme loading 1.92 mg gfabric −1 , 3 h). Dye bath
Before (mg/mL)
After (mg/mL)
%
Controla Modifieda Controlb Modifiedb Controlc Modifiedc
3.786 3.737 3.169 3.321 2.749 2.747
3.462 2.788 1.653 1.689 1.867 1.693
−8.57 −25.41 −47.84 −49.15 −32.09 −38.36
a b c
Dyed with Black Jakazol 133%. Dyed with Deep Cherry S–D. Dyed with Novacron Yellow S-3R.
with the fabric. The K/S values for Black Jakazol 133%, Deep Cherry and Novacron Yellow dyes were 0.02, 0.27 and 0.14, respectively (minus the K/S value of the unmodified fabric). In literature, the K/S values found for enzymatically modified PET fabrics under the conditions of 60 ◦ C, pH 7.5 and enzyme loading 12.5 mg g−1 of fabric, dyed with Reactive Black 5 were 0.11 for modification with a recombinant T. fusca cutinase (Tfu 0883) and 0.26 for T. fusca cutinase after a double mutation (Q 132A/T101A) [27]. The K/S values using a T. fusca cutinase and a lipase from T. lanuginosus were 0.32 and 0.12, for PET fabrics modified at pH 7, 60 and 37 ◦ C and enzyme loading 0.2 and 20 g L−1 respectively, using Astrazone Blue BG dye [10]. Moreover, for enzymatic treatment at 35 ◦ C and pH 7.2 with hydrolases from T. fusca and F. solani f. sp. pisi at 8 and 16 U, respectively, the K/S values were 0.07 and 0.45 respectively after dyeing with Reactive Red 2 [9]. In Fig. 6 the dyed modified fabrics are presented using three dyes in comparison to each control, respectively. Dye concentrations in each dyeing bath before and after dyeing are reported in Table 2. The reported values indicate increased dye absorption by the enzymatically treated fabrics in all cases. The increase of dye absorption is marginal in the case of Deep Cherry dye and higher in the cases of Black Jakazol and Novacron Yellow dyes. Despite the fact that the enzymatically treated PET fabric appears to absorb much more Black Jakazol dye from the dyeing bath compared to the untreated fabric, these samples exhibit very low E. This contradiction is found because during the dyeing procedure excess dye molecules “anchored” on the enzymatically treated fabric without, however, adequately bonding to it. The “anchored” dye molecules which had not reacted with the PET fabric were removed during the washing followed by acidification process. 3.3.2. Mechanical measurements The mechanical properties such as tensile strength of PET fabrics are of great importance and they define the product’s applications [18]. Therefore the enzymatic treatment should not cause any debasement that would render the final product unsuitable to use. According to Table 3, there is no obvious effect on the tensile prop-
Table 3 Results from tensile test on untreated and treated at optimal conditions (40 ◦ C, pH 8, enzyme loading 1.92 mg gfabric −1 , 3 h) PET fabrics.
Control Modified
Load at peak (kgf)
Elongation at break (mm)
Strain at peak (%)
21.52 ± 1.10 20.26 ± 0.70
17.52 ± 1.60 17.43 ± 1.10
34.32 ± 1.65 33.88 ± 2.16
erties of the treated fabrics. This result is desirable and expected, as it has previously been reported that no significant decrease of tensile strength on PET fabrics is presented after enzymatic treatment with a commercial lipase (EC 3.1.1.3) from Candida antarctica and a commercial cutinase (EC 3.1.1.74) [3]. Furthermore, no significant differences on burst strength measurements were reported between untreated and enzymatically treated samples of PET fabrics using a cutinase as biocatalyst [15]. 3.3.3. FT-IR ATR analysis Aiming at the spectroscopic identification of the enzyme mediated modification of PET surface, FT-IR ATR analysis was carried out, as shown in Fig. 7. The ester bond of PET consists a strong band at 1710–1750 cm−1 , which corresponds to the carbonyl bond C O due to stretch vibrations and at 1210–1320 cm−1 , which corresponds to the C O bond due to stretch vibrations as well. As it can be discerned from Fig. 7, the intensity of the C O and C O bands at 1718 cm−1 and 1250 cm−1 for the enzymatically modified fabric is decreased most possibly because of the breakage of the ester groups as a result of the polyester hydrolysis. The FT-IR ATR analysis of amorphous and crystalline oriented PET membranes also presented an undoubted decrease of the bands at 1720 and 1260–1246 cm−1 , after modification with the TEXAZYM EM commercial enzyme formulation [26]. This fact is proved by Du et al. where a software was employed to determine the second derivative spectrum of a PET sample hydrothermally degraded, indicating that the band from 1640 to 1780 cm−1 consisted of overlapped peaks that belonged either to main chain ester bonds or end carboxyl groups. During degradation the ester bonds decreased with a parallel increase of the end groups, causing a small fall in the overall band after 30 days of the sample hydrolysis [29]. 3.3.4. SEM analysis The enzymatic surface modification has the advantage of retaining the surface characteristics and preserving the textile properties, as the enzyme does not interfere with the bulk mass of the fibers. On the contrary, an alkali treatment of a polyester fabric concludes to higher hydrophilicity but also influences the fabric’s strength. As shown in the SEM images of untreated and enzymatically treated PET fibers respectively, the enzymatic treatment is visualized by the presence of small etches and cracks of the treated fibers compared to the untreated ones, nevertheless no significant changes were detected (Fig. 8). However, this is not the case when the polyester surface is treated with NaOH as indicated in literature [10] leading
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Fig. 7. FT-IR ATR spectrum of untreated (black line) and enzymatically modified at optimal conditions PET fabric (gray line).
Fig. 8. Surface SEM depictions of untreated (a) and enzymatically modified at optimal conditions (b) PET fibers.
to clear fiber deterioration. Nevertheless, there is no homogeneity between the treated fibers as the hydrolysis signs appear more vivid in some parts of the fibers and not at all in others. 3.3.5. XPS analysis Besides the characteristic XPS peaks of O and C, the PET samples, mainly the modified one, exhibited a small unexpected N content, probably due to superficial residual enzyme. The shape of the predominant C peak region in all specimens suggested the presence of several chemical states for C1s, corresponding to the different distinct functional groups in the polymer chain. In order to extricate the contributions of various states, a peak fitting was applied according to relevant information from literature [10] for
the binding energies (BEs) and using the same peak-width for all components (Fig. S1). The effect of the modification was indicated by the changes in the relative intensities of the peaks, which yielded the relative abundance of specific C1s components, after subtracting holder and contamination contributions, as presented in Fig. 9. It is clear that the enzymatic modification resulted in a small relative increase of the hydrolyzed groups (12 and 5% for C O C, C OH and C O + HO C O groups, respectively) over the aliphatic/aromatic groups (C C) that decreased about 7%. The same pattern has been reported before, after XPS analysis of PET hydrolysed with a lipase from T. lanuginosus and a cutinase from T. fusca [21].
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Acknowledgements The financial support of general Secretariat of Research and Technology (GSRT) of Greece-ESPA 2007-2013 is gratefully acknowledged. Furthermore, Onassis Institute should be gratefully thanked for the support provided to Maria Kanelli throughout this work. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.procbio.2015.08. 013. References
Fig. 9. Relative abundance (%) of different C1s components from XPS analysis of control (black) and modified at optimal conditions (gray) PET fabric (C C ∼ 285 eV; C O C, C OH ∼ 286 eV; C O ∼ 289 eV; OH C O ∼ 291 eV).
3.3.6. DSC-TGA analysis In order to find any change of the thermal properties of the treated fabrics, DSC and TGA analyses were carried out. The heating–cooling–heating cycle applied during DSC analysis allowed the detection of the first melting point (256 ◦ C), the crystallization point (211 ◦ C) and the second melting point (254 ◦ C) of the polyester textile. No remarkable differences in the thermal properties were observed between the untreated and treated polyester fabrics. From TGA analysis the thermal degradation of the fabric is presented with maximum weight loss at 426 ◦ C for the control and at 424 ◦ C for the enzymatically modified fabric. As it can be discerned, there are no significant changes in the thermal properties of the PET fabrics. That is positive since the product’s degradation is not desired, nevertheless small variations exist and are indicative of the enzymatic modification. 4. Conclusions In the present investigation, the potential of a F. oxysporum cutinase for the enzymatic surface modification of PET textiles was studied, aiming at a sustainable and controlled functionalization of the synthetic product. The enzymatic process is proved to be environmentally friendly due to the mild conditions followed (40 ◦ C, pH 8), without affecting the mechanical and thermal properties of the PET fabric, as proved by tensile tests and DSC-TGA analyses, respectively. The subtle superficial changes were confirmed utilizing a variety of analytical techniques, including FT-IR ATR analysis, XPS, SEM, as well as through dyeability tests using reactive dyes. Both FT-IR ATR and XPS analyses justified the presence of free hydroxyl and carboxyl groups on the outermost layers of the PET polymer, which was further confirmed by the reaction of these free groups with different reactive dyes. The dyeing ability of the PET fabrics improved significantly with an increase in K/S values of 150% and 140% after enzymatic treatment with Deep Cherry and Novacron Yellow dyes, respectively. Therefore, it can be concluded that the enzymatic treatment using the F. oxysporum cutinase, is capable of functionalizing textile surface made of PET, without compromising the polymer bulk properties, such as strength. This asset will be further exploited for the modification of other polyesters with interesting properties, such as biobased polyhydroxyalkanoates or polylactic acid, which are biodegradable and can be applied in numerous fields including biomedical, pharmaceutical and packaging industries.
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