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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.

Present estimates suggest that of the 359 million tons of plastics produced annually worldwide 1 , 150–200 million tons accumulate in landfill or in the natural environment 2 . Poly(ethylene terephthalate) (PET) is the most abundant polyester plastic, with almost 70 million tons manufactured annually worldwide for use in textiles and packaging 3 . The main recycling process for PET, via thermomechanical means, results in a loss of mechanical properties 4 . Consequently, de novo synthesis is preferred and PET waste continues to accumulate. With a high ratio of aromatic terephthalate units—which reduce chain mobility—PET is a polyester that is extremely difficult to hydrolyse 5 . Several PET hydrolase enzymes have been reported, but show limited productivity 6 , 7 . Here we describe an improved PET hydrolase that ultimately achieves, over 10 hours, a minimum of 90 per cent PET depolymerization into monomers, with a productivity of 16.7 grams of terephthalate per litre per hour (200 grams per kilogram of PET suspension, with an enzyme concentration of 3 milligrams per gram of PET). This highly efficient, optimized enzyme outperforms all PET hydrolases reported so far, including an enzyme 8 , 9 from the bacterium Ideonella sakaiensis strain 201-F6 (even assisted by a secondary enzyme 10 ) and related improved variants 11 – 14 that have attracted recent interest. We also show that biologically recycled PET exhibiting the same properties as petrochemical PET can be produced from enzymatically depolymerized PET waste, before being processed into bottles, thereby contributing towards the concept of a circular PET economy. Computer-aided engineering produces improvements to an enzyme that breaks down poly(ethylene terephthalate) (PET) into its constituent monomers, which are used to synthesize PET of near-petrochemical grade that can be further processed into bottles.

Bio-based plastics are increasingly appearing in a range of consumption products, and after use they often end up in technical recycling chains. Bio-based plastics are different from fossil-based ones and could disturb the current recycling of plastics and hence inhibit the closure of plastic cycles, which is undesirable given the current focus on a transition towards a circular economy. In this paper, this risk has been assessed via three elaborated case studies using data and information retrieved through an extended literature search. No overall risks were revealed for bio-based plastics as a group; rather, every bio-based plastic is to be considered as a potential separate source of contamination in current recycling practices. For PLA (polylactic acid), a severe incompatibility with PET (polyethylene terephthalate) recycling is known; hence, future risks are assessed by measuring amounts of PLA ending up in PET waste streams. For PHA (polyhydroxy alkanoate) there is no risk currently, but it will be crucial to monitor future application development. For PEF (polyethylene furanoate), a particular approach for contamination-related issues has been included in the upcoming market introduction. With respect to developing policy, it is important that any introduction of novel plastics is well guided from a system perspective and with a particular eye on incompatibilities with current and upcoming practices in the recycling of plastics.

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.

The extreme durability of polyethylene terephthalate (PET) debris has rendered it a long-term environmental burden. At the same time, current recycling efforts still lack sustainability. Two recently discovered bacterial enzymes that specifically degrade PET represent a promising solution. First, Ideonella sakaiensis PETase, a structurally well-characterized consensus α/β-hydrolase fold enzyme, converts PET to mono-(2-hydroxyethyl) terephthalate (MHET). MHETase, the second key enzyme, hydrolyzes MHET to the PET educts terephthalate and ethylene glycol. Here, we report the crystal structures of active ligand-free MHETase and MHETase bound to a nonhydrolyzable MHET analog. MHETase, which is reminiscent of feruloyl esterases, possesses a classic α/β-hydrolase domain and a lid domain conferring substrate specificity. In the light of structure-based mapping of the active site, activity assays, mutagenesis studies and a first structure-guided alteration of substrate specificity towards bis-(2-hydroxyethyl) terephthalate (BHET) reported here, we anticipate MHETase to be a valuable resource to further advance enzymatic plastic degradation. Plastic polymer PET degrading enzymes are of great interest for achieving sustainable plastics recycling. Here, the authors present the crystal structures of the plastic degrading bacterial enzymes PETase, MHETase in its apo-form and MHETase bound to a non-hydrolyzable substrate analog.

Poly(ethylene terephthalate) (PET) is one of the most abundantly produced synthetic polymers and is accumulating in the environment at a staggering rate as discarded packaging and textiles. The properties that make PET so useful also endow it with an alarming resistance to biodegradation, likely lasting centuries in the environment. Our collective reliance on PET and other plastics means that this buildup will continue unless solutions are found. Recently, a newly discovered bacterium, Ideonella sakaiensis 201-F6, was shown to exhibit the rare ability to grow on PET as a major carbon and energy source. Central to its PET biodegradation capability is a secreted PETase (PET-digesting enzyme). Here, we present a 0.92 Å resolution X-ray crystal structure of PETase, which reveals features common to both cutinases and lipases. PETase retains the ancestral α/β-hydrolase fold but exhibits a more open active-site cleft than homologous cutinases. By narrowing the binding cleft via mutation of two active-site residues to conserved amino acids in cutinases, we surprisingly observe improved PET degradation, suggesting that PETase is not fully optimized for crystalline PET degradation, despite presumably evolving in a PET-rich environment. Additionally, we show that PETase degrades another semiaromatic polyester, polyethylene-2,5-furandicarboxylate (PEF), which is an emerging, bioderived PET replacement with improved barrier properties. In contrast, PETase does not degrade aliphatic polyesters, suggesting that it is generally an aromatic polyesterase. These findings suggest that additional protein engineering to increase PETase performance is realistic and highlight the need for further developments of structure/activity relationships for biodegradation of synthetic polyesters.

Plastics, including poly(ethylene terephthalate) (PET), possess many desirable characteristics and thus are widely used in daily life. However, non-biodegradability, once thought to be an advantage offered by plastics, is causing major environmental problem. Recently, a PET-degrading bacterium, Ideonella sakaiensis , was identified and suggested for possible use in degradation and/or recycling of PET. However, the molecular mechanism of PET degradation is not known. Here we report the crystal structure of I. sakaiensis PETase ( Is PETase) at 1.5 Å resolution. Is PETase has a Ser–His-Asp catalytic triad at its active site and contains an optimal substrate binding site to accommodate four monohydroxyethyl terephthalate (MHET) moieties of PET. Based on structural and site-directed mutagenesis experiments, the detailed process of PET degradation into MHET, terephthalic acid, and ethylene glycol is suggested. Moreover, other PETase candidates potentially having high PET-degrading activities are suggested based on phylogenetic tree analysis of 69 PETase-like proteins. Poly(ethylene terephthalate) (PET) is a widely used plastic and its accumulation in the environment has become global problem. Here the authors report the crystal structure of a Ideonella sakaiensis PET-degrading enzyme and propose a molecular mechanism for PET degradation.

An oligomer is a molecule that consists of a few monomer units. It can be formed during polymer manufacturing and also due to polymer degradation processes or even during use conditions. Since oligomers are not included in chemical databases, their identification is a complex process. In this work, the oligomers present in 20 different PET pellet samples have been determined. Two different sample treatment procedures, solvent extraction and total dissolution, were applied in order to select the most efficient one. The analyses were carried out by UPLC-MS-QTOF. The use of high resolution mass spectrometry allowed the structural elucidation of these compounds and their correct identification. The main oligomers identified were cyclic as well as lineal from the first, second, and third series. All of them were composed of terephthalic acid (TPA), diethylene glycol (DEG), and ethylene glycol (EG). Quantitative values were very different in both procedures. In total dissolution of PET samples, the concentration of oligomers was always, at least, 10 times higher than in solvent extraction; some of the compounds were only detected when total dissolution was used. Results showed that the oligomers with the highest concentration values were dimers and trimers, cyclic, as well as lineal, from the first and second series. The oligomer with the maximum concentration value was TPA2-EG-DEG that was found in all the samples in a concentration range from 2493 to 19,290 ng/g PET. No differences between virgin and recycled PET were found. Migration experiments were performed in two PET bottles, and results showed the transference of most of these oligomers to a fat food simulant (ethanol 95%).Graphical abstractGraphical abstract of the two procedures developd and optimized for identifying oligomers in PET pellets and in migration form PET bottles

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

Plastic waste poses an ecological challenge 1 – 3 and enzymatic degradation offers one, potentially green and scalable, route for polyesters waste recycling 4 . Poly(ethylene terephthalate) (PET) accounts for 12% of global solid waste 5 , and a circular carbon economy for PET is theoretically attainable through rapid enzymatic depolymerization followed by repolymerization or conversion/valorization into other products 6 – 10 . Application of PET hydrolases, however, has been hampered by their lack of robustness to pH and temperature ranges, slow reaction rates and inability to directly use untreated postconsumer plastics 11 . Here, we use a structure-based, machine learning algorithm to engineer a robust and active PET hydrolase. Our mutant and scaffold combination (FAST-PETase: functional, active, stable and tolerant PETase) contains five mutations compared to wild-type PETase (N233K/R224Q/S121E from prediction and D186H/R280A from scaffold) and shows superior PET-hydrolytic activity relative to both wild-type and engineered alternatives 12 between 30 and 50 °C and a range of pH levels. We demonstrate that untreated, postconsumer-PET from 51 different thermoformed products can all be almost completely degraded by FAST-PETase in 1 week. FAST-PETase can also depolymerize untreated, amorphous portions of a commercial water bottle and an entire thermally pretreated water bottle at 50 ºC. Finally, we demonstrate a closed-loop PET recycling process by using FAST-PETase and resynthesizing PET from the recovered monomers. Collectively, our results demonstrate a viable route for enzymatic plastic recycling at the industrial scale. Untreated, postconsumer-PET from 51 different thermoformed products can all be almost completely degraded by FAST-PETase in 1 week and PET can be resynthesized from the recovered monomers, demonstrating recycling at the industrial scale.