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Polybutylene adipate terephthalate (PBAT) is a biodegradable alternative to polyethylene and can be broadly used in various applications. These polymers can be degraded by hydrolases of terrestrial and aquatic origin. In a previous study, we identified tandem PETase-like hydrolases (Ples) from the marine microbial consortium I1 that were highly expressed when a PBAT blend was supplied as the only carbon source. In this study, the tandem Ples, Ple628 and Ple629, were recombinantly expressed and characterized. Both enzymes are mesophilic and active on a wide range of oligomers. The activities of the Ples differed greatly when model substrates, PBAT-modified polymers or PET nanoparticles were supplied. Ple629 was always more active than Ple628. Crystal structures of Ple628 and Ple629 revealed a structural similarity to other PETases and can be classified as member of the PETases IIa subclass, α/β hydrolase superfamily. Our results show that the predicted functions of Ple628 and Ple629 agree with the bioinformatic predictions, and these enzymes play a significant role in the plastic degradation by the consortium.

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.

Enzymatic deconstruction of poly(ethylene terephthalate) (PET) is under intense investigation, given the ability of hydrolase enzymes to depolymerize PET to its constituent monomers near the polymer glass transition temperature. To date, reported PET hydrolases have been sourced from a relatively narrow sequence space. Here, we identify additional PET-active biocatalysts from natural diversity by using bioinformatics and machine learning to mine 74 putative thermotolerant PET hydrolases. We successfully express, purify, and assay 51 enzymes from seven distinct phylogenetic groups; observing PET hydrolysis activity on amorphous PET film from 37 enzymes in reactions spanning pH from 4.5–9.0 and temperatures from 30–70 °C. We conduct PET hydrolysis time-course reactions with the best-performing enzymes, where we observe differences in substrate selectivity as function of PET morphology. We employed X-ray crystallography and AlphaFold to examine the enzyme architectures of all 74 candidates, revealing protein folds and accessory domains not previously associated with PET deconstruction. Overall, this study expands the number and diversity of thermotolerant scaffolds for enzymatic PET deconstruction. Enzymes have potential for recycling plastics such as PET, a polyester used in textiles and single-use packaging. Here, the authors identify and characterize additional PET-active biocatalysts and expand the number and diversity of thermotolerant scaffolds for enzymatic PET deconstruction.

Plastics are highly durable and widely used materials. Current methodologies of plastic degradation, elimination, and recycling are flawed. In recent years, biodegradation (the usage of microorganisms for material recycling) has grown as a valid alternative to previously used methods. The evolution of bioengineering techniques and the discovery of novel microorganisms and enzymes with degradation ability have been key. One of the most produced plastics is PET, a long chain polymer of terephthalic acid (TPA) and ethylene glycol (EG) repeating monomers. Many enzymes with PET degradation activity have been discovered, characterized, and engineered in the last few years. However, classification and integrated knowledge of these enzymes are not trivial. Therefore, in this work we present a summary of currently known PET degrading enzymes, focusing on their structural and activity characteristics, and summarizing engineering efforts to improve activity. Although several high potential enzymes have been discovered, further efforts to improve activity and thermal stability are necessary.

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.

Background The biological degradation of plastics is a promising method to counter the increasing pollution of our planet with artificial polymers and to develop eco-friendly recycling strategies. Polyethylene terephthalate (PET) is a thermoplast industrially produced from fossil feedstocks since the 1940s, nowadays prevalently used in bottle packaging and textiles. Although established industrial processes for PET recycling exist, large amounts of PET still end up in the environment—a significant portion thereof in the world’s oceans. In 2016, Ideonella sakaiensis , a bacterium possessing the ability to degrade PET and use the degradation products as a sole carbon source for growth, was isolated. I.   sakaiensis expresses a key enzyme responsible for the breakdown of PET into monomers: PETase. This hydrolase might possess huge potential for the development of biological PET degradation and recycling processes as well as bioremediation approaches of environmental plastic waste. Results Using the photosynthetic microalga Phaeodactylum tricornutum as a chassis we generated a microbial cell factory capable of producing and secreting an engineered version of PETase into the surrounding culture medium. Initial degradation experiments using culture supernatant at 30 °C showed that PETase possessed activity against PET and the copolymer polyethylene terephthalate glycol (PETG) with an approximately 80-fold higher turnover of low crystallinity PETG compared to bottle PET. Moreover, we show that diatom produced PETase was active against industrially shredded PET in a saltwater-based environment even at mesophilic temperatures (21 °C). The products resulting from the degradation of the PET substrate were mainly terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalic acid (MHET) estimated to be formed in the micromolar range under the selected reaction conditions. Conclusion We provide a promising and eco-friendly solution for biological decomposition of PET waste in a saltwater-based environment by using a eukaryotic microalga instead of a bacterium as a model system. Our results show that via synthetic biology the diatom P.   tricornutum indeed could be converted into a valuable chassis for biological PET degradation. Overall, this proof of principle study demonstrates the potential of the diatom system for future biotechnological applications in biological PET degradation especially for bioremediation approaches of PET polluted seawater.

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) becomes one of the most well-known polyesters and is widely used as packaging material. Recently, polyethylene terephthalate hydrolase (PETase) has emerged as a potential biocatalyst demonstrating the ability to degrade polyethylene terephthalate (PET). We showed that the rate of PETase hydrolysis could be significantly increased in the presence of hydrophobin RolA. Hydrophobins represent a class of small fungal protein that has a high surface-active substance and can spontaneously self-assemble at hydrophilic-hydrophobic interfaces. In this work, a class I hydrophobin named RolA was extracted from the mycelium pellet collected from a fermentation culture of Aspergillus oryzae. The SDS-PAGE analysis of the isolated RolA showed the presence of 11 kDa polypeptide. Recombinant PETase from Ideonella sakaiensis was also successfully expressed in Escherichia coli as a soluble protein with molecular weight approximately 30 kDa. The hydrophobin RolA could enhance the PET hydrolysis in the presence of the recombinant PETase. The hydrolysis of PET bottle by RolA-PETase achieved the highest weight loss of 26% in 4 days. It is speculated that the wetting effect of RolA acts on PET surface converts PET to become hydrophilic that leads PETase easier to contact and attack the surface.

Polyethylene terephthalate (PET) is extensively used in plastic products, and its accumulation in the environment has become a global concern. Being a non-degradable pollutant, a tremendous quantity of PET-bearing plastic materials have already accumulated in the environment, posing severe challenges towards the existence of various endangered species and consequently threatening the ecosystem and biodiversity. While conventional recycling and remediation methodologies so far have been ineffective in formulating a “green” degradation protocol, the bioremediation strategies—though nascent—are exhibiting greater promises towards achieving the target. Very recently, a novel bacterial strain called Ideonella sakaiensis 201-F6 has been discovered that produces a couple of unique enzymes, polyethylene terephthalate hydrolase and mono(2-hydroxyethyl) terephthalic acid hydrolase, enabling the bacteria to utilize PET as their sole carbon source. With a detailed understanding of the protein structure of these enzymes, possibilities for their optimization as PET degrading agents have started to emerge. In both proteins, several amino acids have been identified that are not only instrumental for catalysis but also provide avenues for the applications of genetic engineering strategies to improve the catalytic efficiencies of the enzymes. In this review, we focused on such unique structural features of these two enzymes and discussed their potential as molecular tools that can essentially become instrumental towards the development of sustainable bioremediation strategies.

According to the literature review, microbial degradation of polyethylene terephthalate by PETases has been detected effective and eco‐friendly. However, the number of microorganisms capable of such feats is limited with some undesirable bioprospecting results. BTA‐hydrolase has been already reported capable of degrading polyethylene terephthalate. Therefore, mutation by in silico site‐directed mutagenesis means to introduce current isomer of PETase for polyethylene terephthalate degradative capability as a better approach to resolve this issue. This study aimed to use in silico site‐directed mutagenesis to convert a carboxylesterase from Archaeoglobus fulgidus to BTA‐hydrolase from Thermobifida fusca by replacing six amino acids in specific locations. This work was followed by molecular docking analysis with polyethylene terephthalate and polypropylene to compare their interactions. The best‐docked enzyme‐substrate complex was further subjected to molecular dynamics simulation to gauge the binding quality of the BTA‐hydrolase, wild‐type and mutant‐carboxylesterase with only polyethylene terephthalate as a substrate. Results of molecular docking revealed lowest binding energy for the wild‐type carboxylesterase‐polypropylene complex (‐7.5 kcal/mol). The root‐mean‐square deviation value was observed stable for BTA‐hydrolase. Meanwhile, root‐mean‐square fluctuation was assessed with higher fluctuation for the mutated residue Lys178. Consequently, the Rg value for BTA‐hydrolase‐ligand complex (∼1.68 nm) was the lowest compared to the mutant and wild‐type carboxylesterase. The collective data conveyed that mutations imparted a minimal change in the ability of the mutant carboxylesterase to bind to polyethylene terephthalate.