Metagenome-derived SusD-homologs affiliated with Bacteroidota bind to synthetic polymers.

Starch utilization system (Sus)D-homologs are well known for their carbohydrate-binding capabilities and are part of the sus operon in microorganisms affiliated with the phylum Bacteroidota. Until now, SusD-like proteins have been characterized regarding their affinity toward natural polymers. In this study, three metagenomic SusD homologs (designated SusD1, SusD38489, and SusD70111) were identified and tested with respect to binding to natural and non-natural polymers. SusD1 and SusD38489 are cellulose-binding modules, while SusD70111 preferentially binds chitin. Employing translational fusion proteins with superfolder GFP (sfGFP), pull-down assays, and surface plasmon resonance (SPR) has provided evidence for binding to polyethylene terephthalate (PET) and other synthetic polymers. Structural analysis suggested that a Trp triad might be involved in protein adsorption. Mutation of these residues to Ala resulted in an impaired adsorption to microcrystalline cellulose (MC), but not so to PET and other synthetic polymers. We believe that the characterized SusDs, alongside the methods and considerations presented in this work, will aid further research regarding bioremediation of plastics. IMPORTANCE: SusD1 and SusD38489 can be considered for further applications regarding their putative adsorption toward fossil-fuel based polymers. This is the first time that SusD homologs from the polysaccharide utilization loci (PUL), largely described for the phylum Bacteroidota, are characterized as synthetic polymer-binding proteins.

Efficient biodegradation of Polyethylene terephthalate (PET) plastic by Gordonia sp. CN2K isolated from plastic contaminated environment.

Since we rely entirely on plastics or their products in our daily lives, plastics are the invention of the hour. Polyester plastics, such as Polyethylene Terephthalate (PET), are among the most often used types of plastics. PET plastics have a high ratio of aromatic components, which makes them very resistant to microbial attack and highly persistent. As a result, massive amounts of plastic trash accumulate in the environment, where they eventually transform into microplastic (<5 mm). Rather than macroplastics, microplastics are starting to pose a serious hazard to the environment. It is imperative that these polymer microplastics be broken down. Through the use of enrichment culture, the PET microplastic-degrading bacterium was isolated from solid waste management yards. Bacterial strain was identified as Gordonia sp. CN2K by 16 S rDNA sequence analysis and biochemical characterization. It is able to use polyethylene terephthalate as its only energy and carbon source. In 45 days, 40.43 % of the PET microplastic was degraded. By using mass spectral analysis and HPLC to characterize the metabolites produced during PET breakdown, the degradation of PET is verified. The metabolites identified in the spent medium included dimer compound, bis (2-hydroxyethyl) terephthalate (BHET), mono (2-hydroxyethyl) terephthalate (MHET), and terephthalate. Furthermore, the PET sheet exposed to the culture showed considerable surface alterations in the scanning electron microscope images. This illustrates how new the current work is.

Surface Interaction of Ionic Liquids: Stabilization of Polyethylene Terephthalate-Degrading Enzymes in Solution.

The effect of aqueous solutions of selected ionic liquids solutions on Ideonella sakaiensis PETase with bis(2-hydroxyethyl) terephthalate (BHET) substrate were studied by means of molecular dynamics simulations in order to identify the possible effect of ionic liquids on the structure and dynamics of enzymatic Polyethylene terephthalate (PET) hydrolysis. The use of specific ionic liquids can potentially enhance the enzymatic hydrolyses of PET where these ionic liquids are known to partially dissolve PET. The aqueous solution of cholinium phosphate were found to have the smallest effect of the structure of PETase, and its interaction with (BHET) as substrate was comparable to that with the pure water. Thus, the cholinium phosphate was identified as possible candidate as ionic liquid co-solvent to study the enzymatic hydrolyses of PET.

New Insights into the Function and Global Distribution of Polyethylene Terephthalate (PET)-Degrading Bacteria and Enzymes in Marine and Terrestrial Metagenomes.

Polyethylene terephthalate (PET) is one of the most important synthetic polymers used today. Unfortunately, the polymers accumulate in nature and to date no highly active enzymes are known that can degrade it at high velocity. Enzymes involved in PET degradation are mainly α- and ß-hydrolases, like cutinases and related enzymes (EC 3.1.1). Currently, only a small number of such enzymes are well characterized. In this work, a search algorithm was developed that identified 504 possible PET hydrolase candidate genes from various databases. A further global search that comprised more than 16 Gb of sequence information within 108 marine and 25 terrestrial metagenomes obtained from the Integrated Microbial Genome (IMG) database detected 349 putative PET hydrolases. Heterologous expression of four such candidate enzymes verified the function of these enzymes and confirmed the usefulness of the developed search algorithm. In this way, two novel and thermostable enzymes with high potential for downstream application were partially characterized. Clustering of 504 novel enzyme candidates based on amino acid similarities indicated that PET hydrolases mainly occur in the phyla of Actinobacteria, Proteobacteria, and Bacteroidetes Within the Proteobacteria, the Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria were the main hosts. Remarkably enough, in the marine environment, bacteria affiliated with the phylum Bacteroidetes appear to be the main hosts of PET hydrolase genes, rather than Actinobacteria or Proteobacteria, as observed for the terrestrial metagenomes. Our data further imply that PET hydrolases are truly rare enzymes. The highest occurrence of 1.5 hits/Mb was observed in sequences from a sample site containing crude oil.IMPORTANCE Polyethylene terephthalate (PET) accumulates in our environment without significant microbial conversion. Although a few PET hydrolases are already known, it is still unknown how frequently they appear and with which main bacterial phyla they are affiliated. In this study, deep sequence mining of protein databases and metagenomes demonstrated that PET hydrolases indeed occur at very low frequencies in the environment. Furthermore, it was possible to link them to phyla that were previously not known to harbor such enzymes. This work contributes novel knowledge on the phylogenetic relationships, the recent evolution, and the global distribution of PET hydrolases. Finally, we describe the biochemical traits of four novel PET hydrolases.

Hydrogenation of the benzene rings in PET degraded chemicals over meso-HZSM-5 supported Ru catalyst.

Chemical upcycling of waste polyethylene terephthalate (PET) to value-added products can reduce the emission of CO2, microplastics and toxic chemicals. In this work, mesoporous H-type Zeolite Socony Mobil-5 (HZSM-5) supported Ru catalyst (Ru/m-HZSM-5) was synthesized and tested in the hydrogenation of PET degraded chemicals (bis(2-hydroxyethyl) terephthalate, dimethyl terephthalate, diethyl terephthalate, and terephthalic acid). Characterizations disclosed that Ru/m-HZSM-5 catalyst possesses mesopores (a dominant channel of 5.32 nm), enlarged specific surface area (404 m2·g-1), and Ru NPs dispersed highly (40.6 %) compared to that of Ru/HZSM-5. And also, it was found that Ru/m-HZSM-5 was capable for the hydrogenation of benzene rings in these PET degraded chemicals with large sizes (1.09-1.82 nm). In particular, the conversion of BHET and the selectivity of BHCD over Ru/m-HZSM-5 reached 95.5 % and 95.6 % at 120 °C within 2 h. And Ru/m-HZSM-5 could be recycled at least five times without obvious loss of activity and selectivity.

Optimization of Ti-PA efficiently catalytic the alcoholysis of waste PET using response surface methodology.

A new type of titanium phthalate (Ti-PA) catalyst was prepared by exchange method of phthalic acid and isopropyl titanate, which is never been reported before. The Ti-PA catalyst was characterized by FT-IR, TG, Uv-vis, BET, SEM, and EDS. The Ti-PA catalyst shows good catalytic activity in the alcoholysis reaction of polyethylene terephthalate (PET) and optimal experimental conditions for the alcoholysis process were optimized by response surface methodology; the Ti-PA catalyst provided a BHET yield of 81.98% for reaction lasting 3.98 h at 191 °C of 0.86% catalyst and 13.7 ml ethylene glycol; the model has good reliability. The kinetics and reaction mechanism of the process were explored and apparent activation energy is 75.52 kJ/mol. Finally, the good catalytic activity of Ti-PA was illustrated by comparing it with currently reported catalysts.

Chemical Recycling of PET Using Catalysts from Layered Double Hydroxides: Effect of Synthesis Method and Mg-Fe Biocompatible Metals.

The chemical recycling of poly(ethylene terephthalate) (PET) residues was performed via glycolysis with ethylene glycol (EG) over Mg-Fe and Mg-Al oxide catalysts derived from layered double hydroxides. Catalysts prepared using the high supersaturation method (h.s.c.) presented a higher surface area and larger particles, but this represented less PET conversion than those prepared by the low supersaturation method (l.s.c.). This difference was attributed to the smaller mass transfer limitations inside the (l.s.c.) catalysts. An artificial neural network model well fitted the PET conversion and bis(2-hydroxyethyl) terephthalate (BHET) yield. The influence of Fe in place of Al resulted in a higher PET conversion of the Mg-Fe-h.s.c. catalyst (~95.8%) than of Mg-Al-h.s.c. (~63%). Mg-Fe catalysts could be reused four to five times with final conversions of up to 97% with reaction conditions of EG: PET = 5:1 and catalyst: PET = 0.5%. These results confirm the Mg-Fe oxides as a biocompatible novel catalyst for the chemical recycling of PET residues to obtain non-toxic BHET for further polymerization, and use in food and beverage packaging.

The distinct plastisphere microbiome in the terrestrial-marine ecotone is a reservoir for putative degraders of petroleum-based polymers.

Research into plastic-degrading bacteria and fungi is important for understanding how microorganisms can be used to address the problem of plastic pollution and for developing new approaches to sustainable waste management and bioplastic production. In the present study, we isolated 55 bacterial and 184 fungal strains degrading polycaprolactone (PCL) in plastic waste samples from Dafeng coastal salt marshes, Jiangsu, China. Of these, Jonesia and Streptomyces bacteria also showed potential to degrade other types of petroleum-based polymers. The metabarcoding results proved the existence of plastisphere as a distinct ecological niche regardless of the plastic types where 27 bacterial and 29 fungal amplicon sequence variants (ASVs) were found to be significantly (p < 0.05) enriched, including some belonging to Alternaria (Ascomycota, Fungi) and Pseudomonas (Gammaproteobacteria, Bacteria) that were also mined out by the method of cultivation. Further assembly analyses demonstrated the importance of deterministic processes especially the environmental filtering effect of carbon content and pH on bacteria as well as the carbon and cation content on fungi in shaping the plastisphere communities in this ecosystem. Thus, the unique microbiome of the plastisphere in the terrestrial-marine ecotone is enriched with microorganisms that are potentially capable of utilizing petroleum-based polymers, making it a valuable resource for screening plastic biodegraders.

[Expression, purification and characterization of a novel bis (hydroxyethyl) terephthalate hydrolase from Hydrogenobacter thermophilus].

PET (polyethylene terephthalate) is one of the most important petrochemicals that is widely used in mineral water bottles, food and beverage packaging and textile industry. Because of its stability under environmental conditions, the massive amount of PET wastes caused serious environmental pollution. The use of enzymes to depolymerize PET wastes and upcycling is one of the important directions for plastics pollution control, among which the key is the depolymerization efficiency of PET by PET hydrolase. BHET (bis(hydroxyethyl) terephthalate) is the main intermediate of PET hydrolysis, its accumulation can hinder the degradation efficiency of PET hydrolase significantly, and the synergistic use of PET hydrolase and BHET hydrolase can improve the PET hydrolysis efficiency. In this study, a dienolactone hydrolase from Hydrogenobacter thermophilus which can degrade BHET (HtBHETase) was identified. After heterologous expression in Escherichia coli and purification, the enzymatic properties of HtBHETase were studied. HtBHETase shows higher catalytic activity towards esters with short carbon chains such as p-nitrophenol acetate. The optimal pH and temperature of the reaction with BHET were 5.0 and 55 ℃, respectively. HtBHETase exhibited excellent thermostability, and retained over 80% residual activity after treatment at 80 ℃ for 1 hour. These results indicate that HtBHETase has potential in biological PET depolymerization, which may facilitate the enzymatic degradation of PET.

[Enzymatic properties and degradation characterization of a bis(2-hydroxyethyl) terephthalate hydrolase from Saccharothrix sp.]

The discovery of new enzymes for poly(ethylene terephthalate) (PET) degradation has been a hot topic of research globally. Bis-(2-hydroxyethyl) terephthalate (BHET) is an intermediate compound in the degradation of PET and competes with PET for the substrate binding site of the PET-degrading enzyme, thereby inhibiting further degradation of PET. Discovery of new BHET degradation enzymes may contribute to improving the degradation efficiency of PET. In this paper, we discovered a hydrolase gene sle (ID: CP064192.1, 5085270-5086049) from Saccharothrix luteola, which can hydrolyze BHET into mono-(2-hydroxyethyl) terephthalate (MHET) and terephthalic acid (TPA). BHET hydrolase (Sle) was heterologously expressed in Escherichia coli using a recombinant plasmid, and the highest protein expression was achieved at a final concentration of 0.4 mmol/L of isopropyl-ß-d-thiogalactoside (IPTG), an induction duration of 12 h and an induction temperature of 20 ℃. The recombinant Sle was purified by nickel affinity chromatography, anion exchange chromatography, and gel filtration chromatography, and its enzymatic properties were also characterized. The optimum temperature and pH of Sle were 35 ℃ and 8.0, and more than 80% of the enzyme activity could be maintained in the range of 25-35 ℃ and pH 7.0-9.0 and Co2+ could improve the enzyme activity. Sle belongs to the dienelactone hydrolase (DLH) superfamily and possesses the typical catalytic triad of the family, and the predicted catalytic sites are S129, D175, and H207. Finally, the enzyme was identified as a BHET degrading enzyme by high performance liquid chromatography (HPLC). This study provides a new enzyme resource for the efficient enzymatic degradation of PET plastics.

Metagenomic Screening of a Novel PET Esterase via In Vitro Expression System.

Metagenomic screening is a widely applied biotechnological approach for screening of novel industrial enzymes. The traditional method of metagenomic screening is based on the functional analyses of heterologously expressed environmental genes in a suitable host, which is the bottleneck of this method. To avoid limitation from the clone-dependent system, an in vitro expression technology has been developed in combination with next-generation sequencing and bioinformatics. First, the sequence profile of a target enzyme, e.g., poly(ethylene terephthalate) esterase in this protocol, is constructed according to the sequences of well-characterized enzymes. Then, the sequence screening is performed with this computationally generated profile among all available metagenomic databases. Afterwards, the candidate genes are synthesized and expressed in vitro with RNA polymerase and translation machinery from special cell extract. Finally, such in vitro produced enzymes are directly applied for the functional analyses. Comparing to the traditional screening methods, this in vitro screening technology can not only save time and materials, but also be easily developed for high-throughput screening with an automatic pipetting robot.

Viability of Glycolysis for the Chemical Recycling of Highly Coloured and Multi-Layered Actual PET Wastes.

The chemical recycling of poly(ethylene terephthalate) -PET- fractions, derived from actual household packaging waste streams, using solvolysis, was investigated. This recycling strategy was applied after a previous on-line automatic identification, by near-infrared spectroscopy -NIR-, and a subsequent selective sorting of the different PET materials that were present in the packaging wastes. Using this technology, it was possible to classify fractions exclusively including PET, virtually avoiding the presence of both other plastics and materials, such as paper, cardboard and wood, that are present in the packaging wastes, as they were efficiently recognised and differentiated. The simple PET fractions, including clear and monolayered materials, were adequate to be recycled by mechanical means meanwhile the complex PET fractions, containing highly coloured and multi-layered materials, were suitable candidates to be recycled by chemical routes. The depolymerisation capacity of the catalytic glycolysis, when applied to those complex PET wastes, was studied by evaluating the effect of the process parameters on the resulting formation and recovery of the monomer bis(2-hydroxyethyl) terephthalate -BHET- and the achieved quality of this reaction product. Comparable and reasonable results, in terms of monomer yield and its characteristics, were obtained independently of the type of complex PET waste that was chemically recycled.

Call for biotechnological approach to degrade plastic in the era of COVID-19 pandemic.

Plastic pollution is a global issue and has become a major concern since Coronavirus disease (COVID)-19. In developing nations, landfilling and illegal waste disposal are typical ways to dispose of COVID-19-infected material. These technologies worsen plastic pollution and other human and animal health problems. Plastic degrades in light and heat, generating hazardous primary and secondary micro-plastic. Certain bacteria can degrade artificial polymers using genes, enzymes, and metabolic pathways. Microorganisms including bacteria degrade petrochemical plastics slowly. High molecular weight, strong chemical bonds, and excessive hydrophobicity reduce plastic biodegradation. There is not enough study on genes, enzymes, and bacteria-plastic interactions. Synthetic biology, metabolic engineering, and bioinformatics methods have been created to biodegrade synthetic polymers. This review will focus on how microorganisms' degrading capacity can be increased using recent biotechnological techniques.

Nucleating Agents to Enhance Poly(l-Lactide) Fiber Crystallization during Industrial-Scale Melt Spinning.

The nucleating agent N,N'-bis(2-hydroxyethyl)-terephthalamide (BHET) has promising effects on poly(l-lactide) (PLA) under quiescent conditions and for injection molding applications, but its suitability for industrial-scale fiber melt spinning is unclear. We therefore determined the effects of 1% and 2% (w/w) BHET on the crystallinity, tenacity, and elongation at break of PLA fibers compared to pure PLA and PLA plus talc as a reference nucleating agent. Fibers were spun at take-up velocities of 800, 1400 and 2000 m/min and at drawing at ratios of 1.1-4.0, reaching a final winding speed of 3600 m/min. The fibers were analyzed by differential scanning calorimetry, wide-angle X-ray diffraction, gel permeation chromatography and tensile testing. Statistical analysis of variance was used to determine the combined effects of the spin-line parameters on the material properties. We found that the fiber draw ratio and take-up velocity were the most important factors affecting tenacity and elongation, but the addition of BHET reduced the mechanical performance of the fibers. The self-organizing properties of BHET were not expressed due to the rapid quenching of the fibers, leading to the formation of α'-crystals. Understanding the behavior of BHET in the PLA matrix provides information on the performance of nucleation agents during high-speed processing that will allow processing improvements in the future.

Improving the Efficiency for the Production of Bis-(2-Hydroxyethyl) Terephtalate (BHET) from the Glycolysis Reaction of Poly(Ethylene Terephtalate) (PET) in a Pressure Reactor.

The depolymerization process of PET by glycolysis into BHET monomer is optimized in terms of reaction temperature and time, by carrying out the process under pressure to be faster for reducing the energy required. Almost pure BHET has been obtained by working in a pressure reactor at 3 bar both at 220 and 180 °C after short reaction times, while for longer ones a mixture of oligomers and dimers is obtained. Depending on the potential application required, the obtention of different reaction products is controlled by adjusting reaction temperature and time. The use of a pressure reactor allows work at lower temperatures and shorter reaction times, obtaining almost pure BHET. To the best of our knowledge, except for microwave-assisted procedures, it is the first time in which pure BHET is obtained after such short reaction times, at lower temperatures than those usually employed.

Impact of Enzymatic Degradation on the Material Properties of Poly(Ethylene Terephthalate).

With macroscopic litter and its degradation into secondary microplastic as a major source of environmental pollution, one key challenge is understanding the pathways from macro- to microplastic by abiotic and biotic environmental impact. So far, little is known about the impact of biota on material properties. This study focuses on recycled, bottle-grade poly(ethylene terephthalate) (r-PET) and the degrading enzyme PETase from Ideonella sakaiensis. Compact tension (CT) specimens were incubated in an enzymatic solution and thermally and mechanically characterized. A time-dependent study up to 96 h revealed the formation of steadily growing colloidal structures. After 96 h incubation, high amounts of BHET dimer were found in a near-surface layer, affecting crack propagation and leading to faster material failure. The results of this pilot study show that enzymatic activity accelerates embrittlement and favors fragmentation. We conclude that PET-degrading enzymes must be viewed as a potentially relevant acceleration factor in macroplastic degradation.