Correction to "New Labeled PET Analogues Enable the Functional Screening and Characterization of PET-Degrading Enzymes".

[This corrects the article DOI: 10.1021/acssuschemeng.4c00143.].

Mimicking the enzymatic plant cell wall hydrolysis mechanism for the degradation of polyethylene terephthalate.

Plastic pollution presents a global challenge, impacting ecosystems, wildlife, and economies. Polyethylene terephthalate (PET), widely used in products like bottles, significantly contributes to this issue due to poor waste collection. In recent years, there has been increasing interest in plant biomass-degrading enzymes for plastic breakdown, due to the structural and physicochemical similarities between natural and synthetic polymers. Filamentous fungi involved in hemicellulose degradation have developed a complex mode of action that includes not only enzymes but also biosurfactants; surface-active molecules that facilitate enzyme-substrate interactions. For this reason, this study aimed to mimic the mechanism of biomass degradation by repurposing plant cell wall degrading enzymes including a cutinase and three esterases to cooperatively contribute to PET degradation. Surfactants of different charge were also introduced in the reactions, as their role is similar to biosurfactants, altering the surface tension of the polymers and thus improving enzymes' accessibility. Notably, Fusarium oxysporum cutinase combined with anionic surfactant exhibited a 2.3- and 1.6-fold higher efficacy in hydrolyzing amorphous and semi-crystalline PET, respectively. When cutinase was combined with either of two ferulic acid esterases, it resulted in complete conversion of PET intermediate products to TPA, increasing the overall product release up to 1.9- fold in presence of surfactant. The combination of cutinase with a glucuronoyl esterase demonstrated significant potential in plastic depolymerization, increasing degradation yields in semi-crystalline PET by up to 1.4-fold. The approach of incorporating enzyme cocktails and surfactants emerge as an efficient solution for PET degradation in mild reaction conditions, with potential applications in eco-friendly plastic waste management.

New Labeled PET Analogues Enable the Functional Screening and Characterization of PET-Degrading Enzymes.

The discovery and engineering of novel biocatalysts capable of depolymerizing polyethylene terephthalate (PET) have gained significant attention since the need for green technologies to combat plastic pollution has become increasingly urgent. This study focuses on the development of novel substrates that can indicate enzymes with PET hydrolytic activity, streamlining the process of enzyme evaluation and selection. Four novel substrates, mimicking the structure of PET, were chemically synthesized and labeled with fluorogenic or chromogenic moieties, enabling the direct analysis of candidate enzymes without complex preparatory or analysis steps. The fluorogenic substrates, mUPET1, mUPET2, and mUPET3, not only identify enzymes capable of PET breakdown but also differentiate those with exceptional performance on the polymer, such as the benchmark PETase, LCCICCG. Among the substrates, the chromogenic p-NPhPET3 stands out as a reliable tool for screening both pure and crude enzymes, offering advantages over fluorogenic substrates such as ease of assay using UV-vis spectroscopy and compatibility with crude enzyme samples. However, ferulic acid esterases and mono-(2-hydroxyethyl) terephthalate esterases (MHETases), which exhibit remarkably high affinity for PET oligomers, also show high catalytic activity on these substrates. The substrates introduced in this study hold significant value in the function-based screening and characterization of enzymes that degrade PET, as well as the the potential to be used in screening mutant libraries derived from directed evolution experiments. Following this approach, a rapid and dependable assay method can be carried out using basic laboratory infrastructure, eliminating the necessity for intricate preparatory procedures before analysis.

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.

Triggering and identifying the polyurethane and polyethylene-degrading machinery of filamentous fungi secretomes.

The uncontrollable disposal of plastic waste has raised the concern of the scientific community, which tries to face this environmental burden by discovering and applying new techniques. Regarding the biotechnology field, several important microorganisms possessing the necessary enzymatic arsenal to utilize recalcitrant synthetic polymers as an energy source have been discovered. In the present study, we screened various fungi for their ability to degrade intact polymers, such as ether-based polyurethane (PU) and low-density polyethylene (LDPE). For this, ImpranIil® DLN-SD and a mixture of long-chain alkanes were used as sole carbon sources, indicating not only the most promising strains in agar plate screening but also inducing the secretion of depolymerizing enzymatic activities, useful for polymer degradation. The agar plate screening revealed three fungal strains belonging to Fusarium and Aspergillus genera, whose secretome was further studied for its ability to degrade the aforementioned non-treated polymers. Specifically for ether-based PU, the secretome of a Fusarium species reduced the sample mass and the average molecular weight of the polymer by 24.5 and 20.4%, respectively, while the secretome of an Aspergillus species caused changes in the molecular structure of LDPE, as evidenced by FTIR. The proteomics analysis revealed that the enzymatic activities induced in presence of Impranil® DLN-SD can be associated with urethane bond cleavage, a fact which was also supported by the observed degradation of the ether-based PU. Although, the mechanism of LDPE degradation was not completely elucidated, the presence of oxidative enzymes could be the main factor contributing to polymer modification.

Investigation of Abortiporus biennis lignocellulolytic toolbox, and the role of laccases in polystyrene degradation.

White-rot basidiomycetes are the only microorganisms able to produce both hydrolytic (cellulases and hemicellulases) and oxidative (ligninolytic) enzymes for degrading all lignocellulose constituents. Their enzymatic machinery makes them ideal for the discovery of novel enzymes with desirable properties. In the present work, Abortiporus biennis, a white-rot fungus, was studied in regard to its lignocellulolytic potential. Secretomics and biochemical analyses were employed to study the strain's enzymatic arsenal, after growth in corn stover cultures and xylose-based defined media. The results revealed the presence of all the necessary enzymatic activities for complete breakdown of biomass, while the prominent role of oxidative enzymes in the lignocellulolytic strategy of the strain became evident. Two novel laccases, AbiLac1 and AbiLac2, were isolated from the culture supernatant with ion-exchange chromatography. Characterization of purified laccases revealed their ability to oxidize a wide variety of phenolic and non-phenolic substrates. AbiLac1 was found to oxidize polystyrene powder, showing high depolymerization potential, based on radical chain scission mechanism as evidenced by molecular weight decrease. The results of the present study demonstrate the biotechnological potential of the unexplored enzymatic machinery of white-rot basidiomycetes, including the design of improved lignocellulolytic cocktails, as well as the degradation and/or valorization of plastic waste materials.

A polyesterase from the Antarctic bacterium Moraxella sp. degrades highly crystalline synthetic polymers.

The uncontrolled release of plastics in the environment has rendered them ubiquitous around the planet, threatening the wildlife and human health. Biodegradation and valorization of plastics has emerged as an eco-friendly alternative to conventional management techniques. Discovery of novel polymer-degrading enzymes with diversified properties is hence an important task in order to explore different operational conditions for plastic-waste upcycling. In the present study, a barely studied psychrophilic enzyme (MoPE) from the Antractic bacterium Moraxella sp. was heterologously expressed, characterized and its potential in polymer degradation was further investigated. Based on its amino acid composition and structure, MoPE resembled PET-degrading enzymes, sharing features from both mesophilic and thermophilic homologues. MoPE hydrolyzes non-biodegradable plastics, such as polyethylene terephthalate and polyurethane, as well as biodegradable synthetic polyesters, such as polycaprolactone, polyhydroxy butyrate, polybutylene succinate and polylactic acid. The mass fraction crystallinity of the aliphatic polymers tested ranged from 11% to 64% highlighting the potential of the enzyme to hydrolyze highly crystalline plastics. MoPE was able to degrade different types of amorphous and semi-crystalline PET, releasing water-soluble monomers and showed synergy with a feruloyl esterase of the tannase family for the release of terephthalic acid. Based on the above, MoPE was characterized as a versatile psychrophilic polyesterase demonstrating a broad-range plastics degradation potential.

Progressing Plastics Circularity: A Review of Mechano-Biocatalytic Approaches for Waste Plastic (Re)valorization.

Inspirational concepts, and the transfer of analogs from natural biology to science and engineering, has produced many excellent technologies to date, spanning vaccines to modern architectural feats. This review highlights that answers to the pressing global petroleum-based plastic waste challenges, can be found within the mechanics and mechanisms natural ecosystems. Here, a suite of technological and engineering approaches, which can be implemented to operate in tandem with nature's prescription for regenerative material circularity, is presented as a route to plastics sustainability. A number of mechanical/green chemical (pre)treatment methodologies, which simulate natural weathering and arthropodal dismantling activities are reviewed, including: mechanical milling, reactive extrusion, ultrasonic-, UV- and degradation using supercritical CO2. Akin to natural mechanical degradation, the purpose of the pretreatments is to render the plastic materials more amenable to microbial and biocatalytic activities, to yield effective depolymerization and (re)valorization. While biotechnological based degradation and depolymerization of both recalcitrant and bioplastics are at a relatively early stage of development, the potential for acceleration and expedition of valuable output monomers and oligomers yields is considerable. To date a limited number of independent mechano-green chemical approaches and a considerable and growing number of standalone enzymatic and microbial degradation studies have been reported. A convergent strategy, one which forges mechano-green chemical treatments together with the enzymatic and microbial actions, is largely lacking at this time. An overview of the reported microbial and enzymatic degradations of petroleum-based synthetic polymer plastics, specifically: low-density polyethylene (LDPE), high-density polyethylene (HDPE), polystyrene (PS), polyethylene terephthalate (PET), polyurethanes (PU) and polycaprolactone (PCL) and selected prevalent bio-based or bio-polymers [polylactic acid (PLA), polyhydroxyalkanoates (PHAs) and polybutylene succinate (PBS)], is detailed. The harvesting of depolymerization products to produce new materials and higher-value products is also a key endeavor in effectively completing the circle for plastics. Our challenge is now to effectively combine and conjugate the requisite cross disciplinary approaches and progress the essential science and engineering technologies to categorically complete the life-cycle for plastics.