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Less than 9% of the plastic produced is recycled after use, contributing to the global plastic pollution problem. While polyethylene terephthalate (PET) is one of the most common plastics, its thermomechanical recycling generates a material of lesser quality. Enzymes are highly selective, renewable catalysts active at mild temperatures; however, they lack activity toward the more crystalline forms of PET commonly found in consumer plastics, requiring the energy-expensive melt-amorphization step of PET before enzymatic depolymerization. We report here that, when used in moist-solid reaction mixtures instead of the typical dilute aqueous solutions or slurries, the cutinase from Humicola insolens can directly depolymerize amorphous and crystalline regions of PET equally, without any pretreatment, with a 13-fold higher space-time yield and a 15-fold higher enzyme efficiency than reported in prior studies with high-crystallinity material. Further, this process shows a 26-fold selectivity for terephthalic acid over other hydrolysis products.

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 (Tg) 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.

Polyethylene terephthalate (PET) is the most widely used polyester plastic, with applications in the textile and packaging industry. Currently, re-moulding is the main path for PET recycling, but this eventually leads to an unsustainable loss of quality; thus, other means of recycling are required. Enzymatic hydrolysis offers the possibility of monomer formation under mild conditions and opens up alternative and infinite recycling paths. Here, IsPETase, derived from the bacterium Ideonella sakaiensis, is considered to be the most active enzyme for PET degradation under mild conditions, and although several studies have demonstrated improvements to both the stability and activity of this enzyme, stability at even moderate temperatures is still an issue. In the present study, we have used sequence and structure-based bioinformatic tools to identify mutations to increase the thermal stability of the enzyme so as to increase PET degradation activity during extended hydrolysis reactions. We found that amino acid substitution S136E showed significant increases to activity and stability. S136E is a previously unreported variant that led to a 3.3-fold increase in activity relative to wild type.

Polyethylene terephthalate (PET) degradation by enzymatic hydrolysis is significant for addressing plastic pollution and fostering sustainable waste management practices. Identifying thermophilic and thermostable PET hydrolases is particularly crucial for industrial bioprocesses, where elevated temperatures may enhance enzymatic efficiency and process kinetics. In this study, we present the discovery of a novel thermophilic and thermostable PETase enzyme named Sis, obtained through metagenomic sequence-based analysis. Sis exhibits robust activity on nanoPET substrates, demonstrating effectiveness at temperatures up to 70 °C and displaying exceptional thermal stability with a melting temperature (Tm) of 82 °C. Phylogenetically distinct from previously characterised PET hydrolases, Sis represents a valuable addition to the repertoire of enzymes suitable for PET degradation.

Enzymatic hydrolysis of polyethylene terephthalate (PET) has been the subject of extensive previous research that can be grouped into two categories, viz. enzymatic surface modification of polyester fibers and management of PET waste by enzymatic hydrolysis. Different enzymes with rather specific properties are required for these two processes. Enzymatic surface modification is possible with several hydrolases, such as lipases, carboxylesterases, cutinases, and proteases. These enzymes should be designated as PET surface–modifying enzymes and should not degrade the building blocks of PET but should hydrolyze the surface polymer chain so that the intensity of PET is not weakened. Conversely, management of PET waste requires substantial degradation of the building blocks of PET; therefore, only a limited number of cutinases have been recognized as PET hydrolases since the first PET hydrolase was discovered by Müller et al. ( Macromol Rapid Commun 26:1400–1405, 2005 ). Here, we introduce current knowledge on enzymatic degradation of PET with a focus on the key class of enzymes, PET hydrolases, pertaining to the definition of enzymatic requirements for PET hydrolysis, structural analyses of PET hydrolases, and the reaction mechanisms. This review gives a deep insight into the structural basis and dynamics of PET hydrolases based on the recent progress in X-ray crystallography. Based on the knowledge accumulated to date, we discuss the potential for PET hydrolysis applications, such as in designing waste stream management.

Polyethylene terephthalate (PET) is a major component of plastic waste. Enzymatic PET hydrolysis is the most ecofriendly recycling technology. The biorecycling of PET waste requires the complete depolymerization of PET to terephthalate and ethylene glycol. The history of enzymatic PET depolymerization has revealed two critical issues for the industrial depolymerization of PET: industrially available PET hydrolases and pretreatment of PET waste to make it susceptible to full enzymatic hydrolysis. As none of the wild-type enzymes can satisfy the requirements for industrialization, various mutational improvements have been performed, through classical technology to state-of-the-art computational/machine-learning technology. Recent engineering studies on PET hydrolases have brought a new insight that flexibility of the substrate-binding groove may improve the efficiency of PET hydrolysis while maintaining sufficient thermostability, although the previous studies focused only on enzymatic thermostability above the glass transition temperature of PET. Industrial biorecycling of PET waste is scheduled to be implemented, using micronized amorphous PET. Next stage must be the development of PET hydrolases that can efficiently degrade crystalline parts of PET and expansion of target PET materials, not only bottles but also textiles, packages, and microplastics. This review discusses the current status of PET hydrolases, their potential applications, and their profespectal goals. Key points • PET hydrolases must be thermophilic, but their operation must be below 70 °C • Classical and state-of-the-art engineering approaches are useful for PET hydrolases • Enzyme activity on crystalline PET is most expected for future PET biorecycling Graphical Abstract

High crystallinity leads to low hydrophilicity of fabric made of (poly(ethylene terephthalate)) fibers (PET) causing problems in finishing, washing, and dyeing processes. To improve these properties, the surface of PET fibers is usually modified by hydrolysis. Alkaline hydrolysis is a conventional process usually performed at a temperature higher than 100 °C for more than 1 h. However, the use of strong alkali and high processing temperatures (>100 °C) can lead to fabric damage and a negative impact on the environment. Therefore, in this paper, the possibility of hydrolysis of the PET fibers in the fabric in a sustainable, energy-efficient process was researched. The influence of low temperature (60–100 °C) and an accelerator (a cationic surfactant HDTMAC) to PET alkaline hydrolysis was studied through weight loss, the loss in breaking force, and fiber morphology. The kinetics of PET dissolution in 1.5 mol cm−3 NaOH at low temperature with and without the addition of HDTMAC was determined and the activation energy was calculated according to the theoretical model. It has been confirmed that PET hydrolysis can be carried out in 1.5 mol cm−3 NaOH with the addition of HDTMAC as an accelerator at 80 °C for 10 min. This process is more economically and energetically acceptable than the conventional process, and is therefore more sustainable.

3′-deoxy-3′-[[sup.18]F]fluorothymidine ([[sup.18]F]FLT) is a positron emission tomography (PET) tracer useful for tumor proliferation assessment for a number of cancers, particularly in the cases of brain, lung, and breast tumors. At present [[sup.18]F], FLT is commonly prepared by means of the nucleophilic radiofluorination of 3-N-Boc-5′-O-DMT-3′-O-nosyl thymidine precursor in the presence of a phase-transfer catalyst, followed by an acidic hydrolysis. To achieve high radiochemical yield, relatively large amounts of precursor (20–40 mg) are commonly used, leading to difficulties during purification steps, especially if a solid-phase extraction (SPE) approach is attempted. The present study describes an efficient method for [[sup.18]F]FLT synthesis, employing tetrabutyl ammonium tosylate as a non-basic phase-transfer catalyst, with a greatly reduced amount of precursor employed. With a reduction of the precursor amount contributing to lower amounts of synthesis by-products in the reaction mixture, an SPE purification procedure using only two commercially available cartridges—OASIS HLB 6cc and Sep-Pak Alumina N Plus Light—has been developed for use on the GE TRACERlab FX N Pro synthesis module. [[sup.18]F]FLT was obtained in radiochemical yield of 16 ± 2% (decay-corrected) and radiochemical purity >99% with synthesis time not exceeding 55 min. The product was formulated in 16 mL of normal saline with 5% ethanol (v/v). The amounts of chemical impurities and residual solvents were within the limits established by European Pharmacopoeia. The procedure described compares favorably with previously reported methods due to simplified automation, cheaper and more accessible consumables, and a significant reduction in the consumption of an expensive precursor.

Background For decades, plastic has been a valuable global product due to its convenience and low price. For example, polyethylene terephthalate (PET) was one of the most popular materials for disposable bottles due to its beneficial properties, namely impact resistance, high clarity, and light weight. Increasing demand of plastic resulted in indiscriminate disposal by consumers, causing severe accumulation of plastic wastes. Because of this, scientists have made great efforts to find a way to biologically treat plastic wastes. As a result, a novel plastic degradation enzyme, PETase, which can hydrolyze PET, was discovered in Ideonella sakaiensis 201-F6 in 2016. Results A green algae, Chlamydomonas reinhardtii , which produces PETase, was developed for this study. Two representative strains ( C. reinhardtii CC-124 and CC-503) were examined, and we found that CC-124 could express PETase well. To verify the catalytic activity of PETase produced by C. reinhardtii , cell lysate of the transformant and PET samples were co-incubated at 30 °C for up to 4 weeks. After incubation, terephthalic acid (TPA), i.e. the fully-degraded form of PET, was detected by high performance liquid chromatography analysis. Additionally, morphological changes, such as holes and dents on the surface of PET film, were observed using scanning electron microscopy. Conclusions A PET hydrolyzing enzyme, PETase, was successfully expressed in C. reinhardtii , and its catalytic activity was demonstrated. To the best of our knowledge, this is the first case of PETase expression in green algae.