Exploring the substrate spectrum of phylogenetically distinct bacterial polyesterases.

The rapid escalation of plastic waste accumulation presents a significant threat of the modern world, demanding an immediate solution. Over the last years, utilization of the enzymatic machinery of various microorganisms has emerged as an environmentally friendly asset in tackling this pressing global challenge. Thus, various hydrolases have been demonstrated to effectively degrade polyesters. Plastic waste streams often consist of a variety of different polyesters, as impurities, mainly due to wrong disposal practices, rendering recycling process challenging. The elucidation of the selective degradation of polyesters by hydrolases could offer a proper solution to this problem, enhancing the recyclability performance. Towards this, our study focused on the investigation of four bacterial polyesterases, including DaPUase, IsPETase, PfPHOase, and Se1JFR, a novel PETase-like lipase. The enzymes, which were biochemically characterized and structurally analyzed, demonstrated degradation ability of synthetic plastics. While a consistent pattern of polyesters' degradation was observed across all enzymes, Se1JFR stood out in the degradation of PBS, PLA, and polyether PU. Additionally, it exhibited comparable results to IsPETase, a benchmark mesophilic PETase, in the degradation of PCL and semi-crystalline PET. Our results point out the wide substrate spectrum of bacterial hydrolases and underscore the significant potential of PETase-like enzymes in polyesters degradation.

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.

Computational redesign of a hydrolase for nearly complete PET depolymerization at industrially relevant high-solids loading.

Biotechnological plastic recycling has emerged as a suitable option for addressing the pollution crisis. A major breakthrough in the biodegradation of poly(ethylene terephthalate) (PET) is achieved by using a LCC variant, which permits 90% conversion at an industrial level. Despite the achievements, its applications have been hampered by the remaining 10% of nonbiodegradable PET. Herein, we address current challenges by employing a computational strategy to engineer a hydrolase from the bacterium HR29. The redesigned variant, TurboPETase, outperforms other well-known PET hydrolases. Nearly complete depolymerization is accomplished in 8 h at a solids loading of 200 g kg-1. Kinetic and structural analysis suggest that the improved performance may be attributed to a more flexible PET-binding groove that facilitates the targeting of more specific attack sites. Collectively, our results constitute a significant advance in understanding and engineering of industrially applicable polyester hydrolases, and provide guidance for further efforts on other polymer types.

Analysis of Poly(ethylene terephthalate) degradation kinetics of evolved IsPETase variants using a surface crowding model.

Poly(ethylene terephthalate) (PET) is a major plastic polymer utilized in the single-use and textile industries. The discovery of PET-degrading enzymes (PETases) has led to an increased interest in the biological recycling of PET in addition to mechanical recycling. IsPETase from Ideonella sakaiensis is a candidate catalyst, but little is understood about its structure-function relationships with regards to PET degradation. To understand the effects of mutations on IsPETase productivity, we develop a directed evolution assay to identify mutations beneficial to PET film degradation at 30 °C. IsPETase also displays enzyme concentration-dependent inhibition effects, and surface crowding has been proposed as a causal phenomenon. Based on total internal reflectance fluorescence microscopy and adsorption experiments, IsPETase is likely experiencing crowded conditions on PET films. Molecular dynamics simulations of IsPETase variants reveal a decrease in active site flexibility in free enzymes and reduced probability of productive active site formation in substrate-bound enzymes under crowding. Hence, we develop a surface crowding model to analyze the biochemical effects of three hit mutations (T116P, S238N, S290P) that enhanced ambient temperature activity and/or thermostability. We find that T116P decreases susceptibility to crowding, resulting in higher PET degradation product accumulation despite no change in intrinsic catalytic rate. In conclusion, we show that a macromolecular crowding-based biochemical model can be used to analyze the effects of mutations on properties of PETases and that crowding behavior is a major property to be targeted for enzyme engineering for improved PET degradation.

New combined absorption/1H NMR method for qualitative and quantitative analysis of PET degradation products.

Poly(ethylene terephthalate) (PET) is a very valuable and beneficial material for industrial purposes, with various different applications. Due to the high annual production volume of over 50 million tons worldwide and the indiscriminate disposal by consumers, the polymers accumulate in the environment, causing negative effects on various ecosystems. Biodegradation via suitable enzymes represents a promising approach to combat the plastic waste issue so validated methods are required to measure the efficiency and efficacy of these enzymes. PETase and MHETase from Ideonella sakaiensis are suitable enzymes needed in combination to completely degrade PET into its environmentally friendly monomers. In this project, we compare and combine a previously described bulk absorbance measurement method with a newly established 1H NMR analysis method of the PET degradation products mono(2-hydroxyethyl) terephthalic acid, bis(2-hydroxyethyl) terephthalic acid and terephthalic acid. Both were optimized regarding different solvents, pH values and drying processes. The accuracy of the measurements can be confirmed with sensitivity limits of 2.5-5 µM for the absorption method and 5-10 µM for the 1H NMR analysis. The combination of the described methods therefore allows a quantitative analysis by using bulk absorption coupled with a qualitative analysis through 1H NMR. The methods established in our work can potentially contribute to the development of suitable recycling strategies of PET using recombinant enzymes.

Complete degradation of PET waste using a thermophilic microbe-enzyme system.

Enzymatic degradation has been proposed as a suitable solution for addressing PET pollution, but approximately 10 % of PET is left as nonbiodegradable. Microbes can completely degrade PET at the gram level per year. Based on the complementary benefits of microbes and enzymes, a microbe-enzyme system was created to completely degrade PET. Here, a thermophilic microbe-enzyme (TME) system composed of Bacillus thermoamylovorans JQ3 and leaf-branch compost cutinase variant (ICCG) was used to demonstrate the synergistic degradation of PET, enabling 100 % degradation of PET waste at a high PET loading level (360 g/L). Six endogenous PET hydrolases of strain JQ3 were discovered by employing an ester bond hydrolysis function-first genome mining (EGM) strategy and first successfully expressed in E. coli. These hydrolases could release TPA as the final product from PET and preferentially degraded BHET instead of MHET. Of these, carboxylesterase 39_5 and ICCG could degrade PET in a synergistic manner to generate 50 µM of TPA, which was greater than the sum of the individual treatments. Finally, the degradation pathway of the TME system was speculated to include biofilm formation, PET degradation and utilization. The successful implementation of this study rendered a scale-up degradation feasible of PET at a lower cost.

Applications of waste polyethylene terephthalate (PET) based nanostructured materials: A review.

While polyethylene terephthalate (PET) has enjoyed widespread use, a large volume of plastic waste has also been produced as a result, which is detrimental to the environment. Traditional treatment of plastic waste, such as landfilling and incinerating waste, causes environmental pollution and poses risks to public health. Recycling PET waste into useful chemicals or upcycling the waste into high value-added materials can be remedies. This review first provides a brief introduction of the synthesis, structure, properties, and applications of virgin PET. Then the conversion process of waste PET into high value-added materials for different applications are introduced. The conversion mechanisms (including degradation, recycling and upcycling) are detailed. The advanced applications of these upgraded materials in energy storage devices (supercapacitors, lithium-ion batteries, and microbial fuel cells), and for water treatment (to remove dyes, heavy metals, and antibiotics), environmental remediation (for air filtration, CO2 adsorption, and oil removal) and catalysis (to produce H2, photoreduce CO2, and remove toxic chemicals) are discussed at length. In general, this review details the exploration of advanced technologies for the transformation of waste PET into nanostructured materials for various applications, and provides insights into the role of high value-added waste products in sustainability and economic development.

Enzymatic degradation of poly(ethylene terephthalate) (PET): Identifying the cause of the hypersensitive enzyme kinetic response to increased PET crystallinity.

Plastic pollution poses a significant environmental challenge, with poly(ethylene terephthalate) (PET) being a major contributor due to its extensive use in single use applications such as plastic bottles and other packaging material. Enzymatic degradation of PET offers a promising solution for PET recycling, but the enzyme kinetics in relation to the degree of crystallinity (XC) of the PET substrate are poorly understood. In this study, we investigated the hypersensitive enzyme kinetic response on PET at XC from ∼8.5-12% at 50 °C using the benchmark PET hydrolysing enzyme LCCICCG. We observed a substantial reduction in the maximal enzymatic reaction rate (invVmax) with increasing XC, corresponding to a 3-fold reduction in invVmax when the XC of PET increased from 8.6% to 12.2%. The kinetic analysis revealed that the level of the Mobile Amorphous Fraction (XMAF) was a better descriptor for the enzymatic degradation rate response than XC (or (100%-XC)). By continuous monitoring of the enzymatic reaction progress, we quantified the lag phase prolongation in addition to the steady-state kinetic rates (vss) of the reactions and found that the duration of the lag phase of a reaction could be predicted from the vss and XC by multiple linear regression modeling. The linear correlation between the duration of the lag phase and the vss of the enzymatic PET degradation affirmed that the LCCICCG worked via a random/endo-type enzymatic attack pattern. The longer lag phase at increased XC of PET is proposed to be due to increased substrate entanglement density as well as unproductive enzyme binding to the crystalline regions of PET. The findings enhance our understanding of PET enzymatic degradation kinetics and its dependence on substrate composition, i.e., XMAF and XC.

Seek and you shall find-news on the quest for novel PET-degrading enzymes.

Plastic-degrading enzymes hold immense potential for eco-friendly recycling methods. However, the catalytic rates of current enzymes do not stack up against the mammoth task of degrading millions of tons of plastic waste per year. In the quest for more efficient polyethylene terephthalate (PET)-degrading enzymes, Zhang et al. report the discovery and characterization of PET40, a versatile PET-hydrolyzing esterase that is divergent from most characterized PETases. While PET40 has comparably low hydrolytic activity on PET, Zhang et al. demonstrate its broad activity on an expanded substrate pool. This sheds light on the potential ecological role of these esterases and suggests that PET might be only a recent addition to their substrate spectrum.

Chemical synthesis of a 28 kDa full-length PET degrading enzyme ICCG by the removable backbone modification strategy.

Chemical protein synthesis offers a powerful way to access otherwise-difficult-to-obtain proteins such as mirror-image proteins. Although a large number of proteins have been chemically synthesized to date, the acquisition to proteins containing hydrophobic peptide fragments has proven challenging. Here, we describe an approach that combines the removable backbone modification strategy and the peptide hydrazide-based native chemical ligation for the chemical synthesis of a 28 kDa full-length PET degrading enzyme IGGC (a higher depolymerization efficiency of variant leaf-branch compost cutinase (LCC)) containing hydrophobic peptide segments. The synthetic ICCG exhibits the enzymatic activity and will be useful in establishing the corresponding mirror-image version of ICCG.

Integrating experimental and computational approaches for deep eutectic solvent-catalyzed glycolysis of post-consumer polyethylene terephthalate.

To achieve a sustainable and circular economy, developing effective plastic recycling methods is essential. Despite advances in the chemical recycling of plastic waste, modern industries require highly efficient and sustainable solutions to address environmental problems. In this study, we propose an efficient glycolysis strategy for post-consumer polyethylene terephthalate (PET) using deep eutectic solvents (DESs) to produce bis(2-hydroxyethyl) terephthalate (BHET) with high selectivity. Choline chloride (ChCl)- and urea-based DESs were synthesized using various metal salts and were tested for the glycolysis of PET waste; ChCl-Zn(OAc)2 exhibited the best performance. The DES-containing solvent system afforded a complete PET conversion, producing BHET at a high yield (91.6%) under optimal reaction conditions. The degradation mechanism of PET and its interaction with DESs were systematically investigated using density functional theory-based calculations. Furthermore, an intuitive machine learning model was developed to predict the PET conversion and BHET selectivity for different DES compositions. Our findings demonstrate that the DES-catalyzed glycolysis of post-consumer PET could enable the development of a sustainable chemical recycling process, providing insights to identify the new design of DESs for plastic decomposition.

Influence of UV light, ultrasound, and heat treatment on the migration of bisphenol A from polyethylene terephthalate bottle into the food simulant.

This research examined the impacts of ultrasound, UV light, storage time, and temperature on the leaching of bisphenol A (BPA) from polyethylene terephthalate (PET) drinking water bottles in Turkey. The initial phase of the investigation encompassed the quantification of BPA in two distinct brands of bottled water. Samples were extracted by solid- phase extraction (SPE) and analyzed by high performance liquid chromatography with fluorescence detection (HPLC-FLD). According to the results in the first part, the highest BPA levels were found in bottled water. In the second part of the study, 10 to 30 min of ultrasound treatment increased the BPA migration with increased time in simulants. In the first and second weeks of storage at 25 °C, the effect of storage on BPA migration was below the detection limit (

Biodegradation of Poly(ethylene terephthalate) by Bacillus safensis YX8.

Due to the extensive utilization of poly (ethylene terephthalate) (PET), a significant amount of PET waste has been discharged into the environment, endangering both human health and the ecology. As an eco-friendly approach to PET waste treatment, biodegradation is dependent on efficient strains and enzymes. In this study, a screening method was first established using polycaprolactone (PCL) and PET nanoparticles as substrates. A PET-degrading strain YX8 was isolated from the surface of PET waste. Based on the phylogenetic analysis of 16S rRNA and gyrA genes, this strain was identified as Bacillus safensis. Strain YX8 demonstrated the capability to degrade PET nanoparticles, resulting in the production of terephthalic acid (TPA), mono (2-hydroxyethyl) terephthalic acid (MHET), and bis (2-hydroxyethyl) terephthalic acid (BHET). Erosion spots on the PET film were observed after incubation with strain YX8. Furthermore, the extracellular enzymes produced by strain YX8 exhibited the ability to form a clear zone on the PCL plate and to hydrolyze PET nanoparticles to generate TPA, MHET, and BHET. This work developed a method for the isolation of PET-degrading microorganisms and provides new strain resources for PET degradation and for the mining of functional enzymes.

Computational design of highly efficient thermostable MHET hydrolases and dual enzyme system for PET recycling.

Recently developed enzymes for the depolymerization of polyethylene terephthalate (PET) such as FAST-PETase and LCC-ICCG are inhibited by the intermediate PET product mono(2-hydroxyethyl) terephthalate (MHET). Consequently, the conversion of PET enzymatically into its constituent monomers terephthalic acid (TPA) and ethylene glycol (EG) is inefficient. In this study, a protein scaffold (1TQH) corresponding to a thermophilic carboxylesterase (Est30) was selected from the structural database and redesigned in silico. Among designs, a double variant KL-MHETase (I171K/G130L) with a similar protein melting temperature (67.58 °C) to that of the PET hydrolase FAST-PETase (67.80 °C) exhibited a 67-fold higher activity for MHET hydrolysis than FAST-PETase. A fused dual enzyme system comprising KL-MHETase and FAST-PETase exhibited a 2.6-fold faster PET depolymerization rate than FAST-PETase alone. Synergy increased the yield of TPA by 1.64 fold, and its purity in the released aromatic products reached 99.5%. In large reaction systems with 100 g/L substrate concentrations, the dual enzyme system KL36F achieved over 90% PET depolymerization into monomers, demonstrating its potential applicability in the industrial recycling of PET plastics. Therefore, a dual enzyme system can greatly reduce the reaction and separation cost for sustainable enzymatic PET recycling.

Characterization of a novel esterase and construction of a Rhodococcus-Burkholderia consortium capable of catabolism bis (2-hydroxyethyl) terephthalate.

Bis (2-hydroxyethyl) terephthalate (BHET) is one of the main compounds produced by enzymatic hydrolysis or chemical depolymerization of polyethylene terephthalate (PET). However, the lack of understanding on BHET microbial metabolism is a main factor limiting the bio-upcycling of PET. In this study, BHET-degrading strains of Rhodococcus biphenylivorans GA1 and Burkholderia sp. EG1 were isolated and identified, which can grow with BHET as the sole carbon source. Furthermore, a novel esterase gene betH was cloned from strain GA1, which encodes a BHET hydrolyzing esterase with the highest activity at 30 °C and pH 7.0. In addition, the co-culture containing strain GA1 and strain EG1 could completely degrade high concentration of BHET, eliminating the inhibition on strain GA1 caused by the accumulation of intermediate metabolite ethylene glycol (EG). This work will provide potential strains and a feasible strategy for PET bio-upcycling.

Determination of key factors affecting biofilm formation on the aged Poly(ethylene terephthalate) during anaerobic digestion.

Biofilm formation on plastic surface is a growing concern because it can alter the plastic surface properties and exacerbate the ecological risk. Identifying key factors that affecting biofilm formation is critical for effective pollution control. In this study, the poly (ethylene terephthalate) (PET) was aged in water and air conditions with UV irradiation, then incubated in the digestate of food waste anaerobic digestion to allow biofilm formation. Surface analysis techniques, including scanning electron microscopy (SEM), atomic force microscopy (AFM), and Fourier-transform infrared spectroscopy with attenuated total reflection (FTIR-ATR), were utilized to investigated the changes in the topography, roughness, hydrophily, and functional groups change of the PET surface during the aging process. Confocal laser scanning microscopy (CLSM) was used to determine the distribution of microorganisms on the PET surface after incubation in the digestate. This study focused on understanding the interactions between the PET surface and biofilm to identify critical surface factors that affect biofilm formation. Results showed that the four months aging process decreased the contact angle of the PET surface from 96.92° to 76.08° and 68.97° in water and air conditions, respectively, corresponding to an increase of 44% and 70% in the surface energy. Additionally, aging in air conditions led to a rougher surface compared to water conditions. The arithmetic roughness average (Ra) of the PET-Water was 11.0 nm, comparable to that of the pristine PET, while the value of PET-Air was much higher (43.9 nm). The results further indicated that biofilm formation during anaerobic digestion was more sensitive to roughness than hydrophily. The PET surface aged in air conditions provided a more suitable environment for microbial reproduction, leading to the aggradation of living cells.

Upcycling of PET oligomers from chemical recycling processes to PHA by microbial co-cultivation.

Polyethylene terephthalate (PET) is the most widely consumed polyester plastic and can be recycled by many chemical processes, of which glycolysis is most cost-effective and commercially viable. However, PET glycolysis produces oligomers due to incomplete depolymerization, which are undesirable by-products and require proper disposal. In this study, the PET oligomers from chemical recycling processes were completely bio-depolymerized into monomers and then used for the biosynthesis of biodegradable plastics polyhydroxyalkanoates (PHA) by co-cultivation of two engineered microorganisms Escherichia coli BL21 (DE3)-LCCICCG and Pseudomonas putida KT2440-ΔRDt-ΔZP46C-M. E. coli BL21 (DE3)-LCCICCG was used to secrete the PET hydrolase LCCICCG into the medium to directly depolymerize PET oligomers. P. putida KT2440-ΔRDt-ΔZP46C-M that mastered the metabolism of aromatic compounds was engineered to accelerate the hydrolysis of intermediate products mono-2-(hydroxyethyl) terephthalate (MHET) by expressing IsMHETase, and biosynthesize PHA using ultimate products terephthalate and ethylene glycol depolymerized from the PET oligomers. The population ratios of the two microorganisms during the co-cultivation were characterized by fluorescent reporter system, and revealed the collaboration of the two microorganisms to bio-depolymerize and bioconversion of PET oligomers in a single process. This study provides a biological strategy for the upcycling of PET oligomers and promotes the plastic circular economy.

Engineering the catalytic activity of an Antarctic PET-degrading enzyme by loop exchange.

Several hydrolases have been described to degrade polyethylene terephthalate (PET) at moderate temperatures ranging from 25°C to 40°C. These mesophilic PET hydrolases (PETases) are less efficient in degrading this plastic polymer than their thermophilic homologs and have, therefore, been the subject of many protein engineering campaigns. However, enhancing their enzymatic activity through rational design or directed evolution poses a formidable challenge due to the need for exploring a large number of mutations. Additionally, evaluating the improvements in both activity and stability requires screening numerous variants, either individually or using high-throughput screening methods. Here, we utilize instead the design of chimeras as a protein engineering strategy to increase the activity and stability of Mors1, an Antarctic PETase active at 25°C. First, we obtained the crystal structure of Mors1 at 1.6 Å resolution, which we used as a scaffold for structure- and sequence-based chimeric design. Then, we designed a Mors1 chimera via loop exchange of a highly divergent active site loop from the thermophilic leaf-branch compost cutinase (LCC) into the equivalent region in Mors1. After restitution of an active site disulfide bond into this chimera, the enzyme exhibited a shift in optimal temperature for activity to 45°C and an increase in fivefold in PET hydrolysis when compared with wild-type Mors1 at 25°C. Our results serve as a proof of concept of the utility of chimeric design to further improve the activity and stability of PETases active at moderate temperatures.

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.

Enzyme selection, optimization, and production toward biodegradation of post-consumer poly(ethylene terephthalate) at scale.

Poly(ethylene terephthalate) (PET) is one of the world's most widely used polyester plastics. Due to its chemical stability, PET is extremely difficult to hydrolyze in a natural environment. Recent discoveries in new polyester hydrolases and breakthroughs in enzyme engineering strategies have inspired enormous research on biorecycling of PET. This study summarizes our research efforts toward large-scale, efficient, and economical biodegradation of post-consumer waste PET, including PET hydrolase selection and optimization, high-yield enzyme production, and high-capacity enzymatic degradation of post-consumer waste PET. First, genes encoding PETase and MHETase from Ideonella sakaiensis and the ICCG variant of leaf-branch compost cutinase (LCCICCG ) were codon-optimized and expressed in Escherichia coli BL21(DE3) for high-yield production. To further lower the enzyme production cost, a pelB leader sequence was fused to LCCICCG so that the enzyme can be secreted into the medium to facilitate recovery. To help bind the enzyme on the hydrophobic surface of PET, a substrate-binding module in a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (PBM) was fused to the C-terminus of LCCICCG . The resulting four different LCCICCG variants (LCC, PelB-LCC, LCC-PBM, and PelB-LCC-PBM), together with PETase and MHETase, were compared for PET degradation efficiency. A fed-batch fermentation process was developed to produce the target enzymes up to 1.2 g L-1 . Finally, the best enzyme, PelB-LCC, was selected and used for the efficient degradation of 200 g L-1 recycled PET in a well-controlled, stirred-tank reactor. The results will help develop an economical and scalable biorecycling process toward a circular PET economy.