Enzymatic Functionalization of HMLS-Polyethylene Terephthalate

Austrian Centre of Industrial Biotechnology GmbH, Konrad Lorenz Strasse 20, 3430 Tulln an der Donau, Austria ... Publication Date (Web): June 26, 2017...
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Research Article pubs.acs.org/journal/ascecg

Enzymatic Functionalization of HMLS-Polyethylene Terephthalate Fabrics Improves the Adhesion to Rubber Sara Vecchiato,† Jennifer Ahrens,‡ Alessandro Pellis,§ Denis Scaini,∥ Bernhard Mueller,*,⊥ Enrique Herrero Acero,*,⊥ and Georg M. Guebitz†,§ †

Austrian Centre of Industrial Biotechnology GmbH, Konrad Lorenz Strasse 20, 3430 Tulln an der Donau, Austria Glanzstoff-Textilcord Steinfort s.a., rue de Schwarzenhof, 8452 Steinfort, Luxembourg § University of Natural Resources and Life Sciences, Institute for Environmental Biotechnology, Konrad Lorenz Strasse 20, 3430 Tulln an der Donau, Austria ∥ Department of Life Sciences, University of Trieste, Piazzale Europa 1, 34127 Trieste, Italy ⊥ Glanzstoff Industries GmbH, Herzogenburger Strasse 69, 3100 St. Poelten, Austria ‡

S Supporting Information *

ABSTRACT: Among synthetic thermoplastic fiber materials for reinforcement, high modulus and low shrinkage poly(ethylene terephthalate) (HMLS-PET) became the major carcass material for the low- to medium-end tire segment. Usually cords are coated with a resorcinol−formaldehyde−latex (RFL) dip to achieve acceptable power transmission. However, the low concentration of polar groups on the PET’s surface requires an additional activation with costly and potentially toxic chemicals to create additional nucleophilic groups prior to RFL dipping. Here, a green enzyme based alternative to chemical HMLS-PET activation was investigated. Four different cutinase variants from Thermobif ida cellulosilytica were shown to hydrolyze HMLS-PET cords, creating new carboxylic and hydroxyl groups with distinct exoendo-wise selectivity. The highest degree of enzymatic functionalization reached a concentration of 0.51 nmol mm−2 of COOH with a release of 1.35 mM of soluble products after 72 h. The chemical treatment with 1 M NaOH released more soluble products leading up to a 10% decrease of the tensile strength while the functionalization degree achieved was only 0.21 nmol mm−2. This clearly indicates a more endowise mode of hydrolysis for the enzymatic treatment when compared to chemical hydrolysis. Scanning electron microscopy of the fibers confirmed the aggressiveness of the chemical treatment, whereas the enzymatic approach only led to 0.7% solubilization of the polymer with no loss of mechanical properties and crystallinity changes. The newly created groups were chemically accessible and reactive in the dipping step and led after the vulcanization to a significant improvement of the adhesion between the polymer and a representative carcass rubber compound according to the peel tests. KEYWORDS: HMLS-Poly(ethylene terephthalate), Enzymatic functionalization, Tire reinforcement, Cutinase, Rubber

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INTRODUCTION Poly(ethylene terephthalate) (PET) is a synthetic, thermoplastic polymer that, ever since it was commercially introduced in 1942 by Whinfield and Dickson, has found a wide range of applications, such as packaging material, medical devices,1−3 home furnishing, automobile parts,4−7 and textile fibers.8 Currently, with a global production of almost 25 million tons in 2015,1 PET is the largest synthetic polyester on the market. Due to its high tenacity and Young’s modulus, PET fibers were proposed as an ideal reinforcement material for the tire manufacturing process in 1962.9 Over the past four decades, the mechanical properties of technical PET fibers have been further improved, and with the introduction of high modulus, low shrinkage (HMLS) PET-yarns in the late 1980s, PET become the major carcass reinforcement material for passenger car tires in the new millennium. The development of HMLS© 2017 American Chemical Society

PET arises from the need to combine the desirable characteristics of the regular textile PET, such as the good tenacity and modulus, along with dimensional stability at elevated temperatures of 80 °C and above while avoiding an increase in the process price. Belt cords made from this yarn possess properties such as sufficient dimensional stability below the glasstransition temperature, low elongation, low creep, good tension retention, and high dynamic integrity.10−12 In addition to the dynamometric properties, HMLS-PET facilitates the calendaring (coating with rubber) process as it has a low moisture uptake in the range of 0.5−1% w w−1 when compared to other used fibers, such as Nylon, which has a value Received: February 14, 2017 Revised: May 8, 2017 Published: June 26, 2017 6456

DOI: 10.1021/acssuschemeng.7b00475 ACS Sustainable Chem. Eng. 2017, 5, 6456−6465

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ACS Sustainable Chemistry & Engineering >5% w w−1 and hence does not require an additional dying step. However, the lack of reactive groups on the untreated PET surface limits the nature of the cohesive forces between the fiber surface and the rubber to electrostatic or weak interactions. For most applications, especially as reinforcement material in tires or conveyor belts, the level of adhesion obtained with untreated PET is by far too low and represents an intrinsic technical limitation.9 In order to increase the level of adhesion, polyester tire cord fabrics are chemically activated by coating the fiber surface prior to the resorcinol−formaldehyde−latex (RFL) dipping with a so-called predip, an aqueous based dispersion containing lowmolecular epoxides and blocked isocyanates. In this way, a high amount of hydroxyl and amino functionalities are generated on the cord surface, providing sufficient functionalities for strong covalent bonding between the RFL dip and fiber surface/ cord.13 More in detail, the latex phase within the RFL dip is capable of cross-linking with the rubber during composite molding, while its resorcinol-formaldehyde phase is primarily involved in the textile bonding, taking advantage of its hydroxyl and amino groups.14 However, the highly negative environmental15 and health concerns regarding isocyanates exposure must be taken into great consideration since they are regarded as potential human carcinogens and known to cause cancer in animals.15 Alternatively, plasma or wet chemistry methods have been developed to insert hydrophilic moieties like carboxylic or hydroxyl groups on the surface and amino functionalities as well as double-bond rich molecules in the case of the plasma.6,8,16−21 Plasma treatments21 nevertheless have restrictions when applied to nonplanar surfaces in addition to the unstable nature of the introduced polar groups that can revert to a hydrophobic character.16,17 Other methods normally involve harsh chemicals (e.g., concentrated alkali) that can make the process very difficult to control and typically lead to a decrease of polymer weight, along with a loss of strength.6,19 The use of enzymes to modify the outermost layers of synthetic polymers while avoiding polymer damage22−25 is emerging as a powerful alternative approach to overcome the limitations of traditional methods highlighted above. While both oxidoreductases26,27 and hydrolases28 have been shown to successfully modify different synthetic polymers, the latter typically lead to defined end groups, which facilitate further processing. Different hydrolases are used to increase the biocompatibility of polyester membranes,29 enable the grafting of antimicrobial substances,30 or the creation of superhydrophobic surfaces.31 Particularly regarding PET, cutinases,4,18,32−34 lipases,29,35 and esterases36 have been reported to specifically hydrolyze the polymer backbone, making them suitable candidates for recycling37 and functionalization29,38,39 purposes in an environmentally friendly way. In the present work, the enzymatic generation of carboxylic and hydroxyl groups on the surface of HMLS-PET tire reinforcement material was investigated. The newly created groups would represent novel anchor points to create covalent bonds between the polymer and the rubber material mechanism, which ultimately would lead to improve adhesion.40 Here, different native and engineered cutinases from Thermobif ida cellulosilytica33 were compared regarding the degree of functionalization, material degradation, and rubber adhesion level. Enzymatically activated HMLS-PET cords showed a significantly higher adhesion level without substan-

tially altering material tensile strength when compared with traditional approaches.

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MATERIAL AND METHODS

Chemicals, PET Substrate, and Enzymes. Poly(ethylene terephthalate) fabrics and PET 1440/2 GXD cords, at a thickness of 0.41 ± 0.015 mm and weight of 240 ± 12 g (m2)−1, were provided by Glanzstoff (Lovosice, Czech Republic). All the other chemicals and solvents were purchased from Sigma-Aldrich (Vienna, Austria) at reagent grade and used without further purification if not otherwise specified. All the used enzymes, Thc_Cut1 and Thc_Cut2 from Thermobif ida cellulosilytica, Thc_Cut2_DM (double mutant Arg29Asn_Ala30Val of Thc_Cut2), and Thc_Cut2_TM (triple mutant Arg19Ser_Arg29Asn_Ala30Val of Thc_Cut2), were produced and purified as previously described.4,33 Vector pET26b(+) (Novagen, Germany) was used for the enzyme expression in BL21-Gold(DE3) Escherichia coli competent cells (Agilent Technologies, USA). Enzyme Expression and Purification. Freshly transformed E. coli BL21-Gold (DE3) cells were used to inoculate 250 mL of LB media supplemented with 40 μg mL−1 kanamycin and incubated overnight at 37 °C and 150 rpm in an orbital shaker (INFORS HT Multitron Pro, Switzerland). The overnight culture was used to prepare the main culture; 250 mL of LB media with 40 μg mL−1 kanamycin had to be inoculated with the preculture to reach an optical density (OD600) of 0.1 and incubated at 37 °C and 150 rpm until a final OD600 of 0.6−0.8 was measured. The main culture was cooled down to 20 °C and induced with IPTG at a final concentration of 0.05 mM; afterward, an overnight incubation at 20 °C and 150 rpm was followed. The cells were harvested by centrifugation at 4000 rpm and 4 °C for 30 min. The culture pellets were resuspended in 25 mL of binding buffer (20 mM Na2PO4·2H2O, 500 mM NaCl, 10 mM imidazole, pH 7.4). Afterward, the cell solutions were sonicated three times for 45 s and 60% amplitude under an ice cooling sonicator system. The lysates were centrifuged (40 min, 18000 rpm at 4 °C) and filtered with a 0.22 μm PES membrane. Finally, the cell lysate was purified as previously described,4,32 using the Ion Metal Affinity Chromatography system (IMAC_FPLC − Amersham Pharmacia Biotech, Sweden). Protein Quantification and SDS-PAGE Analysis. Protein concentrations were determined with the BIO-RAD Protein Assay (Bio-Rad Laboratories GmbH, Cat.No: 500-0006) using bovine serum albumin (BSA) as a protein standard. Briefly, 10 μL of the sample was added into the wells of a 96 well microtiter plate (Greiner 96 Flat Bottom Transparent Polystyrene). After all samples were added to the wells, 200 μL of the prepared BioRad reaction solution was added (BioRad Reagent diluted 1:5 with Fresenius water). The plate was incubated for 5 min at 21 °C and 400 rpm. The buffer for protein dilution (0.1 M Tris-HCl, pH 7) was used as a blank. The absorption after 5 min was measured at λ = 595 nm and the concentration calculated from the average of triplicate samples and blanks. SDS-PAGE was performed following the protocol described by Laemmli,41 and all of the proteins were stained with the Comassie Brilliant Blue R-250. Esterase Activity Assay. Esterase activity was measured at 30 °C using p-nitrophenyl butyrate (PNPB) as a substrate, as previously reported by Ribitsch et al. with some modification.42 The final assay mixture was made by mixing 40 μL of the substrate stock solution (86 μL of PNPB in 1000 μL of DMSO) with 1000 μL of 100 mM pH = 7 phosphate buffer. To 200 μL of the final assay mixture was added 20 μL of the enzyme solution to a well from a 96-well microtiter plate.30 The increase of the absorbance at 405 nm due to the release of pnitrophenol (ε 405 nm = 9.36 mL (μmol cm)−1) was measured for 5 min, every 18 s with a plate reader (Tecan INFINITE M200). A blank was included using 20 μL of buffer instead of enzyme dilution. The activity was calculated in units (U), where 1 unit is defined as the amount of enzyme required to hydrolyze 1 μmol of substrate per minute under the given assay conditions. Enzymatic Treatments of PET Fabrics. PET fabrics were cut into 2 cm × 2 cm pieces. Three prewashing steps were carried out to 6457

DOI: 10.1021/acssuschemeng.7b00475 ACS Sustainable Chem. Eng. 2017, 5, 6456−6465

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ACS Sustainable Chemistry & Engineering remove possible impurities from the polymer surface. The fabrics hence were treated with Triton X-100 (5 g L−1), Na2CO3 (100 mM), and distilled water. Each step was carried out for 30 min, at 50 °C, as previously reported by Brueckner et al.6 A subsequent overnight Soxhlet extraction step was carried out in ethanol. This procedure included a cooking phase of 90 min and an extraction phase of 100 min. The cooking phase was carried out at 80 °C in order to not modify the surface. The samples were finally extensively washed with mQ water. Afterward, each PET fabric piece was dried with a constant air flow for 5 min at 21 °C. The fabrics were incubated in phosphate buffer (100 mM, pH 7) with the different enzymes, Thc_Cut1, Thc_Cut2, Thc_Cut2_DM, and Thc_Cut2_TM, at two concentrations (0.5 μM and 5.0 μM) and for different incubation times (24 and 72 h) in 50 mL falcon tubes, allowing the samples to remain fully immersed into the enzymatic solution (10 mL). Incubations were performed at 50, 60 or 70 °C and 130 rpm in an orbital shaker (IKA KS 4000 ic control, Germany). After the enzymatic treatment, the samples were washed again as described above to completely remove the protein from the PET surface. Blank reactions consisted of PET fabrics incubated with buffer only under the same conditions reported above. All of the experiments were performed in triplicate. The enzymatic hydrolysis was compared to an alkali treatment which has previously been described for the hydrolysis of PET films.43,44 PET fabrics were washed as described above and incubated in different concentrations of NaOH (0.05 M, 0.01 M, 0.25 M, 0.5 M, 1 M). All of the reactions (10 mL) were performed for 2 h at 50 °C and 130 rpm in 50 mL Falcon tubes. Thereafter, the samples were washed with three washing steps as described for the enzymatic treatment.6 Enzyme and Alkali Treatments in Larger Scale. Ten neters of the same PET in a cord form was spooled around a circle block; therefore each fiber row was able to homogeneously enter in contact with the enzyme solution. The three washing steps were performed as mentioned before. Thereafter, the support with PET cords was introduced in a beaker and incubated with 400 mL of phosphate buffer (100 mM, pH 7) containing 0.5 μM and 5.0 μM of two different enzymes, Thc_Cut1 and Thc_Cut2_TM. The experiments were carried out at 60 °C, for 24 and 72 h, in a water bath. After the incubation, the cord was washed with the three washing steps as described in the protocol above. The enzymatic hydrolysis was compared to alkali treatment of PET cord with two concentrations of NaOH (0.05 and 1 M) at 50 °C, for 2 h, in a water bath. Analysis of the Soluble Release Products via High Performance Liquid Chromatography (HPLC). After enzymatic and alkali treatments, proteins were precipitated using 1:1 (v/v) ice cold methanol and acidified to pH 3.5 by adding 6 N HCl. Samples were centrifuged (Hettich MIKRO 200 R, Tuttlingen, Germany) at 14 000 rpm and T = 0 °C for 15 min and filtered through a 0.45 μm nylon filter directly into an HPLC vial for measurement. The HPLC used was an Agilent (Hewlett-Packard Series 1050) equipped with a RPC18 reversed phase column (YMC 30, 250 mm × 4.6 mm ID, S- 5 μm). The flow rate was set to 1 mL min−1, and the column was maintained at a temperature of 40 °C. The injection volume was 10 μL. Detection of the analytes was performed with a photodiode array detector (Diode Array Detector) at a wavelength of 241 nm. After the analysis, a post run was carried out for 10 min. Determination of the Surface Carboxylic Groups. The degree of surface modification was determined by measuring the carboxylic groups formed on the PET fabrics surface after the enzymatic and chemical treatments. The surface carboxylic groups (SC) were determined using the Toluidine Blue O (TBO) method as reported by Rödiger et al.45 with some modifications. The incubation of the samples and the blanks was carried out in 0.1% TBO solution in Tris/ HCl buffer (100 mM, pH 8.6) for 15 min at 50 °C and 130 rpm (6 mL). Hence, the samples were washed repeatedly (5 times) in Tris/ HCl (100 mM, pH 8.6) for 5 min until the washing solution got clear. The samples were transferred then to 20 mL vials to desorb the TBO bound to the surface carboxylic groups, by washing with 20% SDS for 30 min, at 50 °C and 130 rpm. The released TBO was quantified spectrophotometrically by measuring the absorbance at 625 nm and 23 °C. From each solution, 200 μL of sample were transferred to a 96-

well plate reader (Greiner, Sigma-Aldrich, Vienna); the results presented are the average of triplicates. The SC was calculated according to the following formula:45,46

SC = (A × V )/(A s × d × ε)

(1)

A is the absorption at 625 nm. V is the volume of desorption solution [L]. As is the area of the PET fabrics surface [mm−2]. d is the light path way [cm]. ε is the extinction coefficient of TBO [= 54 800 L mol−1 cm−1]. SC is the surface carboxylic groups [nmol mm−2]. A calibration curve on PET fabrics after chemical treatments is reported in the Supporting Information (Figures S1 and S2). Hydrophobicity Measurements. Hydrophobicity of the samples was quantified via water contact angle (WCA) measurements. PET fabrics were measured after enzymatic treatments with Thc_Cut1 and Thc_Cut2_TM (0.5 μM and 5.0 μM, 60 °C, 130 rpm). The proteins were washed from the surface using three consecutive washing steps6,33 as described previously. When indicated, Soxhlet extraction (approximately 3 h) was performed in ethanol at 80 °C before the enzymatic incubation as reported elsewhere.6,19,35,42 Afterward, PET fabric samples were rinsed with mQ water and dried. A sample incubated with phosphate buffer (100 mM, pH 7) under the same conditions as above was used as a blank. Afterward, samples enzymatically treated with Thc_Cut1 and Thc_Cut2_TM (0.5−5.0 μM, 60 °C, 24−72 h) were compared. Polymer fabrics were analyzed with the Drop Shape Analysis System DSA 100 (Kruss GmbH, Hamburg, Germany) using ddH2O as test liquid with a drop size of 5 μL and a deposition speed of 60 U min−1. The water contact angle was measured after 1 s from the deposition of the drop, and the data were analyzed using a video recording method. Each treatment was conducted in triplicate for both sample and droplet position in order to allow a homogeneous distribution of the water on the PET fabrics surface. The data were obtained from the average of nine measurements. The surface free energy (SFE) was performed using as reported above the video recording method after Thc_Cut1 and Thc_Cut2_TM (5.0 μM) incubation for 24 and 72 h. A blank incubated only with buffer was used as the control. The analysis was carried out using three different liquid solutions of water, glycerol, and olive oil. The Owens, Wendt, Rabel, and Kaelble (OWRK)47 method was used as a standard way for calculating the surface free energy of PET film from the contact angle. Imaging and Analysis of the Polymer Surface via Scanning Electron Microscopy and Atomic Force Microscopy. Scanning electron microscopy (SEM) was used to investigate the morphology of PET samples treated with Thc_Cut1 and Thc_Cut2_TM (5.0 μM, 60 °C, 72 h, 130 rpm), NaOH (1 M, 50 °C, 2 h and 130 rpm), and only with phosphate buffer (100 mM, pH 7 60 °C, 24 h) as the control. Before analysis samples were washed and dried as previously described. Images were acquired collecting secondary electrons on a Gemini SUPRA 40 SEM (Carl Zeiss NTS GmbH, Oberkochen) working at an acceleration voltage of 5 keV. Prior to SEM imaging, samples were gold metalized in a metal sputter coater (Polaron SC7620). Atomic force microscopy (AFM) was used to obtain high resolution 3D images of PET fabrics samples’ surfaces. Samples were mounted on metallic plates using epoxy glue and subsequently characterized using an MFP-3D AFM (Asylum Research, Oxford Instruments, Santa Barbara, CA, U.S.). Measurements were carried out in the air at room temperature working in dynamic mode. Cantilevers, characterized by a resonant frequency of 320 kHz and force constant of 40 nN/nm (NSG30, NT-MDT, Moscow, RU), were used working at low oscillation amplitudes with a half free-amplitude set point. Images were acquired at 256 × 256 pixels at a one line/s scan speed. All AFM data were analyzed using Gwyddion open-source SPM analysis software;48 in particular three-dimensional reconstructions were used to evaluate surface roughness. Surface roughness was computed as the root-mean-square (RMS) value of the height irregularities of AFM images. Differential Scanning Calorimetry. The thermal analysis of the neat and modified PET fabrics was performed using a PerkinElmer 6458

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Figure 1. Enzymatic hydrolysis and functionalization of PET fabrics using various Thermobif ida cellulosilytica cutinase variants after 24 h (A, C) and 72 h (B, D) varying temperatures and two different concentrations (0.5 and 5.0 μM). (DSC, Diamond) apparatus under a nitrogen flow (20 mL min −1). In order to investigate the effect of treatment on the degree of crystallinity, PET samples of 3−5 mg were heated in aluminum pans from 25 to 270 °C at a heating rate of 10 K min−1. The corresponding data and curves were recorded, and the degree of crystallinity (χc) was calculated by using the equation below:

χc = (ΔHm − ΔHcc)/ΔHm 0

This test determines the force required to separate two cords bonded together with an intermediate rubber layer (QWE 0.6 mm).9 During the peel test, it was necessary to create a weftless fabric of sufficient size to cover the curing mold cavity on a rotatable drum. These two fabrics were laid one against the other, with cords in the same direction, to form the pad. This pad had a nonstick fabric, such as holland cloth, that was able to separate the two fabric layers at one end for a sufficient distance to eventually permit adjacent ends of each fabric layer to be separated after curing. Therefore, the pad was incorporated in the mold and then vulcanized in a curing press at a controlled temperature, pressure, and time. The temperature was set at 160 °C and pressure at 22 kN for 15 min. In the end, the vulcanized specimens were removed from the hot mold and air-cooled for 3 h before testing. After the air-cooling period, the specimen was cut into two straps (duplicate) in the long direction, parallel to that of the cords, and the peel test was performed (ASTM D4393).50 While pulling apart the layers of cords, two types of data are collected and analyzed, the coverage grade (%) and the pull (N/cm). The coverage grade indicates the surface of the rubber that is possible to observe after the test; 100% indicates a complete homogeneous coverage and correlates with a cohesive failure, whereas 0% means a polymer surface without rubber and therefore denotes an adhesive failure. The pull indicates the force that the instrument needs to apply for separating the rubber from the rubber.

(2)

where ΔHm is the enthalpy of melting, ΔHcc is the enthalpy of cold crystallization, and ΔHm0 is the enthalpy of fusion per gram of a perfect crystal of infinite size. The latter is 140 J/g for 100% crystalline PET.49 Rubber Adhesion Test. Adhesion tests were performed according to ASTM D4393. This method is primarily used to evaluate tire cords using a suitable tire cord adhesive and a suitable rubber compound,50 and it is a commonly known and accepted test method within the tire industry. After enzymatic and chemical incubations, PET cord samples were treated with a dipping solution in order to prepare the cord for the rubber adhesion test. The dipping solution was a resorcinol formaldehyde latex (RFL-dip) solution that was an emulsion of a rubber latex in a solution of precondensate resin (resorcinol and formaldehyde in water) and under basic conditions. After the preparation of the dipping emulsion, some properties of the RFL dip were measured, e.g., viscosity, solid content, and pH (Table S1). Thereafter, PET cord (10 m) samples were rolled up on a tube and let through a small scale dipping line which was a copy of the production dipping line used in conversion plants. The machine was composed of a small bath, different rotors, and three ovens with different temperatures. The cord first went through the bath, filled with the RFL dip, where after it went through the three ovens, at the end, it was again wound-up on a tube. The parameters controlled during this process were oven temperature (°C), the tension applied on the cord (N), and the residence time of the cord in each oven (s), which was determined by the speed (m/min) and the number of passes in each oven (#); the conditions for the ovens are reported in the SI (Table S2). After the dipping, PET cord samples were left for 1 h under an ASTM environment (55% humidity and 24 °C) before preparing the samples for the adhesion test, called in a specific way the “Peel test.”

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RESULTS AND DISCUSSION In the particular case of PET, hydrolytic activity has been reported for members of the cutinase,28,51−53 lipase,28,51 and esterase families,28,36,54,55 among them cutinases are recognized to be the most active class. The ability of cutinase variants from Thermobif ida cellulosilytica to hydrolyze PET films and powders was reported,4,33,56 while hydrolysis of HMLS-PET has not been studied before. Hence, in this study, the hydrolysis of HMLSPET fabrics and cords used in rubber composites, in particular passenger car tires and hoses, by two native cutinases, Thc_Cut1 and Thc_Cut2, and two most active mutants, 6459

DOI: 10.1021/acssuschemeng.7b00475 ACS Sustainable Chem. Eng. 2017, 5, 6456−6465

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ACS Sustainable Chemistry & Engineering Thc_Cut2_DM and Thc_Cut2_TM,33 engineered toward amorphous PET as a substrate, was investigated (Figure 1). Enzymatic Hydrolysis: Detection of Carboxylic Groups. The first step of the study was to determine the optimum enzymatic hydrolysis temperature by measuring the degree of functionalization achieved on the material surface and assessing the fabric damage via quantification of the soluble released products, namely terephthalic acid (TA), mono-(2hydroxyethyl) terephthalate (MHET), and bis(2-hydroxyethyl) terephthalate (BHET)37 (Figure 1). Titration,18 liquid chromatography,4,57 or spectrophotometric methods58 are typically used to assess enzymatic PET hydrolysis, especially for recycling purposes, where the knowledge regarding the exact product composition is needed for a successful repolymerization. For functionalization applications, an easy and precise method to quantify the new surface created groups has not been reported in this field. The extent of surface functionalization correlates with the amount of COOH groups generated on the PET fabrics surface (SC, surface carboxylic groups) and was quantified with Toluidine Blue O (TBO), a cationic dye selectively binding to carboxyl funcitonalities.45,46,59,60 In this study, the highest SC for all investigated cutinases, Thc_Cut1 (114.38 U/mg), Thc_Cut2 (109.81 U/mg), Thc_Cut2_DM (104.99 U/mg), and Thc_Cut2_TM (147.60 U/mg) was obtained with an incubation temperature of 60 °C. Incubation at 50 and 70 °C resulted in a much lower SC compared to 60 °C. In line with the SC, also a lower amount of released products was observed (Figure 1). The finding for 70 °C was surprising since higher temperatures increase the enzymatic activity along with an increased polymer chain mobility, which facilitates the polymer accessibility to the enzyme’s active site as recently reported.56 Due to the rather long reaction times at elevated temperatures, the denaturation of the enzymes cannot be neglected as already shown for the native cutinase.61 In this study, Thc_Cut2_DM was particularly more active, achieving the highest SC at 70 °C after 24 h of incubation for both enzyme dosage levels (0.5 μM and 5.0 μM) when compared to native cutinases as well as triple mutant Thc_Cut2_TM. Obviously, the further mutation Arg19Ser did not improve the stability but reduced it even below the values of the native cutinases. Interestingly, a carboxyl amount reduction of 0.06 nmol mm−2 in the case of 0.5 μM and 0.03 nmol mm−2 for 5.0 μM was found for Thc_Cut2_DM after 72 h at 70 °C compared to the values after 24 h at 70 °C. This effect was probably a consequence of the reorganization of the initially generated carboxylic groups after long incubation times or due to solubilization of the outermost fragments generated (Figure S3). Fiber damage as well as the mode of enzyme hydrolysis of the high tenacity PET fabrics, exo vs endo, can be analyzed by combining the SC results with quantitative and qualitative analysis of the soluble released products. The most active enzymes in terms of released products were Thc_Cut1 and Thc_Cut2 DM, releasing 1.44 mM and 1.35 mM of TA and MHET, respectively, at optimal conditions (Figure 2). MHET was the most abundant release product for all the enzymes at a lower enzyme concentration, in the case of Thc_Cut2 and Thc_Cut2_TM, also at 5.0 μM. Previously, for amorphous PET3, Thc_Cut2_DM and Thc_Cut2_TM were the most active enzymes showing 4 and 1.5 times higher amounts of release products when compared with Thc_Cut2 and Thc_Cut1, respectively.

Figure 2. Enzymatic hydrolysis of PET fabrics using various cutinases at 24 and 72 h (T = 60 °C). The amount of the release products TA (black bars) and MHET (white bars) was quantified via HPLC analysis.

The ratio of TA/MHET for the mutants and the native Thc_Cut1 was close to 8:1, whereas the ratio for Thc_Cut2 was nearly 1:1. In this study, 3 and 4 times higher amounts of products were released from PET fabrics for Thc_Cut2_TM and for Thc_Cut2_DM, respectively, when compared to Thc_Cut2. The final ratio between TA and MHET was nearly 1:1 at low enzyme concentrations and nearly 3 for the double mutant at high concentrations (5.0 μM, 72 h). A similar profile with MHET concentrations, being up to 8 times higher when compared to TA, was described for Fusarium solani pisi cutinase.35 Equally, a clear enzyme concentration effect on the product distribution ratio over time can be observed in our case. Thc_Cut1 and Thc_Cut2_DM showed an increased TA to MHET ratio at the highest enzyme concentration, due to a fast MHET hydrolysis. The different product pattern detected for these two enzymes could be either due to changes in specificity for the soluble product MHET due to inhibitory effects as proposed by Zimmerman for TfCut2 from Thermobif ida f usca KW337 or due to different sorption properties onto PET film caused by the enzyme surface mutations.32 Comparing the highest SC values achieved, Thc_Cut1, Thc_Cut2_DM, and Thc_Cut2_TM created comparable amounts of new functional groups on the PET fabrics’ surface, yet Thc_Cut2_TM released 60% less soluble products. This indicates a more endo type hydrolysis mode of action for Thc_Cu2_TM when compared with the more exo acting Thc_Cut1 and Thc_Cut2_DM. However, at the lower concentration level, Thc_Cut1 showed a higher SC than both mutants, while releasing slightly lower amounts of hydrolysis products. The difference between the results obtained at the two concentration levels shows the importance of the ratio of the enzyme to polymer surface, indicating the relevance of the adsorption/desorption steps in the overall process. When compared with alkali treatments, which industrially have been used to create new functional groups on the PET surface,62,63 the highest SC of 0.51 nmol mm−2, obtained for Thc_Cut2_TM (60 °C, 72 h), was more than twice the value obtained after the strongest chemical modification tested (1 M NaOH, 50 °C for 2 h), 0.21 nmol mm−2 (Figure 3). An increase of the duration of the alkali treatment to 24 or 72 h did not lead to a further increase of the amount of carboxylic groups, while PET was structurally considerably damaged after 6460

DOI: 10.1021/acssuschemeng.7b00475 ACS Sustainable Chem. Eng. 2017, 5, 6456−6465

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ACS Sustainable Chemistry & Engineering

Figure 3. Surface carboxylic groups measured after treatment of PET fabrics with different concentrations of NaOH for 2 h at 50 °C and with Thc_Cut1 and Thc_Cut2_TM (5.0 μM) for 72 h at 60 °C. The SC was measured based on derivatization with the TBO method.

72 h of incubation (Figure S4). Hence, all further alkali treatments were conducted for 2 h. The use of concentrated base to hydrolyze polyesters is known to negatively affect the bulk properties of polyester, compromising its mechanical performance.35 The breaking strength, or rather the maximum stress that a material can withstand while being stretched or pulled before failing or breaking, was 169.3 ± 1.50 N after 2 h incubation with NaOH (1 M) and decreased to 168.6 ± 0.65 N after 4 h of incubation. In contrast, after the enzyme treatment (Thc_Cut1, 5.0 μM, 24 h), with a value of 176 ± 0.69 N, there was no significant decrease of the breaking strength when compared to a control without enzyme (Table S3). The 10% decrease in mechanical strength measured for the alkali treatment is an unacceptable value for an application like tires in which safety is one of strongest drivers. A more exowise hydrolytic action of the alkali treatment compared with a more endowise mode of action of the cutinase treatment was confirmed by quantification of the soluble degradation products and is in agreement with the data previously obtained by using XPS analysis.6 After enzymatic incubation with Thc_Cut1 and Thc_Cut2_TM (5.0 μM, 72 h) only 0.7% and 1.2% degradation of the material was measured, respectively. In contrast, the alkali treatment led to a 2.4% material loss after 2 h of incubation with NaOH (1 M) and 11.4% after 24 h, further explaining the reduction in the mechanical strength (Table S4). Scanning electron microscope (SEM) and atomic force microscope (AFM) analysis (Figure 4) of enzymatically treated yarn vs NaOH hydrolyzed yarns revealed significant differences in morphology with a smoother surface for the enzyme treated yarn, while the chemically treated one showed a damaged craterlike surface along all the fiber surface (Figure 4D). Surface roughness of the blank surface measured via AFM was 0.9 nm, considerably different when compared to alkali treated PET fabrics with a value of 3.6 nm. The roughness measured after enzymatic treatment showed intermediate values of 1.7 and 2.5 nm, respectively, for Thc_Cut1 and Thc_Cut2_TM. The effect of the different functionalization treatments on the polymer bulk structure was also investigated by measuring the polymer crystallinity by differential scanning calorimetry (DSC). The samples treated with the buffer as a control had a crystallinity of 36.3% ± 1.2, whereas the PET enzymatically treated under the most effective conditions showed a value of

Figure 4. SEM (left images) and AFM (right images) analysis of PET fabrics surface structure incubated with (A) phosphate buffer (pH 7, 100 mM at 60 °C for 24 h), (B) Thc_Cut1 (5.0 μM at 60 °C for 72 h), (C) Thc_Cut2_TM (5.0 μM at 60 °C for 72 h), and (D) NaOH (1 M at 50 °C for 2 h). The pictures show a clear difference in the samples after the different treatments; NaOH incubation damages the surface, creating holes along the yarn surface.

35.3% ± 0.6 (Thc_Cut1, 5.0 μM, 72 h) and 34.3% ± 0.6 (Thc_Cut2_TM, 5.0 μM, 72 h). The chemical treatment with NaOH (1 M, 2 h) showed a crystallinity value of 37.3 ± 0.6. The crystallinity values obtained after each treatment are shown in the Supporting Information (Table S5). The enzymatic and chemical treatments did not affect the PET fabrics’ bulk crystallinity nor the Soxhlet extraction step used to clean the surface (Table S6). Rubber Adhesion Tests. There are different types of adhesion tests used in the rubber industry. Among these, the peel test, as described in the Material and Methods (ASTM D4393),50 is considered to be the most elaborate but also most informative one. It gives rise to two important aspects of adhesion at the same time, the pull-force and the coverage. The pull-force provides information about the crack mechanism as well as the bonding strength of the investigated reinforcement after rubber vulcanization, whereas the coverage indicates the homogeneity of the dip as well as the interaction of the dip with the fiber surface and rubber matrix. For industrial applications, the aim is to achieve coverage values above 60% and pull forces as high as possible. After PET cord functionalization with Thc_Cut1 and Thc_Cut2_TM for 24 h and subsequent RFL treatment, a 6461

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ACS Sustainable Chemistry & Engineering clear increase in the coverage for the chosen carcass compound was observed (Figure 5).

Figure 6. Comparison of pull-force measurements of vulcanized PET cord after incubation with Thc_Cut1 and Thc_Cut2_TM (5.0 μM) for 24 and 72 h, and with NaOH (0.05 M, 1 M) for 2 h. The enzymatic and chemical treatments increase in the pull compared to the untreated PET cord.

In addition, an excessive amount of polar groups generated on the surface might hinder the wettability of the hydrophobic RFL solution and its polymer penetration. Hence, measurements of water contact angle over time, recording the soaking speed of water drops into the PET fabric, were recorded to assess the wettability after enzymatic treatment (see videos in SI Video S1). The results (Figure 7) showed the same trend for the two different enzymes after 24 h of incubation. Treatment of PET

Figure 5. Coverage measurements of PET cord after incubation with Thc_Cut1 and Thc_Cut2_TM (5.0 μM) for 24 and 72 h and with NaOH (0.05 M, 1 M) for 2 h. After functionalization, the samples were subsequently dipped with a standard RFL dipping protocol; for the “Peel test,” a standard carcass rubber compound was used. The enzymatic and chemical treatments determine an increase in the rubber coverage compared to the PET cord without any treatment. The coverage grade refers to the surface of the rubber (%) that can be observed after the test while 100% indicates a complete homogeneous coverage and correlates with a cohesive failure, whereas 0% means a polymer surface without rubber and therefore denotes an adhesive failure.

Levels of 25% (0.5 μM) and 30% (5.0 μM) in the coverage were measured after Thc_Cut1 treatment, values that increased to 45% (0.5 μM) and 40% (5.0 μM) after treatment with the triple mutant Thc_Cut2_TM. These values represented a significant improvement compared to the blank sample having only 10% coverage. The rubber coverage after NaOH incubation was 20% at both concentrations of 1 and 0.05 M. The pull-force (Figure 6) required to separate the rubber from the PET cord was 76.7 ± 1.4 N/cm (0.5 μM) and 83.3 ± 1.3 N/cm (5.0 μM) for Thc_Cut1 and 89.2 ± 1.7 N/cm (0.5 μM) and 84.0 ± 6.4 N/cm (5.0 μM) for Thc_Cut2_TM. These values were higher than the one found for the untreated PET cord (58.0 ± 9.9 N/cm) reference. Interestingly, the pull values were also higher than after NaOH (1 M) treatment (72.1 ± 4.9 N/cm) despite a 50% lower amount of surface carboxyl groups compared to the enzyme treated cords. In addition, the higher roughness might contribute to an increase in the pull values by creating a mechanical interlock. The pull-force decreased after an extended enzyme treatment of 72 h; regarding the root cause of this observation, the generation of small fragments on the PET cord surface after extended enzyme treatment may reduce intrapolymer bonding strength. Previously, Hudek et al.64 used a plasma treatment method in order to improve the adhesion strength of HMLS-PET cords to rubber. Similarly with our study, they observed that a higher functionalization degree leads to a decrease of the adhesion strength.65

Figure 7. Wettability of PET fabrics after incubation with Thc_Cut1 for 24 h (top) and Thc_Cut2_TM for 24 h (bottom) at 60 °C.

fabrics (0.5 μM and 5.0 μM) with cutinases leads to increasing hydrophilicity. For Thc_Cut1, the curve tended to zero after 5.5 s (0.5 μM) and 2.6 s (5.0 μM). Similarly, after the activation with Thc_Cut2_TM, the water drop completely soaked after 5 s (0.5 μM) and 3 s (5.0 μM). 6462

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ACS Sustainable Chemistry & Engineering The samples incubated with both enzymes for 72 h; it was impossible to measure the water contact angle due to a high hydrophilicity, which made the water drop immediately soaked (Video S1). Blank samples showed a water contact angle of 66.5° ± 2.12°; the water drop was completely soaked into the fabric after 20 s. Hence, this increase of hydrophilicity after excessive enzyme treatment could be responsible for a mismatch between the interfacial tension of the enzymatically activated surface and the RFL dipping solution, limiting the possible increase in adhesion due to repulsion of the RFL dipping. In order to confirm the lack of connection with the latex part of the RFL solution, a surface free energy (SFE) experiment was performed. The Owens, Wendt, Rabel, and Kaelble (OWRK) method was used as a standard way for calculating the surface free energy of treated PET films from the contact angle of three liquids with different interfacial tensions, such as water, glycerol, and olive oil. Due to the soaking properties of the PET fabrics itself, PET films were used to carried out the experiment. As expected, the SFE of the latex solution (42−46 mN/m)65 was considerably different from the value detected on PET films after Thc_Cut1 and Thc_Cut2_TM (5.0 μM) 24 h incubation, reported as 79 ± 2.19 mN/m and 94.67 ± 4.06 mN/m, respectively. All of the values measured after 72 h of incubation are shown in the SI (Table S7). The results of the study demonstrated that enzymatic creation of new functional groups on the PET-cord surface prior to the RFL dipping can significantly increase the adhesion to the rubber matrix, reaching up to 45% in coverage with the carcass compound from QWE. Above a certain carboxyl surface density, the positive impact on adhesion is reduced, due to a clear mismatch of surface energies between the hydrophilic functionalized fiber surface and the RF-resin. Potentially, a weak interaction of the cleaved polymer chains with the underneath polymer is currently under investigation.

is currently investigated and could further enhance the level of adhesion.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b00475. Details of aspect of the HMLS-PET fabrics after toluidine blue treatment, parameters regarding the RFL-dipping solution for HMLS-PET cords, conditions during the dipping step in tires’ manufacturing, the aging of the surface carboxylic groups, breaking strength values after enzymatic and chemical incubation, weight loss after enzymatic and chemical treatment, crystallinity results, and surface free energy on HMLS-PET films (PDF) Water contact angle video A (AVI) Water contact angle video B (AVI)

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +43 664 4195940. E-mail: mueller@glanzstoff.com. *Tel.: +43 664 4195940. E-mail: E.Herrero@glanzstoff.com. ORCID

Sara Vecchiato: 0000-0003-4082-6184 Enrique Herrero Acero: 0000-0001-7122-9353 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work has been supported by the Federal Ministry of Science, Research and Economy (BMWFW); the Federal Ministry of Traffic, Innovation and Technology (bmvit); the Styrian Business Promotion Agency SFG; the Standortagentur Tirol; and ZIT−Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG. The authors are grateful to Gagik Ghazaryan for the support with the differential scanning calorimetry analysis; to Barbara Zartl for the enzymatic production of all the cutinases used; and to Dr. Stepanka Jankova, Olvier Defrain, and Fabian Septon for their support in adhesion sample preparation and testing.

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CONCLUSIONS A new enzymatic process for activation of high modulus low shrinkage HMLS-PET fabrics used for rubber composites was investigated. Mild enzymatic hydrolysis of the outermost layer of fabric/cords led to a 5 fold increase of carboxylic and hydroxyl groups compared to the blank sample. The selectivity of different cutinases from Thermobif ida cellulosilytica in terms of mode of hydrolysis, endo vs exo, and release products was compared with this highly crystalline PET substrate. Compared with alkaline treatment, the enzymatic process did not alter the bulk or mechanical properties of the material as shown by DSC and breaking strength. Confirming these data, imaging of the polymer surface revealed a much more damaged surface in the case of the alkaline treatment when compared with enzymatic surface hydrolysis. Adhesion tests according to ASTM D4393 with enzymatically activated HMLS-PET revealed increased bonding between the rubber and the polymer in terms of coverage (+35%) and pull force (+31.2 N/cm) compared to untreated PET cords. However, the over generation of carboxyl groups on the HMLS-PET surface led to a decreased bonding of the RF-resin with the fiber surface, resulting in lower coverage and pull values when applying standard RFL dipping chemistry. The development of RFL or functionally similar dipping formulations exploiting enzymatically hydrophilized polymeric surfaces

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