Amorphous Poly Ethylene Terephthalate Films Research Articles

Efficient thermophilic PET hydrolase enhanced by cross correlation-based accumulated mutagenesis strategy

Polyethylene terephthalate (PET) has caused significant pollution issues. Compared to chemical degradation with high energy consumption and cost, enzymatic degradation offers a sustainable solution for PET waste recycling. However, the hydrolytic activity of current PET hydrolases still requires improvement. In this study, a cross-correlation-based accumulated mutagenesis (CAM) strategy was developed to enhance the hydrolysis activity. By mitigating epistatic effect and combinational mutations, we achieved a highly active variant LCC-YGA (H183Y/L124G/S29A) with 2.1-fold hydrolytic activity on amorphous PET films of LCC-ICCG. Conformational analysis elucidated how the introduction of distal mutations enhanced activity. The dynamic correlation among different regions facilitated a synergistic effect, enhancing binding pocket flexibility through remote interactions. Totally, this work offers novel insights and methods for PET hydrolases engineering and provides an efficient enzyme for PET degradation and recycling.

Glacier as a source of novel polyethylene terephthalate hydrolases.

Polyethylene terephthalate (PET) is a major component of microplastic contamination globally, which is now detected in pristine environments including Polar and mountain glaciers. As a carbon-rich molecule, PET could be a carbon source for microorganisms dwelling in glacier habitats. Thus, glacial microorganisms may be potential PET degraders with novel PET hydrolases. Here, we obtained 414 putative PET hydrolase sequences by searching a global glacier metagenome dataset. Metagenomes from the Alps and Tibetan glaciers exhibited a higher relative abundance of putative PET hydrolases than those from the Arctic and Antarctic. Twelve putative PET hydrolase sequences were cloned and expressed, with one sequence (designated as GlacPETase) proven to degrade amorphous PET film with a similar performance as IsPETase, but with a higher thermostability. GlacPETase exhibited only 30% sequence identity to known active PET hydrolases with a novel disulphide bridge location and, therefore may represent a novel PET hydrolases class. The present work suggests that extreme carbon-poor environments may harbour a diverse range of known and novel PET hydrolases for carbon acquisition as an environmental adaptation mechanism.

Deep learning guided enzyme engineering of Thermobifida fusca cutinase for increased PET depolymerization

A responsible and sustainable circular economy of polymers requires efficient recycling processes with a low CO2 footprint. Enzymatic depolymerization of polyethylene terephthalate (PET) is a first step to make PET polymers a part of a circular economy of polymers. In this study, a structure-based deep learning model was utilized to identify residues in TfCut2 that are responsible for improved hydrolytic activity and enhanced stability. Machine learning guided design identified novel beneficial positions (L32E, S35E, H77Y, R110L, S113E, T237Q, R245Q, and E253H), which were evaluated and stepwise recombined yielding finally the beneficial variant L32E/S113E/T237Q. The latter TfCut2 variant exhibited improved PET depolymerization when compared with TfCut2 WT (amorphous PET film, 2.9-fold improvement // crystalline PET powder (crystallinity > 40%), 5.3-fold improvement). In terms of thermal resistance the variant L32E/S113E/T237Q showed a 5.7 °C increased half-inactivation temperature (T5060). The PET-hydrolysis process was monitored via a quartz crystal microbalance with dissipation monitoring (QCM-D) in real-time to determine depolymerization kinetics of PET coated onto the gold sensor. Finally, conformational dynamics analysis revealed that the substitutions induced a conformational change in the variant L32E/S113E/T237Q, in which the dominant conformation enabled a closer contact between the catalytic site and PET resulting in increased PET-hydrolysis. Overall, this study demonstrates the potential of deep learning models in protein engineering for identifying and designing efficient PET depolymerization enzymes.

Structure and function of the metagenomic plastic-degrading polyester hydrolase PHL7 bound to its product

The recently discovered metagenomic-derived polyester hydrolase PHL7 is able to efficiently degrade amorphous polyethylene terephthalate (PET) in post-consumer plastic waste. We present the cocrystal structure of this hydrolase with its hydrolysis product terephthalic acid and elucidate the influence of 17 single mutations on the PET-hydrolytic activity and thermal stability of PHL7. The substrate-binding mode of terephthalic acid is similar to that of the thermophilic polyester hydrolase LCC and deviates from the mesophilic IsPETase. The subsite I modifications L93F and Q95Y, derived from LCC, increased the thermal stability, while exchange of H185S, derived from IsPETase, reduced the stability of PHL7. The subsite II residue H130 is suggested to represent an adaptation for high thermal stability, whereas L210 emerged as the main contributor to the observed high PET-hydrolytic activity. Variant L210T showed significantly higher activity, achieving a degradation rate of 20 µm h−1 with amorphous PET films.

Comparison of the enzymatic depolymerization of polyethylene terephthalate and AkestraTM using Humicola insolens cutinase

The enzymatic depolymerization of synthetic polyesters has become of great interest in recycling plastics. Most of the research in this area focuses on the depolymerization of polyethylene terephthalate (PET) due to its widespread use in various applications. However, the enzymatic activity on other commercial polyesters is less frequently investigated. Therefore, AkestraTM attracted our attention, which is a copolymer derived from PET with a partially biobased spirocyclic acetal structure. In this study, the activity of Humicola insolens cutinase (HiCut) on PET and AkestraTM films and powder was investigated. HiCut showed higher depolymerization activity on amorphous PET films than on Akestra™ films. However, an outstanding performance was achieved on AkestraTM powder, reaching 38% depolymerization in 235h, while only 12% for PET powder. These results are consistent with the dependence of the enzymes on the crystallinity of the polymer since Akestra™ is amorphous while the PET powder has 14% crystallinity. On the other hand, HiCut docking studies and molecular dynamic simulations (MD) suggested that the PET-derived mono (hydroxyethyl)terephthalate dimer (MHET)2 is a hydrolyzable ligand, producing terephthalic acid (TPA), while the Akestra™-derived TPA-spiroglycol ester is not, which is consistent with the depolymerization products determined experimentally. MD studies also suggest ligand-induced local conformational changes in the active site.

Efficient depolymerization of polyethylene terephthalate (PET) and polyethylene furanoate by engineered PET hydrolase Cut190

The enzymatic recycling of polyethylene terephthalate (PET) can be a promising approach to tackle the problem of plastic waste. The thermostability and activity of PET-hydrolyzing enzymes are still insufficient for practical application. Pretreatment of PET waste is needed for bio-recycling. Here, we analyzed the degradation of PET films, packages, and bottles using the newly engineered cutinase Cut190. Using gel permeation chromatography and high-performance liquid chromatography, the degradation of PET films by the Cut190 variant was shown to proceed via a repeating two-step hydrolysis process; initial endo-type scission of a surface polymer chain, followed by exo-type hydrolysis to produce mono/bis(2-hydroxyethyl) terephthalate and terephthalate from the ends of fragmented polymer molecules. Amorphous PET powders were degraded more than twofold higher than amorphous PET film with the same weight. Moreover, homogenization of post-consumer PET products, such as packages and bottles, increased their degradability, indicating the importance of surface area for the enzymatic hydrolysis of PET. In addition, it was required to maintain an alkaline pH to enable continuous enzymatic hydrolysis, by increasing the buffer concentration (HEPES, pH 9.0) depending on the level of the acidic products formed. The cationic surfactant dodecyltrimethylammonium chloride promoted PET degradation via adsorption on the PET surface and binding to the anionic surface of the Cut190 variant. The Cut190 variant also hydrolyzed polyethylene furanoate. Using the best performing Cut190 variant (L136F/Q138A/S226P/R228S/D250C-E296C/Q123H/N202H/K305del/L306del/N307del) and amorphous PET powders, more than 90 mM degradation products were obtained in 3 days and approximately 80 mM in 1 day.Graphical

Low Carbon Footprint Recycling of Post-Consumer PET Plastic with a Metagenomic Polyester Hydrolase.

Earth is flooded with plastics and the need for sustainable recycling strategies for polymers has become increasingly urgent. Enzyme‐based hydrolysis of post‐consumer plastic is an emerging strategy for closed‐loop recycling of polyethylene terephthalate (PET). The polyester hydrolase PHL7, isolated from a compost metagenome, completely hydrolyzes amorphous PET films, releasing 91 mg of terephthalic acid per hour and mg of enzyme. Vertical scanning interferometry shows degradation rates of the PET film of 6.8 μm h−1. Structural analysis indicates the importance of leucine at position 210 for the extraordinarily high PET‐hydrolyzing activity of PHL7. Within 24 h, 0.6 mgenzyme gPET −1 completely degrades post‐consumer thermoform PET packaging in an aqueous buffer at 70 °C without any energy‐intensive pretreatments. Terephthalic acid recovered from the enzymatic hydrolysate is then used to synthesize virgin PET, demonstrating the potential of polyester hydrolases as catalysts in sustainable PET recycling processes with a low carbon footprint.

Engineering and evaluation of thermostable IsPETase variants for PET degradation.

Polyethylene terephthalate (PET) is a mass‐produced petroleum‐based synthetic polymer. Enzymatic PET degradation using, for example, Ideonella sakaiensis PETase (IsPETase) can be a more environmentally friendly and energy‐saving alternative to the chemical recycling of PET. However, IsPETase is a mesophilic enzyme with an optimal reaction temperature lower than the glass transition temperature (T g) of PET, where the amorphous polymers can be readily accessed for enzymatic breakdown. In this study, we used error‐prone PCR to generate a mutant library based on a thermostable triple mutant (TM) of IsPETase. The library was screened against the commercially available polyester‐polyurethane Impranil DLN W 50 for more thermostable IsPETase variants, yielding four variants with higher melting points. The most promising IsPETaseTMK95N/F201I variant had a 5.0°C higher melting point than IsPETaseTM. Although this variant showed a slightly lower activity on PET at lower incubation temperatures, its increased thermostability makes it a more active PET hydrolase at higher reaction temperatures up to 60°C. Several other variants were compared and combined with selected previously published IsPETase mutants in terms of thermostability and hydrolytic activity against PET nanoparticles and amorphous PET films. Our findings indicate that thermostability is one of the most important characteristics of an effective PET hydrolase.

Effect of crystallinity on the recovery rate of superhydrophobicity in plasma-nanostructured polymers.

This study explored the optimum conditions to achieve superhydrophobicity in polyethylene terephthalate (PET) in terms of crystallinity and microstructure. Surface superhydrophobicity was achieved by nanostructures induced by oxygen plasma etching and the recovery process of low surface energy through thermal aging of various PETs; semi-crystalline biaxial PET (B-PET) film, amorphous PET (A-PET) film, and semi-crystalline PET (F-PET) fabric. Under the anisotropic plasma etching, the nanostructures on the B-PET film were the longest, followed by the F-PET fabric, which developed a hierarchical micro/nanostructure, then the A-PET film. During thermal aging at 80 °C near Tg, the plasma-treated A-PET film recovered its superhydrophobicity within 3 h, while the plasma-treated B-PET film did not exhibit superhydrophobicity. At 130 °C, higher than Tg, the plasma-treated B-PET film recovered its superhydrophobicity within 1 h, but the plasma-treated A-PET film became opaque as its nanostructures deformed, decreasing its superhydrophobicity. The plasma-treated F-PET fabric exhibited faster recovery and greater superhydrophobicity than the plasma-treated B-PET film, due to its hierarchical micro/nanostructure. In addition, hydrophobic recovery during thermal aging was proved with a decrease in surface polar groups, lowering the surface energy using XPS analysis. Therefore, by designing the ratio of crystal to amorphous regions and surface micro/nanostructures, one can rapidly fabricate superhydrophobic PETs without additional surface finishing.

Molecular dynamics simulation study on the structural properties of poly (ethylene terephthalate) under uniaxial extension and thermal shrinkage processes

In thermal shrinkage process for polyethylene terephthalate (PET) films, PET film is uniaxially stretched and then thermally relaxed. We investigate the effect of heat relaxation temperature on the thermal shrinkage of uniaxially stretched PET film using molecular dynamics simulation method. Through our investigation, we find that the thermal shrinkage ratio of the amorphous PET film is reduced as a function of heat relaxation temperature, whereas the skirt ratio is weakly correlated with the heat relaxation temperature, exhibiting the most negative skirt ratio in the case of the highest thermal shrinkage ratio. Our analysis on the PET film at molecular level further verifies that the trans-gauche transformation observed in ethylene glycol units during the simulated process is a driving force for the thermal shrinkage of uniaxially stretched PET systems.

Effect of tensile speed on mechanical characteristics of preliminary rolled polyethylene terephthalate films

The effect of tensile speed on the mechanical properties of amorphous polyethylene terephthalate films is studied. The films are oriented by rolling on laboratory rollers at room temperature. In the samples with the rolling degree of two, the subsequent tension with the velocity of 5 mm/min does not afford a neck. As the tensile speed increases to 500 mm/min, a neck results from instability of the deformation process, which is caused by the polymer heating. Upon high-speed tension, the temperature of the rolled samples exceeds the glass-transition point of polyethylene terephthalate. Despite the polymer heating, the rolling completely suppresses the formation of pores. The rolling leads to the delocalization of flow over the region of polymer transfer to the neck. While in the nonrolled films the flow occurs in the zone with the length on the order of the film thickness, after the rolling, the size of the flow zone increases, and the region with the length on the order of the film width is deformed.

Formation of shear bands in amorphous polyethylene terephthalate films while rolling

158 Amorphous polyethylene terephthalate (PET) were oriented by rolling on a laboratory scale rolling press under ambient conditions. The effect of rolling on the polymer structure was studied. At early stages of rolling, plastic deformation of the polymer is inhomo geneous. Deformation is localized in the system of nearly perpendicular shear bands forming a zigzag. The formation of the zigzag while rolling can be explained by polymer strain softening. After a shear band has reached the surface, a band that is perpendic ular to it and extends to the opposite surface nucleates in that existing band.

Analysis of thermally stimulated current and effect of rubbery annealing around glass-rubber transition temperature in polyethylene terephthalate

Thermally stimulated currents (TSC) in amorphous polyethylene terephthalate films have been investigated in the temperature range of −180 to 140°C. This material shows a very weak intensity peak at approximately −95°C and another around 80°C originated from dipolar process (α-peak), as evidenced from the variation of polarizing conditions such as applied electric field and polarizing time. The effect of isochronal rubbery annealing starts to appear from a temperature of annealing of 90°C, it then appears in a TSC spectrum two components around 88 and 108°C allotted, respectively to the true and rigid amorphous phases. The first component tends to disappear in an irreversible way to the detriment of the second which implies the establishment of an order within material during annealing by the formation and growth of nodules. The thermostimulated currents technique allowed to calculate with good precision the activation parameters of each process as well as the evaluation of the crystallinity rate by an established empirical formula.

Crystallization orientation and relaxation in uniaxially drawn poly(ethylene terephthalate)

Amorphous polyethylene terephthalate film has been uniaxially drawn below and above the glass transition temperature, T g, and the effect of temperature, strain rate and extent of elongation on the development of crystallinity investigated by differential scanning calorimetry (DSC). The isothermal crystallization time dependence was analysed using the Avrami equation and the mechanism derived was in good agreement with the morphology observed by SEM. Nucleation and growth changed with the degree of strain and there was a change from isotropic spherulites growing uniformly in all directions from randomly oriented nuclei to elliptical spherulites originating from row nuclei and an increased growth rate. The effect of initial orientation on crystallization rate and its dependence on temperature were quantitatively described by Ziabicki's equation. Changes in molecular orientation on drawing have been measured by polarized FT-IR spectroscopy, based on the ratio of the trans to gauche conformers of CH 2 group in glycol segments. A combination of polarized FT-IR spectroscopy and DSC enabled the degree of orientation in the crystalline and non-crystalline phases to be determined separately. Results indicated that orientation and the strain-induced crystallization increased with increasing draw ratio and elongation, but decreased with increasing temperature in the range of 75–85°C due to the relaxation of molecular chains segments. Relaxation of chain segments mainly occurred in non-crystalline region.

The effect of stretching conditions on properties of amorphous polyethylene terephthalate film

AbstractThe effect of stretching condition on stress‐induced crystallization of amorphous polyethylene terephthalate (PET) film has been studied. Cast PET film was stretched at 85°C with stretching rates ranging from 100% per minute to 600% per minute for various stretching ratios. The stretched film samples were annealed at 140°C both under tension as well as under relaxed conditions. Tensile properties were determined using Instron Tensile Tester and results were compared. Tenacity, yield stress, and initial modulus were found to increase while elongation at break decreased with the increase in stretching rate. The degree of crystallinity was measured by x‐ray diffraction as well as density methods. Birefringence and crystalline orientation factor values were determined experimentally and using these, amorphous orientation factor values were calculated. Birefringence, crystalline orientation and amorphous orientation values were found to increase with the increase in stretching rate. These studies showed that at low stretching rates a significant amount of relaxation takes place, resulting in lower orientation and strength. On the other hand, at higher stretching rates relaxation is minimized thus providing high orientation and strength. Upon annealing the crystallinity values increased as expected, however the trend of orientation and mechanical properties with respect to stretching rate was similar to those of unannealed films.

The yield behaviour of amorphous polyethylene terephthalate: An activated rate theory approach

Yield-stress measurements on amorphous polyethylene terephthalate film are presented covering five decades of strain rate at temperatures from just below the glass transition (≈ 60° C) to −160° C. The data were obtained by combining measurements from a conventional Instron machine with data from a high-speed tensile tester capable of applying strain rates of up to 50 sec−1. Five different failure modes have been identified over this extensive range of test conditions. All data from tests which show a clear yield point can be described accurately by an extension of the Ree Eyring approach to include two processes. The process which is only important at low temperatures correlates well with theβ-process observed in dynamic mechanical experiments.

The crystallization of polyethylene terephthalate by organic liquids

The development of crystallinity in essentially amorphous polyethylene terephthalate film following immersion in a range of organic liquids at 25°C has been studied. The rate of development of crystallinity varies markedly with the liquid and although molecular size of the liquid may be a contributory factor it is not the only one. Both the equilibrium degree of crystallinity and swelling of the film vary with the liquid and in a similar manner and it would seem that polarity, type and solubility parameter of the liquid are important factors governing both equilibrium crystallinity and swelling. It is suggested that solvation of the polymer occurs and that interaction of solvated polymer and liquid leads to conditions favourable to crystallization.