Explorations of Polyethylene Terephthalate (PET) Hydrolase for addressing PET Plastic Pollution

Biodegradation of terephthalate (TPA) is a highly desired catabolic process for the bacterial utilization of this Polyethylene terephthalate (PET) depolymerization product, but to date, the structure of terephthalate dioxygenase (TPDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of TPA to a cis -diol is unavailable. In this study, we characterized the steady-state kinetics and first crystal structure of TPDO from Comamonas testosteroni KF1 (TPDO KF1 ). The TPDO KF1 exhibited the substrate specificity for TPA ( k cat / K m = 57 ± 9 mM −1 s −1 ). The TPDO KF1 structure harbors characteristics RO features as well as a unique catalytic domain that rationalizes the enzyme’s function. The docking and mutagenesis studies reveal that its substrate specificity to TPA is mediated by Arg309 and Arg390 residues, two residues positioned on opposite faces of the active site. Additionally, residue Gln300 is also proven to be crucial for the activity, its substitution to alanine decreases the activity ( k cat ) by 80%. Together, this study delineates the structural features that dictate the substrate recognition and specificity of TPDO. Importance The global plastic pollution has become the most pressing environmental issue. Recent studies on enzymes depolymerizing polyethylene terephthalate plastic into terephthalate (TPA) show some potential in tackling this. Microbial utilization of this released product, TPA is an emerging and promising strategy for waste-to-value creation. Research from the last decade has discovered terephthalate dioxygenase (TPDO), as being responsible for initiating the enzymatic degradation of TPA in a few Gram-negative and Gram-positive bacteria. Here, we have determined the crystal structure of TPDO from Comamonas testosteroni KF1 and revealed that it possesses a unique catalytic domain featuring two basic residues in the active site to recognize TPA. Biochemical and mutagenesis studies demonstrated the crucial residues responsible for the substrate specificity of this enzyme.

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Clean Enzymatic depolymerization of highly crystalline polyethylene terephthalate in moist-solid reaction mixtures

10.1101/2020.07.06.189720 ◽

2020 ◽

Cited By ~ 1

Author(s):

Sandra Kaabel ◽

J. P. Daniel Therien ◽

Catherine E. Deschênes ◽

Dustin Duncan ◽

Tomislav Friščić ◽

...

Keyword(s):

Polyethylene Terephthalate ◽

Metal Organic Framework ◽

Direct Synthesis ◽

Plastic Pollution ◽

Solid Reaction ◽

Pollution Problem ◽

Metal Organic ◽

Dilute Aqueous Solutions ◽

Space Time Yield ◽

Crystalline Forms

AbstractLess 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, the current consensus is that they lack activity towards the more crystalline forms of PET. We report here that when used in moist-solid reaction mixtures instead of the typical dilute aqueous solutions, enzymes can directly depolymerize high crystallinity PET in 13-fold higher space-time yield and a 15-fold higher enzyme efficiency than prior reports. Further, this process shows a 26-fold selectivity for terephthalic acid over other hydrolysis products, which allows the direct synthesis of UiO-66 metal-organic framework.

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Current Advances in the Biodegradation and Bioconversion of Polyethylene Terephthalate

Microorganisms ◽

10.3390/microorganisms10010039 ◽

2021 ◽

Vol 10 (1) ◽

pp. 39

Author(s):

Xinhua Qi ◽

Wenlong Yan ◽

Zhibei Cao ◽

Mingzhu Ding ◽

Yingjin Yuan

Keyword(s):

Polyethylene Terephthalate ◽

Microbial Consortium ◽

Tricarboxylic Acid Cycle ◽

Tca Cycle ◽

Tricarboxylic Acid ◽

Microbial Consortia ◽

Plastic Pollution ◽

One Step ◽

Complex Polymers ◽

The One

Polyethylene terephthalate (PET) is a widely used plastic that is polymerized by terephthalic acid (TPA) and ethylene glycol (EG). In recent years, PET biodegradation and bioconversion have become important in solving environmental plastic pollution. More and more PET hydrolases have been discovered and modified, which mainly act on and degrade the ester bond of PET. The monomers, TPA and EG, can be further utilized by microorganisms, entering the tricarboxylic acid cycle (TCA cycle) or being converted into high value chemicals, and finally realizing the biodegradation and bioconversion of PET. Based on synthetic biology and metabolic engineering strategies, this review summarizes the current advances in the modified PET hydrolases, engineered microbial chassis in degrading PET, bioconversion pathways of PET monomers, and artificial microbial consortia in PET biodegradation and bioconversion. Artificial microbial consortium provides novel ideas for the biodegradation and bioconversion of PET or other complex polymers. It is helpful to realize the one-step bioconversion of PET into high value chemicals.

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Microbial Degradation of Plastics and Approaches to Make it More Efficient

Microbiology ◽

10.1134/s0026261721060084 ◽

2021 ◽

Vol 90 (6) ◽

pp. 671-701

Author(s):

I. B. Kotova ◽

Yu. V. Taktarova ◽

E. A. Tsavkelova ◽

M. A. Egorova ◽

I. A. Bubnov ◽

...

Keyword(s):

Polyethylene Terephthalate ◽

Microbial Degradation ◽

Microscopic Fungi ◽

Slow Process ◽

Plastic Pollution ◽

Degradation Processes ◽

Worldwide Production ◽

Plastic Degradation

Abstract— The growing worldwide production of synthetic plastics leads to increased amounts of plastic pollution. Even though microbial degradation of plastics is known to be a very slow process, this capacity has been found in many bacteria, including invertebrate symbionts, and microscopic fungi. Research in this field has been mostly focused on microbial degradation of polyethylene, polystyrene, and polyethylene terephthalate (PET). Quite an arsenal of different methods is available today for detecting processes of plastic degradation and measuring their rates. Given the lack of generally accepted protocols, it is difficult to compare results presented by different authors. PET degradation by recombinant hydrolases from thermophilic actinobacteria happens to be the most efficient among the currently known plastic degradation processes. Various approaches to accelerating microbial plastic degradation are also discussed.

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Engineering Microbes to Bio-Upcycle Polyethylene Terephthalate

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.656465 ◽

2021 ◽

Vol 9 ◽

Author(s):

Lakshika Dissanayake ◽

Lahiru N. Jayakody

Keyword(s):

Polyethylene Terephthalate ◽

Metabolic Pathways ◽

Aquatic Ecosystems ◽

Value Added ◽

Building Blocks ◽

Chemical Recycling ◽

Plastic Pollution ◽

Microbial Cell Factories ◽

Cell Factories ◽

Value Added Products

Polyethylene terephthalate (PET) is globally the largest produced aromatic polyester with an annual production exceeding 50 million metric tons. PET can be mechanically and chemically recycled; however, the extra costs in chemical recycling are not justified when converting PET back to the original polymer, which leads to less than 30% of PET produced annually to be recycled. Hence, waste PET massively contributes to plastic pollution and damaging the terrestrial and aquatic ecosystems. The global energy and environmental concerns with PET highlight a clear need for technologies in PET “upcycling,” the creation of higher-value products from reclaimed PET. Several microbes that degrade PET and corresponding PET hydrolase enzymes have been successfully identified. The characterization and engineering of these enzymes to selectively depolymerize PET into original monomers such as terephthalic acid and ethylene glycol have been successful. Synthetic microbiology and metabolic engineering approaches enable the development of efficient microbial cell factories to convert PET-derived monomers into value-added products. In this mini-review, we present the recent progress of engineering microbes to produce higher-value chemical building blocks from waste PET using a wholly biological and a hybrid chemocatalytic–biological strategy. We also highlight the potent metabolic pathways to bio-upcycle PET into high-value biotransformed molecules. The new synthetic microbes will help establish the circular materials economy, alleviate the adverse energy and environmental impacts of PET, and provide market incentives for PET reclamation.

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Fusion of Chitin-Binding Domain From Chitinolyticbacter meiyuanensis SYBC-H1 to the Leaf-Branch Compost Cutinase for Enhanced PET Hydrolysis

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.762854 ◽

2021 ◽

Vol 9 ◽

Author(s):

Rui Xue ◽

Yinping Chen ◽

Huan Rong ◽

Ren Wei ◽

Zhongli Cui ◽

...

Keyword(s):

Catalytic Activity ◽

Enzymatic Hydrolysis ◽

Polyethylene Terephthalate ◽

Binding Domain ◽

Plastic Pollution ◽

Pet Waste ◽

Chitin Binding Domain ◽

C Terminus ◽

Measurable Activity ◽

Hydrolysis Of

Polyethylene terephthalate (PET) is a mass-produced petroleum-based non-biodegradable plastic that contributes to the global plastic pollution. Recently, biocatalytic degradation has emerged as a viable recycling approach for PET waste, especially with thermophilic polyester hydrolases such as a cutinase (LCC) isolated from a leaf-branch compost metagenome and its variants. To improve the enzymatic PET hydrolysis performance, we fused a chitin-binding domain (ChBD) from Chitinolyticbacter meiyuanensis SYBC-H1 to the C-terminus of the previously reported LCCICCG variant, demonstrating higher adsorption to PET substrates and, as a result, improved degradation performance by up to 19.6% compared to with its precursor enzyme without the binding module. For compare hydrolysis with different binding module, the catalytic activity of LCCICCG-ChBD, LCCICCG-CBM, LCCICCG-PBM and LCCICCG-HFB4 were further investigated with PET substrates of various crystallinity and it showed measurable activity on high crystalline PET with 40% crystallinity. These results indicated that fusing a polymer-binding module to LCCICCG is a promising method stimulating the enzymatic hydrolysis of PET.

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Salt leverages polyethylene terephthalate hydrolase (PETase) enzymatic activity via the predicted conformational switch

10.1101/2021.09.23.461413 ◽

2021 ◽

Author(s):

Sergey A. Shiryaev ◽

Piotr Cieplak ◽

Andrei V. Chernov

Keyword(s):

Polyethylene Terephthalate ◽

Conformational Changes ◽

Carbon Sources ◽

Catalytic Triad ◽

High Salt ◽

Plastic Pollution ◽

Conformational Switch ◽

Global Challenge ◽

Hydrolyzing Enzymes ◽

Dynamics Simulations

AbstractPlastic pollution spawned a global challenge caused by the environmental accumulation of polyethylene terephthalate (PET) plastics. Ongoing remediation efforts using microbial and engineered PET hydrolyzing enzymes (PETases) are hindered by slow depolymerization activities. Here, we report the optimized reaction conditions that leveraged the PETase hydrolase activity 2 to 3.8-fold in the presence of high NaCl concentrations. Molecular dynamics simulations (MDS) were applied to model salt-dependent conformational changes of the PETase enzyme bound to a 3-unit PET polymer. MDS demonstrated that PETase interaction with flanking polymer units exhibited a striking structural disparity at low and high salt concentrations. At low salt concentrations, flanking polymer units displayed significant bending. In contrast, flanking units extended at high salt concentrations, thus residues D206, H237, and S160 of the catalytic triad positioned in close vicinity of the scissile ester bond of the polymer substrate. The resulting high salt-specific enzyme/substrate geometry can potentially facilitate hydrolysis. We theorized that a salt-dependent conformational switch could attenuate the enzyme to a broad range of natural and artificial polymers consumed as carbon sources. Altogether, new knowledge may advance the engineering of PET hydrolase enzymes and benefit bioconversion programs.

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

Applied and Environmental Microbiology ◽

10.1128/aem.02773-17 ◽

2018 ◽

Vol 84 (8) ◽

pp. e02773-17 ◽

Cited By ~ 41

Author(s):

Dominik Danso ◽

Christel Schmeisser ◽

Jennifer Chow ◽

Wolfgang Zimmermann ◽

Ren Wei ◽

...

Keyword(s):

Polyethylene Terephthalate ◽

Search Algorithm ◽

Global Distribution ◽

Sequence Information ◽

Microbial Conversion ◽

Thermostable Enzymes ◽

Content Type ◽

Bacterial Phyla ◽

Degrading Bacteria ◽

Pet Hydrolase

ABSTRACTPolyethylene 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 β-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 ofActinobacteria,Proteobacteria, andBacteroidetes. Within theProteobacteria, theBetaproteobacteria,Deltaproteobacteria, andGammaproteobacteriawere the main hosts. Remarkably enough, in the marine environment, bacteria affiliated with the phylumBacteroidetesappear to be the main hosts of PET hydrolase genes, rather thanActinobacteriaorProteobacteria, 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.IMPORTANCEPolyethylene 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.

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Yeast cell surface display of bacterial PET hydrolase as a sustainable biocatalyst for the degradation of polyethylene terephthalate

Methods in Enzymology - Enzymatic Plastic Degradation ◽

10.1016/bs.mie.2020.12.030 ◽

2021 ◽

pp. 457-477

Author(s):

Zhuozhi Chen ◽

Yunjie Xiao ◽

Gert Weber ◽

Ren Wei ◽

Zefang Wang

Keyword(s):

Cell Surface ◽

Yeast Cell ◽

Polyethylene Terephthalate ◽

Surface Display ◽

Cell Surface Display ◽

Yeast Cell Surface ◽

Pet Hydrolase

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Fungal Enzymes as Catalytic Tools for Polyethylene Terephthalate (PET) Degradation

Journal of Fungi ◽

10.3390/jof7110931 ◽

2021 ◽

Vol 7 (11) ◽

pp. 931

Author(s):

Seyedehazita Ahmaditabatabaei ◽

Godfrey Kyazze ◽

Hafiz M. N. Iqbal ◽

Tajalli Keshavarz

Keyword(s):

Polyethylene Terephthalate ◽

Catalytic Mechanism ◽

Environmental Fate ◽

Plastic Waste ◽

Environmental Crisis ◽

Environmental Matrices ◽

Plastic Pollution ◽

Fungal Enzymes ◽

Bacterial Enzyme ◽

Human Health Effects

The ubiquitous persistence of plastic waste in diverse forms and different environmental matrices is one of the main challenges that modern societies are facing at present. The exponential utilization and recalcitrance of synthetic plastics, including polyethylene terephthalate (PET), results in their extensive accumulation, which is a significant threat to the ecosystem. The growing amount of plastic waste ending up in landfills and oceans is alarming due to its possible adverse effects on biota. Thus, there is an urgent need to mitigate plastic waste to tackle the environmental crisis of plastic pollution. With regards to PET, there is a plethora of literature on the transportation route, ingestion, environmental fate, amount, and the adverse ecological and human health effects. Several studies have described the deployment of various microbial enzymes with much focus on bacterial-enzyme mediated removal and remediation of PET. However, there is a lack of consolidated studies on the exploitation of fungal enzymes for PET degradation. Herein, an effort has been made to cover this literature gap by spotlighting the fungi and their unique enzymes, e.g., esterases, lipases, and cutinases. These fungal enzymes have emerged as candidates for the development of biocatalytic PET degradation processes. The first half of this review is focused on fungal biocatalysts involved in the degradation of PET. The latter half explains three main aspects: (1) catalytic mechanism of PET hydrolysis in the presence of cutinases as a model fungal enzyme, (2) limitations hindering enzymatic PET biodegradation, and (3) strategies for enhancement of enzymatic PET biodegradation.

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