Inhalable Textile Microplastic Fibers Impair Airway Epithelial Differentiation.

Rationale: Microplastics are a pressing global concern, and inhalation of microplastic fibers has been associated with interstitial and bronchial inflammation in flock workers. However, how microplastic fibers affect the lungs is unknown. Objectives: Our aim was to assess the effects of 12 × 31 µm nylon 6,6 (nylon) and 15 × 52 µm polyethylene terephthalate (polyester) textile microplastic fibers on lung epithelial growth and differentiation. Methods: We used human and murine alveolar and airway-type organoids as well as air-liquid interface cultures derived from primary lung epithelial progenitor cells and incubated these with either nylon or polyester fibers or nylon leachate. In addition, mice received one dose of nylon fibers or nylon leachate, and, 7 days later, organoid-forming capacity of isolated epithelial cells was investigated. Measurements and Main Results: We observed that nylon microfibers, more than polyester, inhibited developing airway organoids and not established ones. This effect was mediated by components leaching from nylon. Epithelial cells isolated from mice exposed to nylon fibers or leachate also formed fewer airway organoids, suggesting long-lasting effects of nylon components on epithelial cells. Part of these effects was recapitulated in human air-liquid interface cultures. Transcriptomic analysis revealed upregulation of Hoxa5 after exposure to nylon fibers. Inhibiting Hoxa5 during nylon exposure restored airway organoid formation, confirming Hoxa5's pivotal role in the effects of nylon. Conclusions: These results suggest that components leaching from nylon 6,6 may especially harm developing airways and/or airways undergoing repair, and we strongly encourage characterization in more detail of both the hazard of and the exposure to microplastic fibers.

Microplastics increase cadmium absorption and impair nutrient uptake and growth in red amaranth (Amaranthus tricolor L.) in the presence of cadmium and biochar.

Microplastic (MP) pollution in terrestrial ecosystems is gaining attention, but there is limited research on its effects on leafy vegetables when combined with heavy metals. This study examines the impact of three MP types-polyethylene (PE), polyethylene terephthalate (PET), and polystyrene (PS)-at concentrations of 0.02, 0.05, and 0.1% w/w, along with cadmium (Cd) and biochar (B), on germination, growth, nutrient absorption, and heavy metal uptake in red amaranth (Amaranthus tricolor L.). We found that different MP types and concentrations did not negatively affect germination parameters like germination rate, relative germination rate, germination vigor, relative germination vigor, and germination speed. However, they increased phytotoxicity and decreased stress tolerance compared to an untreated control (CK1). The presence of MPs, particularly the PS type, reduced phosphorus and potassium uptake while enhancing Cd uptake. For example, treatments PS0.02CdB, PS0.05CdB, and PS0.1CdB increased Cd content in A. tricolor seedlings by 158%, 126%, and 44%, respectively, compared to the treatment CdB (CK2). Additionally, MP contamination led to reduced plant height, leaf dry matter content, and fresh and dry weights, indicating adverse effects on plant growth. Moreover, the presence of MPs increased bioconcentration factors and translocation factors for Cd, suggesting that MPs might act as carriers for heavy metal absorption in plants. On the positive side, the addition of biochar improved several root parameters, including root length, volume, surface area, and the number of root tips in the presence of MPs, indicating potential benefits for plant growth. Our study shows that the combination of MPs and Cd reduces plant growth and increases the risk of heavy metal contamination in food crops. Further research is needed to understand how different MP types and concentrations affect various plant species, which will aid in developing targeted mitigation strategies and in exploring the mechanisms through which MPs impact plant growth and heavy metal uptake. Finally, investigating the potential of biochar application in conjunction with other amendments in mitigating these effects could be key to addressing MP and heavy metal contamination in agricultural systems.

Reversible Sensing Technologies Using Upcycled TPEE: Crafting pH and Light Responsive Materials towards Sustainable Monitoring.

Multiresponsive materials with reversible and durable characteristics are indispensable because of their promising applications in environmental change detections. To fabricate multiresponsive materials in mass production, however, complex reactions and impractical situations are often involved. Herein, a dual responsive (light and pH) spiropyran-based smart sensor fabricated by a simple layer-by-layer (LbL) assembly process from upcycled thermoplastic polyester elastomer (TPEE) materials derived from recycled polyethylene terephthalate (r-PET) is proposed. Positively charged chitosan solutions and negatively charged merocyanine-COOH (MC-COOH) solutions are employed in the LbL assembly technique, forming the chitosan-spiropyran deposited TPEE (TPEE-CH-SP) film. Upon UV irradiation, the spiropyran-COOH (SP-COOH) molecules on the TPEE-CH-SP film undergo the ring-opening isomerization, along with an apparent color change from colorless to purple, to transform into the MC-COOH molecules. By further exposing the TPEE-CH-MC film to hydrogen chloride (HCl) and nitric acid (HNO3) vapors, the MC-COOH molecules can be transformed into protonated merocyanine-COOH (MCH-COOH) with the simultaneous color change from purple to yellow.

Metagenome-derived SusD-homologs affiliated with Bacteroidota bind to synthetic polymers.

Starch utilization system (Sus)D-homologs are well known for their carbohydrate-binding capabilities and are part of the sus operon in microorganisms affiliated with the phylum Bacteroidota. Until now, SusD-like proteins have been characterized regarding their affinity toward natural polymers. In this study, three metagenomic SusD homologs (designated SusD1, SusD38489, and SusD70111) were identified and tested with respect to binding to natural and non-natural polymers. SusD1 and SusD38489 are cellulose-binding modules, while SusD70111 preferentially binds chitin. Employing translational fusion proteins with superfolder GFP (sfGFP), pull-down assays, and surface plasmon resonance (SPR) has provided evidence for binding to polyethylene terephthalate (PET) and other synthetic polymers. Structural analysis suggested that a Trp triad might be involved in protein adsorption. Mutation of these residues to Ala resulted in an impaired adsorption to microcrystalline cellulose (MC), but not so to PET and other synthetic polymers. We believe that the characterized SusDs, alongside the methods and considerations presented in this work, will aid further research regarding bioremediation of plastics. IMPORTANCE: SusD1 and SusD38489 can be considered for further applications regarding their putative adsorption toward fossil-fuel based polymers. This is the first time that SusD homologs from the polysaccharide utilization loci (PUL), largely described for the phylum Bacteroidota, are characterized as synthetic polymer-binding proteins.

Exploring yeast biodiversity and process conditions for optimizing ethylene glycol conversion into glycolic acid.

Plastics have become an indispensable material in many fields of human activities, with production increasing every year; however, most of the plastic waste is still incinerated or landfilled, and only 10% of the new plastic is recycled even once. Among all plastics, polyethylene terephthalate (PET) is the most produced polyester worldwide; ethylene glycol (EG) is one of the two monomers released by the biorecycling of PET. While most research focuses on bacterial EG metabolism, this work reports the ability of Saccharomyces cerevisiae and nine other common laboratory yeast species not only to consume EG, but also to produce glycolic acid (GA) as the main by-product. A two-step bioconversion of EG to GA by S. cerevisiae was optimized by a design of experiment approach, obtaining 4.51 ± 0.12 g L-1 of GA with a conversion of 94.25 ± 1.74% from 6.21 ± 0.04 g L-1 EG. To improve the titer, screening of yeast biodiversity identified Scheffersomyces stipitis as the best GA producer, obtaining 23.79 ± 1.19 g L-1 of GA (yield 76.68%) in bioreactor fermentation, with a single-step bioprocess. Our findings contribute in laying the ground for EG up-cycling strategies with yeasts.

An Atomic-Level Perspective on the interactions between Organic Pollutants and PET particles: A Comprehensive Computational Investigation.

Microplastics (MPs) have recently attracted a lot of attention worldwide due to their abundance and potentially harmful effects on the environment and on human health. One of the factors of concern is their ability to adsorb and disperse other harmful organic pollutants in the environment. To properly assess the adsorption capacity of MP for organic pollutants in different environments, it is pivotal to understand the mechanisms of their interactions in detail at the atomic level. In this work, we studied interactions between polyethylene terephthalate (PET) MP and small organic pollutants containing different functional groups within the framework of density functional theory (DFT). Our computational outcomes show that organic pollutants mainly bind to the surface of a PET model via weak non-bonding interactions, mostly hydrogen bonds. The binding strength between pollutant molecules and PET particles strongly depends on the adsorption site while we have found that the particle size is of lesser importance. Specifically, carboxylic sites are able to form strong hydrogen bonds with pollutants containing hydrogen bond donor or acceptor groups. On the other hand, it is found that in such kind of systems π-π interactions play a minor role in adsorption on PET particles.

Plastic Quantification and Polyethylene Overestimation in Agricultural Soil Using Large-Volume Pyrolysis and TD-GC-MS/MS.

Quantification of microplastics in soil is needed to understand their impact and fate in agricultural areas. Often, low sample volume and removal of organic matter (OM) limit representative quantification. We present a method which allows simultaneous quantification of microplastics in homogenized, large environmental samples (>1 g) and tested polyethylene (PE), polyethylene terephthalate (PET), and polystyrene (PS) (200-400 µm) overestimation by fresh and diagenetically altered OM in agricultural soils using a new combination of large-volume pyrolysis adsorption with thermal desorption-gas chromatography-tandem mass spectrometry (TD-GC-MS/MS). Characteristic MS/MS profiles for PE, PET, and PS were derived from plastic pyrolysis and allowed for a new mass separation of PET. Volume-defined standard particles (125 × 125 × 20 µm3) were developed with the respective weight (PE: 0.48 ± 0.12, PET: 0.50 ± 0.10, PS: 0.31 ± 0.08 µg), which can be spiked into solid samples. Diagenetically altered OM contained compounds that could be incorrectly identified as PE and suggest a mathematical correction to account for OM contribution. With a standard addition method, we quantified PS, PET, and PEcorrected in two agricultural soils. This provides a base to simultaneously quantify a variety of microplastics in many environmental matrices and agricultural soil.

Effect of different antibacterial agents doping in PET-based electrospun nanofibrous membranes on air filtration and antibacterial performance.

In order to reduce the particulate matter pollution to human health in producing environments, series of polyethylene terephthalate/polyvinyl alcohol (PET/PVA) based nanofibrous membranes were fabricated and investigated the dust collection and antibacterial activity. Silver nanoparticles (AgNPs), berberine (Ber) and titanium oxide nanoparticles (TiO2NPs) were selected as antibacterial agents. These novel membranes were well-characterized using SEM, FTIR, TG, etc. techniques. Results of the dust filtration showed that PET/PVA/Ag membrane had the best filtration efficiency of 99.87% for sodium chloride (NaCl) and 99.89% for dioctyl sebacate (DEHS), held low pressure drop of 160.1 Pa for NaCl and 165.3 Pa for DEHS, and posed a high tensile strength of 4.91 MPa. The bacteriostasis studies exhibited that PET/PVA/TiO2 and PET/PVA/Ag membrane showed the highest bacteriological effect on Escherichia coli (98.7%) and Staphylococcus aureus (95.9%), respectively. Meanwhile, in vitro cytotoxicity test indicated no potential cytotoxicity existed in the cell culture process of these two antibacterial membranes. Moreover, the charge distribution in the nanofibers was increased by these antibacterial agents to improve the filtration performance. The dust filtration process synergistically promoted with the antibacterial process in the antibacterial membranes. It was expected that these membranes could be efficient filter medias with broad application prospects in the field of individual protection.

The effect of Dunaliella salina extracts on the adhesion of Pseudomonas aeruginosa to 3D printed polyethylene terephthalate and polylactic acid.

Polyethylene terephthalate (PET) and polylactic acid (PLA) are among the polymers used in the food industry. In this study, crude extracts of Dunaliella salina were used to treat the surface of 3D printed materials studied, aiming to provide them with an anti-adhesive property against Pseudomonas aeruginosa. The hydrophobicity of treated and untreated surfaces was characterized using the contact angle method. Furthermore, the adhesive behavior of P. aeruginosa toward the substrata surfaces was also studied theoretically and experimentally. The results showed that the untreated PLA was hydrophobic, while the untreated PET was hydrophilic. It was also found that the treated materials became hydrophilic and electron-donating. The total energy of adhesion revealed that P. aeruginosa adhesion was theoretically favorable on untreated materials, while it was unfavorable on treated ones. Moreover, the experimental data proved that the adhesion to untreated substrata was obtained, while there was complete inhibition of adhesion to treated surfaces.

Molecular engineering of PETase for efficient PET biodegradation.

The widespread utilization of polyethylene terephthalate (PET) has caused a variety of environmental and health problems. Compared with traditional thermomechanical or chemical PET cycling, the biodegradation of PET may offer a more feasible solution. Though the PETase from Ideonalla sakaiensis (IsPETase) displays interesting PET degrading performance under mild conditions; the relatively low thermal stability of IsPETase limits its practical application. In this study, enzyme-catalysed PET degradation was investigated with the promising IsPETase mutant HotPETase (HP). On this basis, a carbohydrate-binding module from Bacillus anthracis (BaCBM) was fused to the C-terminus of HP to construct the PETase mutant (HLCB) for increased PET degradation. Furthermore, to effectively improve PET accessibility and PET-degrading activity, the truncated outer membrane hybrid protein (FadL) was used to expose PETase and BaCBM on the surface of E. coli (BL21with) to develop regenerable whole-cell biocatalysts (D-HLCB). Results showed that, among the tested small-molecular weight ester compounds (p-nitrophenyl phosphate (pNPP), p-Nitrophenyl acetate (pNPA), 4-Nitrophenyl butyrate (pNPB)), PETase displayed the highest hydrolysing activity against pNPP. HP displayed the highest catalytic activity (1.94 µM(p-NP)/min) at 50 °C and increased longevity at 40 °C. The fused BaCBM could clearly improve the catalytic performance of PETase by increasing the optimal reaction temperature and improving the thermostability. When HLCB was used for PET degradation, the yield of monomeric products (255.7 µM) was ∼25.5 % greater than that obtained after 50 h of HP-catalysed PET degradation. Moreover, the highest yield of monomeric products from the D-HLCB-mediated system reached 1.03 mM. The whole-cell catalyst D-HLCB displayed good reusability and stability and could maintain more than 54.6 % of its initial activity for nine cycles. Finally, molecular docking simulations were utilized to investigate the binding mechanism and the reaction mechanism of HLCB, which may provide theoretical evidence to further increase the PET-degrading activities of PETases through rational design. The proposed strategy and developed variants show potential for achieving complete biodegradation of PET under mild conditions.

Efficient biodegradation of Polyethylene terephthalate (PET) plastic by Gordonia sp. CN2K isolated from plastic contaminated environment.

Since we rely entirely on plastics or their products in our daily lives, plastics are the invention of the hour. Polyester plastics, such as Polyethylene Terephthalate (PET), are among the most often used types of plastics. PET plastics have a high ratio of aromatic components, which makes them very resistant to microbial attack and highly persistent. As a result, massive amounts of plastic trash accumulate in the environment, where they eventually transform into microplastic (<5 mm). Rather than macroplastics, microplastics are starting to pose a serious hazard to the environment. It is imperative that these polymer microplastics be broken down. Through the use of enrichment culture, the PET microplastic-degrading bacterium was isolated from solid waste management yards. Bacterial strain was identified as Gordonia sp. CN2K by 16 S rDNA sequence analysis and biochemical characterization. It is able to use polyethylene terephthalate as its only energy and carbon source. In 45 days, 40.43 % of the PET microplastic was degraded. By using mass spectral analysis and HPLC to characterize the metabolites produced during PET breakdown, the degradation of PET is verified. The metabolites identified in the spent medium included dimer compound, bis (2-hydroxyethyl) terephthalate (BHET), mono (2-hydroxyethyl) terephthalate (MHET), and terephthalate. Furthermore, the PET sheet exposed to the culture showed considerable surface alterations in the scanning electron microscope images. This illustrates how new the current work is.

Discrimination and simultaneous quantification of poly(ethylene terephthalate) and poly(butylene terephthalate) microplastics in environmental samples via gas chromatography-tandem mass spectrometry.

A method has been developed to quantify PET and PBT microplastics (MPs) based on depolymerization and detection of depolymerization products by gas chromatography-tandem mass spectrometry (GC-MS/MS) without a complex separation process from environmental samples. Under the optimal depolymerization conditions, PET and PBT were efficiently converted to ethylene glycol (78%) and 1,4-butanediol (87%), respectively. Subsequently, the linear curves were constructed between signal intensities of depolymerization products and polymer masses by GC-MS/MS, and the correlation coefficients of PET and PBT were 0.996 and 0.997, respectively. The spiking and recovery experiments of PET and PBT in the environmental samples showed that the recovery was stable in the range 89-100%, and the limit of detection was 4.95 µg and 1.39 µg of PET and PBT, respectively. The method has been proven to be capable of simultaneous identification and quantification of PBT and PET MPs in real environmental water samples without complex separation process, which provided a scheme for the determination of microplastics.

Pressure-Driven Piezoelectric Sensors and Energy Harvesting in Biaxially Oriented Polyethylene Terephthalate Film.

This study reports the possibility of using biaxially oriented polyethylene terephthalate (BOPET) plastic packaging to convert mechanical energy into electrical energy. Electricity is generated due to the piezoelectricity of BOPET. Electricity generation depends on the mechanical deformation of the processing aids (inorganic crystals), which were found and identified by SEM and EDAX analyses as SiO2. BOPET, as an electron source, was assembled and tested as an energy conversion and self-powered mechanical stimuli sensor using potential applications in wearable electronics. When a pressure pulse after pendulum impact with a maximum stress of 926 kPa and an impact velocity of 2.1 m/s was applied, a voltage of 60 V was generated with short-circuit current and charge densities of 15 µAcm-2 and 138 nCm-2, respectively. Due to the orientation and stress-induced crystallization of polymer chains, BOPET films acquire very good mechanical properties, which are not lost during their primary usage as packaging materials and are beneficial for the durability of the sensors. The signals detected using BOPET sensors derived from pendulum impact and sieve analyses were also harvested for up to 80 cycles and up to 40 s with short-circuit voltages of 107 V and 95 V, respectively. In addition to their low price, the advantage of sensors made from BOPET plastic packaging waste lies in their chemical resistance and stability under exposure to oxygen, ultraviolet light, and moisture.

Molecular Insights into the Enhanced Activity and/or Thermostability of PET Hydrolase by D186 Mutations.

PETase exhibits a high degradation activity for polyethylene terephthalate (PET) plastic under moderate temperatures. However, the effect of non-active site residues in the second shell of PETase on the catalytic performance remains unclear. Herein, we proposed a crystal structure- and sequence-based strategy to identify the key non-active site residue. D186 in the second shell of PETase was found to be capable of modulating the enzyme activity and stability. The most active PETaseD186N improved both the activity and thermostability with an increase in Tm by 8.89 °C. The PET degradation product concentrations were 1.86 and 3.69 times higher than those obtained with PETaseWT at 30 and 40 °C, respectively. The most stable PETaseD186V showed an increase in Tm of 12.91 °C over PETaseWT. Molecular dynamics (MD) simulations revealed that the D186 mutations could elevate the substrate binding free energy and change substrate binding mode, and/or rigidify the flexible Loop 10, and lock Loop 10 and Helix 6 by hydrogen bonding, leading to the enhanced activity and/or thermostability of PETase variants. This work unraveled the contribution of the key second-shell residue in PETase in influencing the enzyme activity and stability, which would benefit in the rational design of efficient and thermostable PETase.

Lattice Matching and Microstructure of the Aromatic Amide Fatty Acid Salts Nucleating Agent on the Crystallization Behavior of Recycled Polyethylene Terephthalate.

To solve the decrease in the crystallization, mechanical and thermal properties of recycled polyethylene terephthalate (rPET) during mechanical recycling, the aromatic amide fatty acid salt nucleating agents Na-4-ClBeAmBe, Na-4-ClBeAmGl and Na-4-ClAcAmBe were synthesized and the rPET/nucleating agent blend was prepared by melting blending. The molecular structure, the thermal stability, the microstructure and the crystal structure of the nucleating agent were characterized in detail. The differential scanning calorimetry (DSC) result indicated that the addition of the nucleating agent improved the crystallization temperature and accelerated the crystallization rate of the rPET. The nucleation efficiencies (NE) of the Na-4-ClBeAmBe, Na-4-ClBeAmGl and Na-4-ClAcAmBe were increased by 87.2%, 87.3% and 41.7% compared with rPET which indicated that Na-4-ClBeAmBe and Na-4-ClBeAmGl, with their long-strip microstructures, were more conducive to promoting the nucleation of rPET. The equilibrium melting points (Tm0) of rPET/Na-4-ClBeAmBe, rPET/Na-4-ClBeAmGl and rPET/Na-4-ClAcAmBe were increased by 11.7 °C, 18.6 °C and 1.9 °C compared with rPET, which illustrated that the lower mismatch rate between rPET and Na-4-ClBeAmGl (0.8% in b-axis) caused Na-4-ClBeAmGl to be the most capable in inducing the epitaxial crystallization and orient growth along the b-axis direction of the rPET. The small angle X-ray diffraction (SAXS) result proved this conclusion. Meanwhile, the addition of Na-4-ClBeAmGl caused the clearest increase in the rPET of its flexural strength and heat-distortion temperature (HDT) at 20.4% and 46.7%.

Optimizing Antimony Speciation Analysis via Frontal Chromatography-ICP-MS to Explore the Release of PET Additives.

Antimony (Sb) contamination poses significant environmental and health concerns due to its toxic nature and widespread presence, largely from anthropogenic activities. This study addresses the urgent need for an accurate speciation analysis of Sb, particularly in water sources, emphasizing its migration from polyethylene terephthalate (PET) plastic materials. Current methodologies primarily focus on total Sb content, leaving a critical knowledge gap for its speciation. Here, we present a novel analytical approach utilizing frontal chromatography coupled with inductively coupled plasma mass spectrometry (FC-ICP-MS) for the rapid speciation analysis of Sb(III) and Sb(V) in water. Systematic optimization of the FC-ICP-MS method was achieved through multivariate data analysis, resulting in a remarkably short analysis time of 150 s with a limit of detection below 1 ng kg-1. The optimized method was then applied to characterize PET leaching, revealing a marked effect of the plastic aging and manufacturing process not only on the total amount of Sb released but also on the nature of leached Sb species. This evidence demonstrates the effectiveness of the FC-ICP-MS approach in addressing such an environmental concern, benchmarking a new standard for Sb speciation analysis in consideration of its simplicity, cost effectiveness, greenness, and broad applicability in environmental and health monitoring.

Polyethylene terephthalate nanoplastics cause oxidative stress induced cell death in Saccharomyces cerevisiae.

Polyethylene terephthalate (PET) is a common plastic widely used in food and beverage packaging that poses a serious risk to human health and the environment due to the continual rise in its production and usage. After being produced and used, PET accumulates in the environment and breaks down into nanoplastics (NPs), which are then consumed by humans through water and food sources. The threats to human health and the environment posed by PET-NPs are of great concern worldwide, yet little is known about their biological impacts. Herein, the smallest sized PET-NPs so far (56 nm) with an unperturbed PET structure were produced by a modified dilution-precipitation method and their potential cytotoxicity was evaluated in Saccharomyces cerevisiae. Exposure to PET-NPs decreased cell viability due to oxidative stress induction revealed by the increased expression levels of stress response related-genes as well as increased lipid peroxidation. Cell death induced by PET-NP exposure was mainly through apoptosis, while autophagy had a protective role.

Enzymatic Upcycling of PET Waste to Calcium Terephthalate for Battery Anodes.

Biotechnological recycling offers a promising solution to address the environmental concerns associated with waste plastics, particularly polyethylene terephthalate (PET), widely utilized in packaging materials and textiles. To advance the development of a bio-based circular plastic economy, innovative upcycling strategies capable of generating higher-value products are needed. In this study, we enhanced the enzymatic depolymerization of waste PET by incorporating highly concentrated calcium ions (up to 1 m) to the hydrolytic reaction catalyzed by the best currently known enzyme LCCICCG . The presence of calcium ions not only improved the thermal stability and activity of the biocatalyst but also significantly reduced the consumption of base required to maintain optimal pH levels. Employing optimized conditions at 80 °C for 12 h, we successfully converted ≈84 % of the waste PET (200 g L-1 ) into solid hydrated calcium terephthalate (CaTP ⋅ 3H2 O) as the primary product instead of soluble terephthalate salt. CaTP ⋅ 3H2 O was easily purified and employed as a raw material for battery electrode production, exhibiting an initial reversible specific capacity of 164.2 mAh g-1 . Through techno-economic analysis, we conclusively demonstrated that the one-pot biocatalysis-based synthesis of CaTP is a superior PET upcycling strategy than the secondary synthesis method employing recycled terephthalic acid.

Counter-intuitive enhancement of degradation of polyethylene terephthalate through engineering of lowered enzyme binding to solid plastic.

Degradation of solid polyethylene terephthalate (PET) by leaf branch compost cutinase (LCC) produces various PET-derived degradation intermediates (DIs), in addition to terephthalic acid (TPA), which is the recyclable terminal product of all PET degradation. Although DIs can also be converted into TPA, in solution, by LCC, the TPA that is obtained through enzymatic degradation of PET, in practice, is always contaminated by DIs. Here, we demonstrate that the primary reason for non-degradation of DIs into TPA in solution is the efficient binding of LCC onto the surface of solid PET. Although such binding enhances the degradation of solid PET, it depletes the surrounding solution of enzyme that could otherwise have converted DIs into TPA. To retain a subpopulation of enzyme in solution that would mainly degrade DIs, we introduced mutations to reduce the hydrophobicity of areas surrounding LCC's active site, with the express intention of reducing LCC's binding to solid PET. Despite the consequent reduction in invasion and degradation of solid PET, overall levels of production of TPA were ~3.6-fold higher, due to the partitioning of enzyme between solid PET and the surrounding solution, and the consequent heightened production of TPA from DIs. Further, synergy between such mutated LCC (F125L/F243I LCC) and wild-type LCC resulted in even higher yields, and TPA of nearly ~100% purity.

Computation-Based Design of Salt Bridges in PETase for Enhanced Thermostability and Performance for PET Degradation.

Polyethylene terephthalate (PET) is one of the most widely used plastics, and the accumulation of PET poses a great threat to the environment. IsPETase can degrade PET rapidly at moderate temperatures, but its application is greatly limited by the low stability. Herein, molecular dynamics (MD) simulations combined with a sequence alignment strategy were adopted to introduce salt bridges into the flexible region of IsPETase to improve its thermal stability. In the designed variants, the Tm values of IsPETaseI168R/S188D and IsPETaseI168R/S188E were 7.4 and 8.7 °C higher than that of the wild type, respectively. The release of products degraded by IsPETaseI168R/S188E was 4.3 times that of the wild type. Tertiary structure characterization demonstrated that the structure of the variants IsPETaseI168R/S188D and IsPETaseI168R/S188E became more compact. Extensive MD simulations verified that a stable salt bridge was formed between the residue R168 and D186 in IsPETaseI168R/S188D , while in IsPETaseI168R/S188E an R168-D186-E188 salt bridge network was observed. These results confirmed that the proposed computation-based salt bridge design strategy could efficiently generate variants with enhanced thermal stability for the long-term degradation of PET, which would be helpful for the design of enzymes with improved stability.