Environment International 145 (2020) 106144

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Environment International

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Review article Recent advances in biocatalysts engineering for terephthalate plastic waste green recycling

Nadia A. Samak a,b,c,1, Yunpu Jia a,b,1, Moustafa M. Sharshar a,b, Tingzhen Mu a, Maohua Yang a, Sumit Peh a,b, Jianmin Xing a,b,* a CAS Key Laboratory of Green Process and Engineering & State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China b College of Chemical Engineering, University of Chinese Academy of Sciences, 19 A Yuquan Road, Beijing 100049, PR China c Processes Design and Development Department, Egyptian Petroleum Research Institute, Nasr City, 11727 Cairo, Egypt

ARTICLE INFO ABSTRACT

Handling Editor: Guo-ping Sheng The massive waste of poly(ethylene terephthalate) (PET) that ends up in the landfills and oceans and needs hundreds of years for degradation has attracted global concern. The poor stability and productivity of the Keywords: available PET biocatalysts hinder their industrial applications. Active PET biocatalysts can provide a promising Plastic waste avenue for PET bioconversion and recycling. Therefore, there is an urgent need to develop new strategies that Poly(ethylene terephthalate) could enhance the stability, catalytic activity, solubility, productivity, and re-usability of these PET biocatalysts Recycling under harsh conditions such as high temperatures, pH, and salinity. This has raised great attention in using Biocatalysts ’ Bioengineering bioengineering strategies to improve PET biocatalysts robustness and catalytic behavior. Herein, historical and forecasting data of plastic production and disposal were critically reviewed. Challenges facing the PET degra­ dation process and available strategies that could be used to solve them were critically highlighted and sum­ marized. In this review, we also discussed the recent progress in bioengineering approaches used for discovering new PET biocatalysts, elucidating the degradation mechanism, and improving the catalytic perfor­ mance, solubility, and productivity, critically assess their strength and weakness and highlighting the gaps of the available data. Discovery of more potential PET and studying their molecular mechanism extensively via solving their crystal structure will widen this research area to move forward the industrial application. A deeper knowledge of PET molecular and degradation mechanisms will give great insight into the future iden­ tification of related . The reported bioengineering strategies during this review could be used to reduce PET crystallinity and to increase the operational temperature of PET hydrolyzing enzymes.

1. Introduction: Plastic waste “A massive environmental crisis” concern, as noted in the Paris agreement on Climate Change, to decouple plastics production from fossil resources and to reduce greenhouse gas The massive production of plastics has been started since the year of emissions (Fig. 1a). Plastic materials, especially PET, provide economic 1950. Subsequently plastics have been massively utilized in different benefits as they are widely spread in our daily life, and its global pro­ sectors and started to be absolutely necessary for modern society duction is increasing rapidly due to the simple synthesis, low-priced (Thompson et al. 2009). Recently, the improvement of plastic design and production, robustness, and durability that are beneficial for the pack­ production to facilitate its re-use, repair, and recycling is the major aging industry (Lebreton and Andrady 2019). The world plastic

Abbreviations: PET, poly(ethylene terephthalate); PE, polyethylene; BHET, bis(2-hydroxyethyl) terephthalate; MHET, mono(2-hydroxyethyl) terephthalate; BA, benzoic acid; HEB, 2-hydroxyethyl benzoate ; PHAs, polyhydroxyalkanoates; EG, Ethylene glycol; TPA, ; PETG, polyethylene tere­ phthalate glycol; PCA, protocatechuate; DCD, 1,6-dihydroxycyclohexa-2,4-diene dicarboxylate; LCC, leaf-branch compost ; IsPETase, sakaiensis 201- F6 PETase; SEM, scanning electron microscopy; Tg, glass transition temperature; HPLC, high performance liquid chromatography; TPA-Na, disodium terephthalate; 3D, three-dimensional; GFP, green fluorescence protein; AP, alkaline ; PAA, polyacrylamide. * Corresponding author at: CAS Key Laboratory of Green Process and Engineering& State Key Laboratory of Biochemical Engineering, Institute of Process Engi­ neering, Chinese Academy of Sciences, 1, Zhongguancun Bei-er-tiao, Haidian District, Beijing 100190, PR China. E-mail address: [email protected] (J. Xing). 1 Both authors contributed equally to this work. https://doi.org/10.1016/j.envint.2020.106144 Received 25 June 2020; Received in revised form 10 September 2020; Accepted 13 September 2020 0160-4120/© 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/). N.A. Samak et al. Environment International 145 (2020) 106144 production is increased rapidly and expected to reach over 34 billion this waste could re-enter the economy as valuable goods to retain their metric tons in 2050 (Geyer et al. 2017). In Europe, the main applications economic value and reduce their waste. of the plastics industry are represented in the automation industry Herein, we critically review the bioprocessing of plastic degradation (~9.9%), packaging industry (~39.9%), construction (~19.8%), and biocatalysts, including the biotechnological processes that have been electronics (6.2%) (Facts 2019). Utilization of plastic in the packaging applied for enhancing plastic waste degradation, with a focus on the industry has risen a massive environmental problem due to the difficult most recent strategies for improving the production, catalytic activity, of many synthetic plastics such as polyethylene (PE), thermal stability, and re-usability of plastic degradation biocatalysts. poly(vinyl chloride), polystyrene, polypropylene, and PET which remain This could be performed through protein engineering intensification in the environment for hundred years (Eriksen et al., 2014; Lechner approaches such as mutagenesis strategies, the discovery of new PET et al., 2014). Besides, the plastic fabrication process consumes about 8% biocatalysts candidates, using native and alternative chassis for of the world’s fossil fuel (Andrady 2015). These environmental problems enhancing the biocatalysts production, incorporation of metal ions to became more critical because of the massive disposal of plastic wastes, stimulate enzymes activity, and enzyme immobilization. The trends and which negatively affects the ecosystems and marine life in rivers and challenges facing the PET biodegradation process were also summarized oceans (Chen et al., 2008; Seo and Park, 2020). Driven by the rapid in this review. We also detailed the biodegradation mechanism using urbanization and the rapid increase in population, the world accumu­ these biocatalysts, as the major hurdle in PET degradation is the initial lated plastic waste is predicted to reach 3.4 billion tons by the year of cleavage of the insoluble macromolecule into smaller ones to facilitate 2050, up from 360 million tons in 2018 (Kaza et al. 2018) (Fig. 1b). its uptake by microorganisms for further utilization. During the year of 2015, about 9% of plastic waste had been recycled, while 12% had been burnt, and 79% had been accumulated in landfills 2. Plastic waste: The search for persistence removal and (Geyer et al. 2017) (Fig. 1c). Landfilling is a less favoured alternative, recycling continues and currently, it is gradually phased out in Europe. Effective strategies for increasing the recycling rate of plastic waste need to be developed, so via chemical, mechanical, and biological processes

Fig. 1. a) Global plastic production, consumption, and end of life-fate. b) Historical data of plastic production from 2010 to 2018 according to the 2018 global plastic production report. C) Historical data and forecasting of cumulative plastic waste and disposal from 2010 to 2050, updated from (Geyer et al. 2017).

2 N.A. Samak et al. Environment International 145 (2020) 106144 are the main conventional methods used until now (Table 1). Elimina­ environment (Azapagic et al. 2003). The recycling sites are commonly tion of plastics in a landfill is the easiest and common way followed in unhealthy as they are the main source for spreading infectious diseases the world, but it is hazardous and has many disadvantages due to the and toxic volatile organic compounds that harm plant and animal life slow degradation rate of plastic wastes because of the anaerobic con­ (d’Ambri`eres 2019). ditions surrounding the landfill. Furthermore, UV and solar radiation Environment-friendly recycling strategies for solving the ubiquitous have no effect on the plastic degradation process (Abdel-Shafy and pollution of the environment with plastic waste are in high demand. Mansour, 2018; Cleary, 2014; Qasaimeh et al., 2016). These causes Biodegradation of plastic waste using microorganisms is a cost- plastic wastes aggregation in landfills, rivers, oceans, and terrestrial effective eco-friendly strategy that could be applied through microor­ environments and contaminate the groundwater (Gewert et al. 2015). ganisms’ cultivation or via their enzymes extraction (Restrepo-Florez´ This pollution has a lethal effect on marine animals via ingestion or et al. 2014). Herein, the enzymatic activity of these enzymes catalyzes being trapped in plastic debris (Nelms et al., 2016; Wilcox et al., 2015). the bond cleavage into . While the hydrophilicity of Countries that suffer from the scarcity of land such as Japan head for plastic surface allows microorganisms to form biofilmsover their surface using plastic incineration as an energy source though this process is and use them as a carbon and energy source, therefore, the degradation usually harmful to the environment due to the liberated toxic materials process will be enhanced (Kale et al. 2015). Aliphatic showed such as furans and dioxins (Li et al. 2001). Besides, the cleaning of high biodegradation efficiency when compared with the aromatic one liberated gases from the incineration process is usually challenging (Cerda-Cu` ´ellar et al. 2004). Intriguingly, a synergistic chemo-enzymatic (Burnley et al., 2015; Levchik and Weil, 2004). hydrolysis process for PET depolymerization, was recently reported Plastic recycling, converting plastics waste into reusable materials, is (Quartinello et al. 2017). Firstly, the chemical pretreatment was con­ an alternative method to overcome plastics waste contamination. Plastic ducted in an eco-friendly way without using harsh chemicals and pro­ recycling using the mechanical process is an economical standard duced powder composed of 85% terephthalic acid (TPA) under neutral ◦ method that usually does not change the basic structure of the material conditions (250 C and 39 bar). After that, the remaining oligomers were and can be performed in two stages. Plastic wastes during mechanical furtherly hydrolyzed enzymatically via cutinase producing TPA with recycling have to be sorted first,shredded, melted, then granulated, and high purity of 97%. Subsequently, more research investigations need to used with the virgin plastic for industry (Grigore, 2017; Ragaert et al., be achieved in this field. Recently, biodegradable plastics with a short 2017). The primary stage of this recycling process returns the plastic to lifespan in the environment have been developed. This kind of plastic its original purpose, and the cleanest waste will be chosen for this stage, called “oxo-degradable plastics” because they contain some additives such as the PET bottles. In the second stage, the low molecular weight that accelerate the oxidation process, but unfortunately, they end up recycled PET is utilized for fiber production. The high-density poly­ with a rapid fragmentation into massive amounts of micro plastics upon ethylene, low-density polyethylene, and PET are processed in the second their exposure to the sunlight and oxygen (Ammala et al., 2011; Kubo­ stage via several melt and remould cycles. One of the main disadvan­ wicz and Booth, 2017). The produced micro plastics take a long time for tages of mechanical recycling is that it is limited to the monolayer complete biodegradation, and the environmental problem will remain plastics than the multilayer one. Furthermore, temperature-sensitive (Chae et al., 2019; Ruan et al., 2018). plastics cannot be handled mechanically (Scalenghe 2018). Chemical recycling is usually performed via alkaline, acidic, and neutral ap­ 3. Production of PET biocatalysts proaches for plastic de-polymerization to its monomers or other useful products using hydrolysis, glycolysis, and methanolysis. This chemical 3.1. Potential native chassis for PET biocatalysts production process needs selective and active catalysts that are costly and energy- consuming and require high temperatures to give a product mixture PET is aromatic polyester, and its degradation difficultyowes to the that is difficult to be separated which limits its industrial applications presence of the non-hydrolyzable covalent bonds that have a subunit (Garcia and Robertson 2017). Although mechanical and chemical substrate called diethylene glycol terephthalate (Kawai et al. 2019). recycling methods are widely used, they are very risky for the Ethylene glycol (EG) and TPA are the main building units of PET.

Table 1 Comparison table showing the differences between PET degradation methods.

Method Mechanical degradation Chemical degradation Biodegradation References

Process • Re-use of plastic products Plastic de-polymerization to Plastic polymer bond cleavage into (Grigore 2017) requirement without structure alteration monomers via hydrolysis, glycolysis, monomers via enzymes produced by • Re-melting and re-processing and methanolysis microorganisms of thermoplastic Temperature High High Not required (Ragaert et al. 2017) Degradation rate Fast Fast Moderate (Carta et al. 2003) Degradation Applicable Difficult Applicable (Grigore, 2017; Ragaert et al., Product 2017) separation Advantages • Commonly used • Simple method • Environmentally friendly (Fatima, 2014; Hopewell et al., • Effective method • Operational for PET • 3 Kg enzyme required for 1 ton PET 2009; Paszun and Spychaj, 1997; • Cost-effective (~250 €/ton) • cost ~500 €/ton degradation (cost ~63€) Tournier et al., 2020) • Byproducts could be used for different applications Disadvantages • Environmentally unfriendly • Environmentally unfriendly • Time consuming when compared (Al-Sabagh et al., 2016; • Source of infectious diseases • Source of infectious diseases and with chemical and mechanical Karayannidis and Achilias, 2007; and toxic volatile organic toxic volatile organic compounds methods Paszun and Spychaj, 1997) compounds • Costly & energy consuming • Deterioration of product • Uncommonly used characteristics • Limited to condensed polymers • limited to monolayer plastics • multilayer and temperature sensitive plastics are not applicable

3 N.A. Samak et al. Environment International 145 (2020) 106144

Plastics biodegradation is a quite slow process and easily affected by degradation rate (Welzel et al. 2002). Bacterial are more effective temperature, pH, humidity, and ultraviolet rays. After that, microor­ in the bioconversion of PET to the intermediate product MHET by 50- ganisms will be adopted for the complete degradation of the remaining fold higher when compared with the fungal one which needs the addi­ plastics. Biodegradation of PET usually causes shortening of the polymer tional presence of plasticizers (Eberl et al. 2009). A consortium chain, which subsequently reduces the molecular weight of the polymer. composed of three Pseudomonas spp. and two Bacillus spp. were reported ◦ After that, oligomers, dimers, water-soluble monomers will be produced for effective PET degradation at 30 C after six weeks incubation (Leon-´ and can pass the microbial cell membrane to be used as a carbon source. Zayas et al. 2019). PET crystallinity, high molecular weight, strong C-C bonds, and Leaf-branch compost (LCC) are lipolytic esterolytic en­ extremely hydrophobic surface play an essential role in the biodegra­ zymes which belonging to the lipases family, but the two enzyme fam­ dation process. Crystallites act as barriers to moisture diffusion (Calleja ilies are showing different catalytic behavior toward PET degradation. et al. 1994). Generally, amorphous polymers with a linear structure can Cutinases have at their catalytic triad which is not buried under be degraded faster than crystalline polymers with branched structure an amphipathic loop unlike lipases (Longhi and Cambillau 1999). (Mierzwa-Hersztek et al. 2019). Recently, some fungal and bacterial Cutinases are more active toward the soluble and emulsified substrates strains have been discovered for PET biodegradation into low molecular (Sulaiman et al. 2012). and fungi are the major sources of weight oligomers or monomers such as Bis(2-hydroxyethyl) tere­ cutinases. It has been demonstrated that cutinases from Aspergilllus phthalate (BHET) and mono(2-hydroxyethyl) terephthalate (MHET) oryzae, Aspergilllus nidulans (Bermúdez-García et al. 2017), Penicillium (Wei et al. 2016). Saprophytic scavengers are bacteria having the ability citrinum (Liebminger et al. 2007), Humicola insolens (Ronkvist et al. to remineralize wastes’ organic carbon and hydrocarbons that are 2009), Thermobifida fusca (Brueckner et al. 2008), considered the building blocks of plastics. Actinobacteria, Gram-positive (Alisch-Mark et al. 2006), F. solani pisi (Vertommen et al. 2005), and phylum, was reported as a promising bacterial strain for whole-cell Thermobifidacellulolysitica (Herrero Acero et al., 2011) have hydrolyzing of PET, especially the genera Thermomonospora and Thermobi­ activity toward low-crystallinity PET. Thielavia terrestris was reported for fida( Herrero Acero et al., 2011; Ribitsch et al., 2012a). PET degradation TtcutA cutinase production with a molecular weight of 25.3 kDa, ◦ based on whole-cell biocatalyst has excellent advantages over utilizing showed optimal activity at pH 4 and 50 C, and effectively degraded PET the free enzymes by reducing the degradation process time and material- and polycaprolactone with a hydrolytic rate of 1.1 mg.h 1mg 1 protein intensive protein purification process (de Carvalho 2017). Comamonas and 203.6 mg.h 1mg 1 protein, respectively (Yang et al. 2013). Cuti­ testosteroni F4 and alkali-resistant C. testosteroni F6 were used as whole- nase from Humicola insolens is more active toward PET films than cuti­ cell catalysis for micro sized-PET degradation (Gong et al. 2018). The nase produced from Thermobifidacellulolysitica. Crystallinity in the PET alkali-resistant C. testosteroni F6 showed better degradation of micro films of 10 and 20% sharply decreased the enzymatic hydrolysis effi­ ◦ sized-PET at high alkaline condition (pH 12) after 48 h at 37 C. The ciency. This drop in enzymatic hydrolysis was not observed when the degradation products using both strains were composed of BHET, same enzyme was used for poly(ethylene furanoate) due to its less MHET, TPA, and methyl acrylate (MA). crystallinity (Weinberger et al. 2017). Despite that, the enzymatic hy­ Yoshida et al, 2016 (Yoshida et al. 2016) discovered a bacterial strain drolysis of PET oligomers is much faster than the long-chain polymers called that belongs to the genus Ideonella and the (Ribitsch et al. 2011). Besides, it has been reported that cutinase from family . This bacterium showed effective biodegrada­ Pseudomonas mendocina and Fusarium solani showed about 10-fold ac­ tion of PET polyester, which was extracted from the plastic recycling tivity on low crystalline PET (7% crystallinity) than on higher crystalline plant in Sakai, Japan, and used the PET as a sole carbon source. PET (35%) (Donelli et al. 2009). I. sakaiensis secretes two enzymes, PETase, and MHETase enzymes, that Recently, it has been reported that some native microorganisms are involved in the conversion of PET polyester into simple and non- producing PET biocatalysts could be found in invertebrates such as in­ harmful recyclable monomers. PETase first hydrolyzed the PET poly­ sects, which can hydrolyze PET via mechanical grinding and mer into MHET that was furtherly hydrolyzed into TPA and EG. These shredding (Yang et al., 2014, 2015b). Thus will produce smaller plastic finalproducts were furtherly utilized by the bacterium as a food source pieces that will increase the surface area and allow better attachment of (Austin et al. 2018). PETase from I. sakaiensis whole-cell catalysis the microorganisms with the surface. The high degradation rate of PE by showed 5.5, 88, 120 fold higher hydrolyzing activity toward PET poly­ wax moth’s larvae, , was reported via producing holes when compared with low-crystallinity cutinase, Fusarium solani in PE film( Bombelli et al. 2017). The genera Citrobacter was found to be cutinase, and Thermobifida fusca at low temperature (Müller responsible for PE degradation in Tenebrio molitor’s gut (Brandon et al. et al. 2005). 2018). While Bacillus sp. Strain YP1 was found to be the responsible is an enzyme family that can cleave the ester bond (short bacterium for PE degradation in mealworms (Yang et al. 2015a). In chain acyl ester) found in PET monomers with observed surface modi­ 2011, Shen et al. (Shen 2011) made a preliminary plastic degrada­ fication.Bacillus and Nocardia were reported for the initial degradation tion study on the mechanism of mealworms, and isolated 8 strains of of PET via (Sharon and Sharon 2012). Esterase from Thermo­ bacteria with degradation characteristics from the intestinal tract of bifidaThh_Est showed effective surface hydrolysis for PET polyester, and mealworms. In 2017, zhang et al. (Yang et al. 2018) raised yellow its effect was similar to cutinases from the same genus (Ribitsch et al., mealworms with the plastic film A (30% starch, 70% PE and auxiliary) 2012a). and B (more than 95% PE and A small amount of auxiliary) as the only Lipases are widely known for their catalytic hydrolysis of the long- food, and yellow mealworms could completely degrade both plastic chain (more than C10) water-insoluble triglycerides, and they are char­ films. The above studies showed that there were PE degrading bacteria acterized by the interfacial activation phenomenon. Lipases were re­ in the intestinal tract of insects, which provided a new way for the ported for degradation of PET fabrics to some extent through enhancing biodegradation of PE. In the oligotrophic deep-sea environment, mi­ their wettability, dye-ability, and absorbent characteristics (Gupta et al. croorganisms in the intestines of marine molluscs, that can degrade 2015). Different organisms have been reported for lipases production as plastics, attracted a great concern. More discoveries of invertebrate PET whole-cell catalyst such as Thermomyces lanuginosus (Eberl et al. species will help in plastic waste reduction through the ingestion and 2009), Candida Antarctica (Vertommen et al. 2005), Triticum aestivum, degradation of plastics in their guts via enzymes. Even though the dis­ and Burkholderia spp. (Nechwatal et al. 2006). Lipases showed a sig­ covery of native strains, the findingof a robust strain that can replicate nificant higher hydrolysis rate toward aliphatic polyester nanoparticles and produce PET biocatalysts in high yield under harsh conditions, such (100 nm) than the polyester biofilm, and the same effect was also as high temperature and pH, is challenging. Therefore, the over­ observed for the aromatic one (Müller et al. 2005). It is supposed that the production of these enzymes via dynamic alternative chassis would be low crystallinity of the polyester nanoparticles is the reason for the high an effective and promising way to promote the large-scale

4 N.A. Samak et al. Environment International 145 (2020) 106144 biodegradation process of PET polyester. crystallinity polyethylene terephthalate glycol (PETG) when compared ◦ with the bottle-grade PET at 30 C. Furthermore; the expressed PETase 3.2. Potential alternative chassis for PET biocatalysts production was active toward shredded pieces of PET at the high saline condition ◦ and mesophilic temperatures, 21 C resulting in a hydrolysate mainly 3.2.1. Escherichia coli consists of TPA and MHET (Moog et al. 2019). The green algae, Chla­ Recently, the use of model organisms for the overexpression of in­ mydomonas reinhardtii CC-124, was recently reported for the effective dustrial enzymes has shown an effective and economical outcome. E. coli heterologous production of PETase. The produced PETase was able to ◦ is the most desired fermentative microorganism for industrial bio­ degrade PET filmat 30 C after incubation of 4 weeks (Kim et al. 2020). catalysts production on a large scale due to its easy handling and genetic The main advantages of microalgae for biocatalyst production can be manipulation (Olajuyin et al. 2016). Recombinant protein production summarized as follows: (1) the phototrophic growth in photo- using E. coli imparts a metabolic burden on the microorganism, and this bioreactors, which hinders the transgenes from releasing into the envi­ decrease the duplication time, within 24 h, with a high cell density ronment (Gong et al. 2011). (2) Expression of the recombinant bio­ (Samak et al. 2018a). Besides, E. coli growth condition requires inex­ catalysts through the chloroplast genomes (Lauersen et al. 2013a). (3) pensive, available components and the target DNA transformation pro­ The ability to perform post-translational modifications, which lead to tocol is very fast and handy (Rosano and Ceccarelli 2014). In the last the production of glycosylated biocatalysts extracellularly (Lauersen four years, E. coli was used extensively for PET biocatalyst production, et al. 2013b). (4) Robust cell duplication in a short time, within 24 h, and which helped in solving the crystal structure of these enzymes such as can reach to 3 h as in the case of Chlorella sorokiniana (Mayfield et al. PETase, MHETase, and LCC and provided great insight about the 2007). To date, there is a big gap in using these advantages of micro­ degradation mechanism and substrate affinity toward PET (Almeida algae for PET biocatalysts production. Using all the available data tools et al., 2019; Fecker et al., 2018; Han et al., 2017; Joo et al., 2018; of microalgae bioengineering will open the field toward cost-effective Sowdhamini et al., 1989; Tournier et al., 2020). Two esterases encoding production of PET biocatalyst with catalytic performance enhancement. genes amplifiedfrom Clostridium botulinum ATCC3502 and expressed in ◦ E. coli were able to hydrolyze PET at 50 C, and their catalytic activity 3.2.4. Clostridium thermocellum was furtherly improved at same temperature via mutation introduction C. thermocellum has attracted considerable attention in recent years in the zinc-binding domain (Perz et al. 2016). Table 2 summarizes all as a heterologous expression chassis due to its thermophilic and anaer­ PET biocatalysts heterologously expressed in E. coli. obic characteristics even though the genetic manipulation difficulties. ◦ E. coli can also be used for whole-cell catalysis by expressing PETase The optimal growth conditions of C. thermocellum can reach 60 C. extracellularly via secretion systems. Sec-dependent and SRP-dependent Scientists put great efforts to facilitate the genetic manipulation of signal peptides were fused with IsPETase and heterologously expressed C. thermocellum and avoid gene disruption by developing efficient from E. coli using pET22b-SPMalE:IsPETase and pET22b-SPLamB:IsPETase transformation tools for this bacterium. A transformation protocol for expression systems. The extracellularly secreted IsPETase successfully C. thermocellum via electroporation has been developed and enhanced ◦ degraded the PET film after 72 h at 30 C and the degradation process the transformation efficiency (Olson and Lynd 2012). This allowed can be directly applied in the culture medium (Seo et al. 2019). C. thermocellum to be utilized as a whole-cell biocatalyst in biofuel production and lignocellulose saccharification. (Yan et al. 2020) used 3.2.2. Pichia pastoris C. thermocellum as a microbial chassis for the extracellular expression of ◦ The eukaryotic microorganism P. pastoris, a methylotrophic , is LCC enzyme to degrade PET films at 60 C. This study showed that the characterized by its easy genetic manipulation and high cell density expressed LCC enzyme converted 60% of PET film into soluble mono­ growth. P. pastoris is very robust in the production of soluble and mers after 14 days incubation. An important direction for future correctly folded recombinant proteins. Furthermore, the cloning pro­ expression of PET biocatalyst using C. thermocellum is desperately cedure of the target DNA gene depends on the linearization of the DNA, needed. followed by its transformation into P. pastoris via homologous recom­ bination. This process facilitates the replication of the target gene and 3.3. Biocatalysts for the recycling of PET hydrolysates allows its overexpression. Expression of PET biocatalysts via P. pastoris provides a dynamic growth rate with high cell density, and facilitate the TPA and EG, the main constituent monomers of PET, are produced as purification process of the produced biocatalyst from the growth me­ the main degradation products of PET with esterases. Microorganisms dium directly due to the extracellular expression. P. pastoris also have having the metabolic pathway for these compounds could degrade these the ability to perform post-translational modificationssuch as acylation, monomers. TPA could be degraded to protocatechuate (PCA) by mi­ glycosylation, and methylation. Intriguingly, the expression of recom­ croorganisms pathway encoded by the tph enzymes. First, TPA dioxy­ binant LCC in P. pastoris leads to the production of correctly-folded genase TphA1A2A3 converts TPA to 1,6-dihydroxycyclohexa-2,4-diene ◦ glycosylated LCC that showed a higher thermostability by 10 C when dicarboxylate (DCD). After that, DCD will be converted to PCA by the compared with the native one (Shirke et al. 2018). Glycosylated Ther­ action of 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate dehy­ mobifida cellulosilytica cutinase, Thc_Cut1, mutant strains expressed in drogenase TphB (Salvador et al. 2019). These biocatalysts were found in P. pastoris showed effective degradation of poly(butylene succinate) and β- Comamonas testosteroni YZW-D, actinomycete Rhodo­ poly(3-hydroxybutyrate-co-3- hydroxyvalerate) within 96 h (Gamerith coccus sp. strain DK17, and Comamonas sp. strain E6 (Choi et al., 2005; et al. 2017). Sasoh et al., 2006; Wang et al., 1995). Acetogens, organisms that utilize the acetyl-CoA pathway to synthesize acetate, can oxidize EG to ethanol 3.2.3. Microalgae and acetaldehyde that will be converted after that to acetate via acetyl- Microalgae is a promising potential economic cell factory for PET CoA like in Acetobacterium woodii (Trifunovi´c et al. 2016). Microor­ biocatalyst production. Recently, algal genome data and transformation ganisms such as Pseudomonas aeruginosa and Pseudomonas putida can protocols became widely available with rapid development of their ge­ convert EG to glyoxylate via the action of the periplasmic alcohol de­ netic tools, and this can facilitate their usage for biocatalysts production hydrogenase funneled directly to the krebs cycle via isocitrate or via enhancement. Over 40 different microalga strains such as P. tricornutum propanediol oxidoreductase in case of E. coli (Luo and Lee, 2017; and C. reinhardtii became very easy and successful for genetic manipu­ Mückschel et al., 2012). It has been reported that most organisms that lation so far (Gangl et al. 2015). Recently, the photosynthetic microalga have the ability to degrade TPA are also able to degrade EG, which P. tricornutum was used as chassis for PETase extracellular expression means that effective usage of PET hydrolysis products could be per­ and the expressed PETase showed 80-fold higher turnover of low formed using the same microorganism. Recently, the bioconversion of

5 N.A. Samak et al. Environment International 145 (2020) 106144 ) ) and al. al. al. al., al., al., ) ) ) ) et al., et 2014 2014 al. al. et et et et Acero Acero Acero al. al. et al., et al. al. et Yoshida et ) ) 2016 2011 2011 2011 et ) et et et ) ) ) ) ) ) ) ) products al., al., al., al., Ribitsch Thumarat Kawai Wei Han Wei Sulaiman Herrero Herrero Ribitsch Ribitsch Silva Müller Ribitsch Herrero Roth Biundo ( 2011 ( 2012 ( 2014 ( Ref. ( 2017; et ( ( 2012 ( et ( et ( 2012b ( 2012b ( 2011 ( 2005 ( 2012a ( et ( 2014 ( 2018 ) h M ± ± μ m C 1/2 K 24 ◦ degradation t its (pH, 88.8 83.1 100 C, of 60 for ◦ 55 ± M C mM at µ mM mM ◦ at 60% 86.2 mM 65 : (pNPB): (pNPB): 2.10 213 2500 – min substrate, 50 (pNPB): stable lost m m 1/2 min h stable 50 50 T K K 2.10 60 parameters m h at K at at 60 48 M, 1 mM, μ mM, thermostability, 8.5, 6, 8.5, 8.5, (85 = (pNPB):1.48 (pNPB): (pNPB):1.93 (pNPB): (pNPB): , (MW), for after for ) m m m m m m 1/2 3.41 ℃ ND 12.8 11.1 activity ℃ K ND ND Stable pH: ℃ ND ND ND K K K K pH: Stable ℃ t pH: (pNPB): pH: Kinetic K weight ) m /K molecular cat 2 2 2 6 8 4 C C ND C ND C C ND ND ND ND ND ND ND C ND ND pNP-Ester Specificity (k code, ) ℃ ℃ (PDB) bank 45 70 65 70 45 – – – – – 50 20 50 50 50 50 50 55 65 65 60 50 55 ND 50 50 40 Degradation temperature( data EG EG protein HEB, HEB, MHET indicated. TPA, BA, BA, TPA TPA TPA, MHET BA, HEB BA, BA, EG MHET HEB MHET, BA, HEB code, were TPA TPA, MHET, BA, TPA, HEB, MHET, TPA, TPA, MHET TPA, MHET TPA, ND TPA, ND ND MHET, TPA, MHET, MHET, MHET, TPA, Degradation product bank (pNPB) ct.) ct.) PET gene ct.) PET PET ’ PET PET ct), ct.) (37% (37% butyrate (7% (1.9% ct.) used film (35% film film nanospheres nanospheres film bottle-grade PET lcPET and hcPET PET lcPET PET PET 3PET 3PET Bottle-grade (10% Bottle-grade amorphous package-grade PET PET Amorphous film LcPET Nanoparticles lcPET Nanoparticles PET 3PET PET Biocatalysts p-nitrophenyl sources. 30 28.6 29.4 29.6 28.1 ND ND 28.3 30.3 35 35 30.8 28 ND ND MW (kDa) toward terephthalate. microbial 4CG3 their 4CG2, code parameters and EB0 4OYY 3VIS 5XG0,5XFY, 5XFZ,5XH2, 5XH3,5XJH, 5YNS6ANE 5LUI 4CG3 ND ND ND 5ZOA 4WFI ND 4CG1, 4 5AH1 ND ND PDB coli kinetic E. in bis(benzoyloxyethyl) and code 3PET, , expressed BAK48590.1 GAP38373.1 ADV92526.1 ADV92528.1 ADV92525.1 AFA45122.1 WP_011291330.1 AJ810119.1 AB728484 ACY96861.1 ACY95991.1 CBY05529.1 CBY05530.1 HQ704839 KP859619 ADH43200.1 Genbank PET; KW3 KW3 p-nitrophenyl 4P3-11 alba fusca alba fusca fusca crystallinity fusca fusca heterologously DSM44931 botulinum source insolens sakaiensis subtilis DSM43183 DSM43183 toward high AHK190 Thermobifida AHK119 Ideonella Thermobifida cellulosilytica DSM44535 Humicola Thermobifida DSM44342 Thermobifida DSM43185 Thermobifida halotolerans Thermobifida DSM43793 Thermobifida DSM43793 Saccharomonospora viridis Thermobifida Thermobifida Leaf-branch compost metagenome Thermomonospora curvata Thermomonospora curvata Clostridium ATCC3502 Bacillus Microbial biocatalysts hcPET, specificity PET of 2 cutinase crystallinity; Est119 sakaiensis PETase Cut190 cutinase Ideonella Thc_Cut1 HiC Thf42_cut1 Tha_Cut1 Thh_Est Tfh BTA-1 cutinase TfCut1 TfCut2 LCC Tcur1278 Tcur0390 Cbotu_EstA BsEstB Enzyme Table Summary Ct., temperatures,

6 N.A. Samak et al. Environment International 145 (2020) 106144 re-cycled PET monomers to value-added bioplastic poly­ its use in the industrial applications such as of polluted hydroxyalkanoate by P. putida KT2440 was demonstrated (Wierckx et al. sites, degradation, and recycling of plastics waste. This could be ach­ 2015). This opens the field toward the production of bio-plastic and ieved by enhancing enzymes expression level, increasing the Kcat of the other value-added products from PET’s enzymatic hydrolysates. enzyme, discovering more biosynthetic pathways and reaction mecha­ nisms. In fact, this is not an easy task as the low expression level; poor 4. Challenges in PET biodegradation process solubility and thermal stability are the main properties of the heterol­ ogously expressed enzymes. Furthermore, the synthetic gene circuit One of the main bottlenecks in PET biodegradation is the hydro­ components and the genomic context need to be optimized and modi­ phobic force between the PET surface and substrate which hinders this fied, respectively. Mutagenesis strategies (i.e. random mutagenesis and process (Kawai et al. 2019). Therefore, surface hydrophilization of PET site-directed mutagenesis), genome editing, simultaneous amino acid is a significant process required for enzymatic PET degradation. substitutions, computational genomics, structural biology and directed Enhancement of rapid degradation rate, anti-pilling behavior, wetta­ evolution are the recent protein engineering strategies that have been bility, and dyeability could be performed via PET surface modification. adopted to address a wide range of enzyme engineering. Artificial The ends of polymer chains on the PET surface are expected to protrude, scaffolding is another promising tool for enhancing the catalytic activity or part of it may form a loop (Oda et al. 2018). Surface hydrophilicity of enzymes by creating enzyme clustering and substrate channels. could be increased through the hydrolysis of these loops to carboxylic Furthermore, proteins fusion is essential for enhancing the catalytic acid and hydroxyl residues. PET hydrolyzing enzymes could be divided activity of PET biocatalysts. Herein, we will critically discuss furtherly into PET hydrolases and PET surface modifying enzymes (Müller et al. how protein engineering tools could enhance plastic degradation en­ 2005). PET inner block can be significantlydegraded via PET hydrolases zymes expression, catalytic activity, and thermostability (Fig. 2). with a definite change using Scanning Electron Microscopy (SEM). While surface hydrophilization level of PET using PET surface modifying 5.2. Experimental approaches for PET biocatalysts candidate discovery enzymes cannot be observed by SEM. In brief, PET biocatalysts can be used for surface degradation while the PET building block cannot be The discovery of enzyme candidates and microorganisms with PET significantlydegraded via PET surface modifying enzymes (Kawai et al. biodegradation abilities and strategies for developing an efficientsystem 2019). to enhance the enzyme’s catalytic activity is in need. The PET bottle- Crystallinity, hydrophobicity, PET molecular weight, the flexibility recycling factories and landfills are enriched sites with PET polymers of the polymer chain, surface topography, and hydrolysis reaction and could be a primary source for the discovery of PET degrading mi­ temperature are considered the most aspects affecting PET degradation croorganisms. Applying microbial biotechnology strategies are prom­ (Tokiwa et al., 2009; Wei and Zimmermann, 2017). The stiffness of PET, ising for identifying novel PET biocatalysts from microbial biodiversity- produced by the aromatic TPA building blocks, is a primary reason of rich environments. Herein, the microbial communities that utilize the PET low biodegradability. The glass transition temperature value (Tg) of PET as the main carbon source will stimulate the microbial cell to ex­ ◦ PET at which polymer chain can dissociate is about 80 C (Kikkawa et al. press enzymes for PET degradation and reaction intermediate hydroly­ 2004). The Tg value of PET during the enzymatic hydrolysis reaction is sis. Whole-genome analysis, RNA-Seq analysis, heterologous expression ◦ about 65 C (Kawai et al. 2014) due to the involvement of water mol­ of hypothesized genes, phylogenetic analysis, protein purification, and ecules between polymer chains; this will cause flexible, random, and identification are the main approaches used to discover the enzymes enzyme accessible polymer chains. Therefore, it is believed that faster responsible for PET biodegradation. The PET degradation process can PET degradation rates could be performed by increasing the tempera­ then be monitored using SEM and Reverse-phase high-performance tures of the enzymatic hydrolysis reaction (Oda et al. 2018). For the liquid chromatography (HPLC). If successful, the identified microor­ previous reasons, strategies for producing thermos-stable PET hydro­ ganisms could be promising candidates for a future sustainable closed- lyzing enzymes are required. Furthermore, the minimal reactive C-C loop PET waste recycling. bonds in the backbone of polyesters are considered as one of the major Yoshida et al. (Yoshida et al. 2016) collected two hundred fifty obstacles of the biodegradation process. Recently, many PET hydrolyz­ environmental specimens from plastic bottles recycling site. The ing enzymes have been discovered, but their ability to utilize PET research team screened the microbial communities from the enriched monomers as a carbon source for the microbial microorganisms had not samples using PET film as a major carbon source. SEM showed been approved. morphological changes in PET filmafter twenty days with the microbial Improving PET surface properties via its functionalization using PET communities isolated from the environmental samples. Idonella biocatalysts is very promising for plastic waste management. These sakaiensis 201-F6 was the responsible microorganism for degrading and could be achieved by introducing functional groups to PET surface in assimilating PET. Transcriptomic analysis based on homologous en­ order to increase its hydrophilicity and subsequently can be used for zymes to identify the responsible enzymes of PET degradation was adhesion with specific materials or covalent binding with specific mol­ performed by growing I. sakaiensis 201-F6 on different substrates such as ecules. Single and double T. fusca cutinases were used for surface hy­ disodium terephthalate (TPA-Na), BHET, PET film, or maltose. TPA drolysis of PET fabrics. One of these mutants created ample space catabolic genes were efficientlyexpressed when the substrates, TPA-Na, around the of cutinase while the other mutant increased the BHET, or PET film, were used in the culture medium, separately. hydrophobicity around the binding site with improvement in the sub­ However, the highest transcriptional level was observed for PETase strate attachment. This PET modification enhanced the degradation encoding gene when PET film used in the culture medium. rate, and the TPA concentration was elevated in the reaction medium The discovery of PETase produced by Ideonella sakaiensis 201-F6 when compared with the activity of the wild type (Silva et al. 2011). (IsPETase) (Yoshida et al. 2016) encourages scientists to search for more potential homologs genes due to its efficientand environmentally 5. Bioengineering intensification strategies for improving friendly characteristics. Almeida et al, (Almeida et al. 2019) developed production and catalytic activity of PET biocatalysts an in silico-based screening approach to search for PETase homologs among some terrestrial and marine isolates. Three potential PETase-like 5.1. Protein engineering-Based PET biocatalysts improvement enzymes from the marine-sponge associated Streptomyces isolates were successfully identified. The amino acid and biochemical properties Protein engineering is playing an essential role in enhancing and similarities between SM14est-PETase and IsPETase were 41% and 19%, constructing biological systems using modern biological tools to in­ respectively. The three-dimensional (3D) structure of SM14est-PETase crease our knowledge in basic life sciences that consequently will widen was compared with IsPETase. This revealed that IsPETase has a

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Fig. 2. Scheme of the workflow of bioengineering approaches role in the discovery, degradation mech­ anism elucidation and catalytic properties improve­ ment of PET degrading enzymes. PET biocatalysts’ discovery depends on the clustering analysis of genes isolated from enriched PET cultures, rational design, and the heterologous expression of these genes for enhanced production and purification steps. The degradation mechanism elucidation includes directing different mutations to target biocatalyst gene, protein crystallization, and molecular docking. Mutagenesis, insertion of DisulfideBridge or metal ions, and protein fusion are well developed to enhance biocatalysts’ catalytic activity. Direct enzyme immobilization or whole-cell immobilization is necessary for biocatalyst re-usability and its cost reduction.

Fig. 3. Schematic diagram showing complete PET degradation mechanism using PETase and MHETase enzymes. PET interacts with the active site of the biocatalyst via PET’s carbonyl group and its aromatic ring that stacks with tryptophan residue of the biocatalyst. This interaction causes the release of TPA, BHET, and MHET. After that, MHET can be hydrolyzed by the MHETase enzyme giving EG and TPA hydrolysates.

8 N.A. Samak et al. Environment International 145 (2020) 106144 disulfidebond between Cys273 and Cys289 and another disulfidebond As mentioned before, the PETase enzyme is characterized by the between Cys203 and Cys239, which is responsible for the enzyme ac­ existence of Trp 185 adjacent to the active site, and this adopts the B tivity because it is very adjacent to the enzyme’s active site (Liu et al. conformation of the biocatalyst as Trp 185 movement opens the active 2018), while SM14est-PETase did not have any of them. These disulfide site cleft (Austin et al. 2018). The serine residue in PETase was replaced bonds are very characteristic for IsPETase and could not be observed at with histidine residue in other PET biocatalysts, causing block for the other PETase-like cutinases, which already showing high thermosta­ tryptophan residue in the C conformation. The replacement of the native bility and catalytic activity without those bonds (Kawai et al. 2014). serine to a histidine residue in PETase confirmedthe importance of this Conversely, the SM14est-PETase and IsPETase sharing a similar mode of residue in this enzyme’s catalytic activity as the histidine variant action toward plastic as their substrate. showed a significant loss of the activity (Austin et al., 2018; Han et al., 2017). Therefore, residues that are not directly attached to the substrate 5.3. Mutagenesis: An effective approach for degradation mechanism could also play a crucial role in enzyme catalysis, such as S185 (Chen elucidation of newly discovered PET biocatalysts et al. 2018).

The discovery of PET hydrolyzing enzymes recently draw re­ 5.4. Mutagenesis: An effective approach for dynamic and thermostable searchers’ interest in establishing effective strategies for enhancing the PET biocatalysts production catalytic activity and thermostability of these biocatalysts. The strong binding between the active site of PET hydrolyzing enzymes that have a Enzyme design is a powerful tool depend on the structural similarity substrate-binding cleft, and PET surface using its flat hydrophobic sur­ between enzymes to take the favorable characteristics from one enzyme face is the crucial point for effective PET biodegradation. IsPETase fol­ to another using site-directed mutagenesis. Mutagenesis has several lows the same mechanism of cutinases for PET degradation. However, effective approaches for enhancing the production, thermostability, and the difference comes from the presence of Trp185 residue near the catalytic activity of PET hydrolyzing enzymes. Directing specific muta­ catalytic triad, which facilitates the binding of the enzyme with PET and tion or new catalytic residues at the catalytic active site of enzymes or the release of the degradation product. The π–staking force of Trp 185 in replacing their divalent metal binding site with a disulfide bridge can B conformation stabilizes the hydrophobic interaction between PETase alter the enzyme mechanism and function. Furthermore, this process and PET surface. The degradation process occurs in two steps, nick became very handy due to the availability of computer-aided enzyme generation step and final digestion step (Fig. 3). During the reaction of engineering. Solving the crystal structures of LC cutinases (LCC), Tf the enzyme with the PET substrate, the PET carbonyl group will be cutinase, and IsPETase opened the field toward thermostable- adjacent to the catalytic cleft of the enzyme. This will stimulate the engineered biocatalysts (Fig. 4). Tournier et al, (Tournier et al. 2020) nucleophilic attack between them. At the same time, the oxyanion hole investigated the mode of binding of LCC enzyme with 2-HE(MHET)3 stabilizes the tetrahedral intermediate. One ester bond will be cleaved substrate via molecular docking and enzyme contact surface analysis. during the nick generation step causing the formation of a nick in PET. Subsequently, he discovered that the substrate prefers the binding with After that, a strong binding between the benzoic group of PET and the an elongated and predominantly hydrophobic groove. In the case of Trp 185 residue of the enzyme will occur via π-stacking interaction, IsPETase enzyme, the substrate binds to a shallow cleft of the active site causing the release of the product from the active center. Furthermore, of the biocatalyst via hydrophobic interaction that is stabilized by the two PET chains will be generated with different edges, one with TPA- π-staking force with the Trp 185 residue present near the catalytic triad. edge liberated from subunit I and MHET-edge liberated from subunit To increase the thermostability of the LCC enzyme and reduce the re­ II. After that, the two PET chains will be degraded to MHET monomers action cost of the enzyme, the divalent metal binding site could be during the finaldigestion step. Continuous degradation of TPA-edge and replaced with Disulfide Bridge. This replacement was performed by MHET-edge will produce the four degradation products: MHET, TPA, Tournier et al, (Tournier et al. 2020) and found that the D238C/S283C ◦ ◦ BHET, and EG. BHET will be furtherly degraded into MHET and EG, and variants are showing a melting temperature (94.5 C) higher by 9.8 C finally, a significant amount of TPA will be accumulated beside the than the wild type. Furthermore, to enhance the specificity of the LCC MHET and EG due to PET degradation. After that, MHET can be enzyme, the mutations F243I and F243W were separately introduced to degraded by the MHETase enzyme. the enzyme causing activity to restore by 122% and 98%, respectively, PETase enzyme belongs to the α/β hydrolase superfamily, and its compared with the wild type. After that, LCC enzyme scale-up produc­ active site consists of Ser160, His237, and Asp206 residues. Herein, tion and PET degradation rate were enhanced by introducing the Y127G during the degradation process, the nucleophilic attack occurs between mutation to the previously mentioned variants causing 90% PET Ser160 and the carbonyl group of PET, which offer a more binding af­ degradation in 10.5 h and 9.3 h for F243W/ D238C/S283C/Y127G and finitywith PET plus the firstbinding with Trp 185. In addition, the core F243I/ D238C/S283C/Y127G variants, respectively. structure of PETase consists of seven α-helices and nine β-strands (Joo The replacement of Arg280 with Ala enhanced the degradation rate et al. 2018). This was elucidated by replacing Ser160, His237, and of PET using IsPETase by 22.4% and 32.4% in 18 h and 36 h, respec­ Asp206 residues with Ala that interestingly showed a complete loss of tively (Joo et al. 2018). Mutations in IsPETase binding pockets have the catalytic activity of the three variants (Joo et al. 2018). different influences on BHET and PET polymers due to the high acces­ The ability of two intramolecular disulfide bridges formation (DS1 sibility of the PET polymers toward the binding pocket of IsPETase when and DS2) is distinguished for the PETase enzyme. Mutagenesis studies compared with BHET. Directing the specific mutations, Y58A, W130A, revealed that the integrity of PETase catalytic triad is correlated with W130H, and A180I, to widen the binding center space in IsPETase or DS1 for improving the activity of the enzyme. Furthermore, DS1 allows enhancing the aromaticity on the edge of the binding cleft using the loop close proximity to the PETase binding site (Austin et al., 2018; specificmutation S185H showed a significantenhancement in IsPETase ◦ Fecker et al., 2018). This loop is very flexibleand three residues longer catalytic activity at 30 C and pH 9 (Liu et al. 2018). Mutations around than other PET biocatalysts causing the superior activity of PETase at the substrate-binding groove, R61A, L88F, and I179F, were elucidated room temperature and the formation of subsite for the effective binding to be effective for enhancing PETase catalytic activity by 1.4, 2.1, and with PET surface (Han et al. 2017). On the other hand, the DS2 disulfide 2.5 fold higher than the PETase wild type, respectively. In another study, bond is adjacent to the C-terminus of the enzyme and far from its active the authors enhanced the thermal stability of PETase enzyme by site causing the biocatalyst’s structural stability. Therefore, modifying directing S121E/D186H/R280A mutations to stabilize β6- β7 connecting amino acid residues which are not adjacent to the enzyme’s active site loop and to extend subsite Ilc. These mutations enhanced the thermal ◦ could significantly enhance PET-hydrolytic activity by more than 30% stability by 8.81 C and the PET degradation process was more effective ◦ (Joo et al. 2018). by 14 times at 40 C when compared with the wild type (Son et al. 2019).

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Fig. 4. (a) Overall X-ray crystal struc­ tures of IsPETase from Idonella sakaien­ ses (PDB ID:5XH3, golden) (Han et al. 2017) bound with 1-(2-hydroxyethyl) 4- methyl TPA, Tfcut2 from Thermobifida fusca kw3 (PDB ID: 4CG2, maginta) (Roth et al. 2014), and LCC from leaf- branch compost cutinase (PDB ID: 4 EB0, light blue) (Tournier et al. 2020). IsPETase is distinguished by an addi­ tional sulfide bond and Tryptophan res­ idue adjacent to Serine residue. The catalytic triad of IsPETase constitutes of Ser131, His208, and Asp176. (b) Sub­ strate binding site in IsPETase is open and broad with an extended loop but very narrow and deep in Tfcut2 and LCC. (For interpretation of the refer­ ences to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5 indicates the mutation effect of replacing the Isoleucine amino subjecting double mutation (Q132A/T101A) to the active pocket of acid (Ile179) by the more hydrophobic phenylalanine residue (Ile179/ Tfu_0883 cutinase due to the affinity enhancement of cutinase toward Phe) that significantly enhanced the catalytic efficiency of PETase the hydrophobic substrate (Silva et al. 2011). Moreover, PET degrada­ ◦ enzyme toward the 2PET substrate at 30 C. The phenylalanine residue tion was enhanced by tailoring the fungal polyester hydrolase for wider allowed stronger binding between the PETase and the substrate by substrate active pocket and hydrophobicity improvement (Araújo et al. enhancing the aromaticity on the edge of the binding pocket (Ma et al. 2007). To achieve maximum exploitation of PET biocatalysts, conserved 2018). amino acid residues, and electrostatic surface are other rationales that One of the most effective strategies in protein engineering is the need further investigations. The replacement of amino acid residues in active site modification via site-directed mutagenesis. This strategy the active pocket of TfCut2 cutinase with the highly conserved amino showed a significant effect in substrate reshaping, co-factor specificity, acid residues of LCC cutinase significantlyimproved PET degradation at ◦ and reactivity (Cedrone et al. 2000). Active site modification of 65 C (Wei et al. 2016). Intriguingly, after the modelling and comparison Tfu_0883 elevated the enzyme hydrophobicity and enhanced the of Thc_Cut1 and Thc_Cut2 cutinases from Thermobifida cellulosilytica biodegradation of PET (Silva et al. 2011). Herrero Acero et al. (Herrero DSM44535, Acero et al. (Herrero Acero et al. 2013) discovered that the Acero et al. 2013) have found that site-directed mutagenesis has electrostatic and hydrophobic properties of the surface near the active increased the catalytic activity of T. cellulosilytica cutinase by three-fold pocket of both enzymes are different and this is the main reason of the higher than the wild type. Mutagenesis not only enhanced the catalytic variation in their degradation efficiency. The replacement of A19 and activity of cutinase, from T. fusca, 2.7 fold higher than the wild type but A29 amino acid residues, positively charged, with uncharged Ser and also mitigated the simultaneous product inhibition by MHET (Wei et al. Asp amino acid residues improved the degradation efficiency for 3PET 2016). Table 3 summarizes the recent achievements in enhancing the and PET. At the same time, the replacement of the uncharged Glu65 by catalytic properties of PET hydrolyzing enzymes via mutagenesis. the negatively charged glutamic acid caused a massive loss in the en­ zyme’s catalytic activity. Pretreatment strategies of PET using anionic 5.5. Rational design of biocatalyst mutants surfactants have been recently studied (Furukawa et al., 2018; Sagong et al., 2020). The activity of PETase was enhanced 120 times after the The rational design of biocatalysts is a promising future trend in the treatment of the low-crystalline PET film with anionic surfactants. The thickness of the PET filmwas decreased by 22% after 36 h degradation enzyme-engineering field. It depends on the in-depth knowledge of ◦ enzyme structure, computational simulations, and enzyme’s catalytic process at 30 C. During this condition, surfactants enhanced the bind­ performance. Recently, different tools were developed to engineer PET ing of the hydrophilic PETase with the hydrophobic surface of PET as biocatalysts rationally and to re-shape enzyme specificities such as ho­ shown in Fig. 6. The surfactant concentration used through this method mology modelling and molecular docking. Hydrophobic interaction of degradation was extremely low (0.005%). Mutagenesis also played an between the enzyme’s surface and the space around the active pocket of important role in the previous mentioned reaction. Furukawa and his the enzyme is crucial for enzyme attachment and reaction activity team (Furukawa et al. 2018) introduced single mutations in the sub­ (Araújo et al. 2007). PET fiber degradation rate was improved by strate binding pocket of PETase by replacing the single cationic amino

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Fig. 5. Comparison between the structure and inter­ action of (a) the wild-type PETase Ile179 and (b) the mutant Ile179/Phe with docked 2PET in the binding site. The mutation of Ile179 with the more hydro­ phobic and larger branch chains phenylalanine res­ idue significantly improved the catalytic efficiency of PETase enzyme due to its strong binding with the substrate. The Km of the mutant enzyme was reduced by 3.8 times, while the Kcat/Km value was elevated by 15.6 fold higher than the wild type. (updated from (Ma et al. 2018).

acid by an anionic one such as R53E, R90E, and K95E. These mutations Intriguingly, the secretion amount of the recombinant protein differs effectively enhanced and accelerated PET hydrolysis by the aid of significantlyaccording to the signal peptide fused with the recombinant anionic surfactant. The surfactant helped in the alignment of PET sur­ protein (Peng et al. 2019). Moog et al, (Moog et al. 2019) used protein face toward the active site of the enzyme. Although the degradation engineering to convert the diatom Phaeodactylum tricornutum into a ◦ process was performed at 30 C, the degradation result was equal to beneficial alternative chassis for PET biodegradation. PETaseR280A ◦ those that were performed with thermostable enzymes at 70 C or enzyme was fused with a signal peptide of P. tricornutum alkaline greater. So, designing more surfactant molecules is urgently needed for phosphatase (AP) for PETase expression in photosynthetic microalga enhancing PET biodegradation at ambient temperatures. P. tricornutum diatom into the surrounding culture medium. This fusion enhanced the secretion and production rate of PETaseR280A significantly from the diatom. The produced PETaseR280A enhanced the degradation 5.6. Enzymes fusion for higher catalytic performance rate and decreased the crystallinity level of PETG by 80 fold compared ◦ with the PET bottle at 30 C and salt water-based environment. These Despite of the recent advances in genetic engineering and enzyme findingspromote the generation of microbial cells capable of using PET fusion strategies, using them in enhancing the catalytic performance of as a carbon source. It can also develop further robust biocatalysts for PET hydrolyzing enzymes is not developed so far. Fusion enzymes is a PET degradation in the ocean. promising strategy for reducing substrate diffusion and eliminating the toxicity of reaction intermediates. This strategy can be performed through linking a set of enzymes covalently at the same macromolecular 5.7. Dual enzyme system for complete PET degradation scaffold or via direct cross-linking (Ricca et al., 2011; Schoffelen and van Hest, 2013; Sharshar et al., 2019). Moreover, PET hydrolyzing enzymes Complete degradation of the crystalline PET thick layer in an fusion with signal peptides, short peptides directing recombinant pro­ environment-friendly manner needs further investigations. MHET is a teins toward the secretory pathway, is a significant strategy for strong and competitive enzyme inhibitor, and its accumulation in the enhancing their production and catalytic activity. NusA, E. coli reaction medium during the degradation process is the main obstacle transcription-termination anti-termination factor, maltose-binding pro­ hindering the complete degradation of PET film. Physical methods to tein, and glutathione-S-transferase (GST) are solubility enhancing fusion solve this problem by using an ultrafiltration membrane reactor was proteins, which were reported for their significant effect in improving performed for the repeated elimination of the produced MHET (Andri´c the expression, and solubility of a large number of insoluble recombi­ et al., 2010; Barth et al., 2015; Kim and Song, 2006). However, this nant proteins (Shih et al. 2002). NusA was found to improve, especially method was disadvantageous due to the massive demand of medium the solubility of more abundant passenger proteins located at organelles buffer to keep a constant dilution rate in the reactor, and the degrada­ (Korf et al. 2005). This strategy can solve the problem of biocatalyst tion rate was still unsatisfactory (Ohlson et al. 1984). Thus, finding an expression in the form of inclusion bodies, one of the main obstacles in alternative solution to avoid the inhibitory effect and to enhance enzyme the heterologous expression in E. coli (Schilling et al. 2007). thermostability via protein engineering is urgently needed. The dual

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Table 3 Examples of different directed mutations to improve the catalytic performance and productivity of PET hydrolases.

Enzyme Chassis Substrate Product Mutation(s) Biological effects (results) Ref (ligand)

IsPETase E. coli PET MHET, S160A D206A H237A Y87A R280A increased PETase catalytic activity by (Joo et al. EG, W185A M161A I208A W159A 22.4% and 32.4% in 18 h and 36 h, respectively 2018) TPA N241A S238A R280A C203A/ compared with IsPETase wild type C239A IsPETase E. coli PET MHET, W185A The absolute crystallinity loss is 4.13% higher (Austin et al. EG, S238F/W159H than IsPETase wild type 2018) TPA leaf-branch compost E. coli PET TPA, F243I/D238C/S283C/Y127G Achieved 90% of PET degradation into (Tournier cutinase EG F243I/D238C/S283C/N246M monomers, with a productivity of 16.7 g of et al. 2020) F243W/D238C/S283C/Y127G terephthalate/L/h over 10 h. ◦ F243W/D238C/S283C/N246M Improved melting temperatures by 9.8 C higher than wild type LCC IsPETase E. coli PET MHET, Y58A, W130A, W130H, A180I, Enhanced the catalytic activity of IsPETase at 30 (Liu et al. ◦ EG, S185H C and pH 9. 2018) TPA

IsPETase E. coli PET MHET, R61A, L88F, I179F Enhanced PETase catalytic activity by 1.4, 2.1, (Ma et al. EG, and 2.5 folds higher than the wild type. I179F 2018) TPA mutant showed the highest degradation rate (22.5 mg.µmol/L) ◦ Saccharomonospora viridis E. coli PET TPA, S226P/R228S Enhanced cutinase activity at 70 C, pH 6.5–8 (Kawai et al. AHK190 Cut190 EG 2019) Saccharomonospora viridis E. coli PET TPA, Q138A/D250C-E296C/Q123H/ Enhanced the melting temperatures of the (Oda et al. AHK190 Cut190 EG N202H mutant enzyme and the degradation rate by 2018) ◦ more than 30% at 70 C Saccharomonospora viridis E. coli PET TPA, Q138A, I224A Q138A:Significantly increased the enzyme (Kawabata AHK190 Cut190 EG affinity (lower Km) and activity (higher Vmax et al. 2017) and kcat) I224A: Significantly increased the enzyme activity (Vmax), but decreased its affinity (increased Km) MHETase E. coli MHET TPA, S419G, S419G_F424N, W397A S419G, S419G_F424N: Enhanced the enzyme (Palm et al. BHET EG affinity and activity toward BHET, W397A: 2019) enhanced the enzyme activity toward MHET carboxylic ester E. coli Amorphous PET BHET, Y250S Re-arranged the active site conformation. (Bollinger hydrolase from MHET, TA Enhanced the enzymatic activity toward et al. 2020) Pseudomonas aestusnigri amorphous PET VGXO14 ◦ IsPETase E. coli semicrystalline BHET, S214H-I168R15-W159H-S188Q- Enhanced IsPETase thermostability at 60 C for (Cui et al., PET MHET, TA R280A-A180I-G165A-Q119Y- 3 days. 2019) L117F-T140D (DuraPETase)

Fig. 6. Scheme showing the effect of PET surface hydrophilization by anionic surfactant for enhancing the binding with PETase enzyme. Updated from (Furukawa et al. 2018). enzyme system has shown to be a beneficial approach for complex rate from Tfcut2 only. Tfcut2 in the dual system continuously degraded polymer hydrolysis (Schoffelen and van Hest 2013). The recent dis­ the inhibitory product MHET proving the synergistic effect of this covery of the bacterial strain Ideonella sakaiensis 201-F6, indicates that enzyme with LC cutinase for efficient PET degradation (Barth et al. PET film can be degraded completely via two steps using PETase and 2016). B from Candida antarctica (CALB) and cutinase from MHETase enzymes together. The synergistic activity of the two enzymes Humicola insolens (HiC) together showed a complete PET degradation to ◦ is needed for efficient PET biodegradation yielding TPA and EG, which TPA with high thermostability at 70 C. In contrast; HiC showed a re­ can be used as a building block for a new round of PET synthesis (Palm striction for the last step of the reaction when used alone (Carniel et al. et al. 2019). Tfcut2 from T. fusca KW3 and LC cutinase together showed 2017). 2.4 fold higher degradation rate of PET compared with the degradation

12 N.A. Samak et al. Environment International 145 (2020) 106144

5.8. Incorporation of metal ions hydrolysis at temperatures near to the Tg of amorphous PET (Alves et al., 2002; Roth et al., 2014). Then et al. (Then et al. 2015) confirmed that + Divalent metal-binding sites play a crucial role in improving hy­ Asp174, Asp204, and Glu253 residues are the binding residues for Ca2 + drolases thermostability and catalytic behavior. The presence of calcium and Mg2 cations in TfCut2 enzyme. Moreover, the melting points of ◦ ion improves large conformational changes in the enzyme structure, TfCut1 and TfCut2 enzymes were increased by 11 and 14 C after adding which maintain the substrate-binding groove in an open conformation, calcium and magnesium ions. Semi-crystalline PET filmswere efficiently and facilitates the enzyme binding with its substrate (Miyakawa et al. degraded by showing a weight loss 12 and 9% after adding 10 mM ◦ 2015). Furthermore, molecular dynamic simulations demonstrated that calcium ion and 10 mM magnesium ion at 65 C. Polyester esterase the higher thermo-stability of calcium ion-bound enzymes is due to less Est119 from Thermobifidaalba showed thermo-stability enhancement in structure fluctuation of the bound enzyme than the unbound enzymes the presence of 500 mM calcium ion and Glu292, Glu213, and Asp243 (Numoto et al. 2018). The side chains of three amino acid residues in residues were responsible for major Calcium ion binding site (Kitado­ cutinases usually form these binding sites with calcium ions. However, koro et al. 2012). Further investigations are required to examine this binding site in LC-Cutinase enzyme is formed of one neutral residue whether metal ions are relevant to PET degradation by other hydrolases. (S283) and two acidic residues (E208 and D238). The thermal stability ◦ of the LC-Cutinase enzyme was significantly enhanced by 9.3 C after 5.9. Enzyme immobilization strategies for stable and re-usable industrial adding 35 mm CaCl2 (Sulaiman et al. 2014). The presence of calcium ◦ ◦ PET biocatalysts ions enhanced the T1/2 value of LC-cutinase by 8.4 C and reached 98 C by increasing calcium ion concentration (Sulaiman et al. 2014). In the + case of Cut190 cutinase from Saccharomonospora viridis AHK190, Ca2 Immobilization technology is a powerful tool for the biocatalysts ions bind with acidic amino acid surfaces and not with the active-site industry due to its significant advantages in the industrial applications + amino acids. Ca2 ions were very crucial for enhancing the thermosta­ (Samak et al. 2018b). Immobilization tools are very effective for + bility of the wild-type and mutant Cut190. 300 mM Ca2 showed enhancing the catalytic activity, stability, specificity, and selectivity of ◦ enzyme stability at pH 9 and 65 C for 24 h and efficiently degraded enzymes. This enhancement in enzyme characteristics is related to the different aromatic PET films (Kawai et al. 2014). Calcium ion, 10 and alteration in enzyme structure, causing favorable structural modifica­ 100 mM, stabilized the tertiary structure of Bacillus stearothermophilus tions. Furthermore, the immobilization process can significantly lower lipase (Kim et al. 2000). The presence of calcium ion significantly the production cost due to the re-usability of the immobilized bio­ enhanced the catalytic activity and showed improvement in protein catalysts, as shown in Fig. 7. Immobilization methods are variants ac­ refolding of lipase from Pseudomonas Sp. MIS38. The calcium ion- cording to the type of immobilization support, linkers, and binding motifs are located at the C-terminal domain of the enzyme immobilization strategies such as entrapment, adsorption, covalent and (Amada et al. 2001). Two aspartic acid residues, Asp354 and Asp357, non-covalent interaction. are also required for calcium ion binding in the case of lipase from To date, the immobilization supports and techniques used for PET Staphylococcus hyicus (Simons et al. 1999). TfCut2 enzyme from Ther­ biocatalysts are very limited, which hindering taking the benefits of mobifida fusca showed a significant loss of its catalytic activity after in­ these biocatalysts for PET waste green recycling. Immobilization sup­ ◦ cubation at 65.6 C for 60 min, which hinders its application for PET ports such as reduced and non-reduced graphene oxide nanosheets, Fe3O4 nanoparticles, and nanoflowers and linkers such as

Fig. 7. Direct biocatalyst immobilization (upper part) enables enzymes re-usability for many cycles by keeping or enhancing their activity toward PET. Immobi­ lization support is sequentially incubated, and reacted with the specific linker to enable the binding of the enzyme with the support. Subsequently, the enzyme immobilization is performed by incubating the enzyme with functionalized support. In contrast, whole-cell immobilization (down part) is performed by reacting the microbial cell with the target support via entrapment method. Therefore, immobilized cells can be used for biocatalysts production for many cycles.

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Polyethylenimine, Nα, Nα-Bis(carboxymethyl)-L-lysine hydrate (NTA- viable strains have to be isolated first,then purifiedand immobilized to NH2) and carbodiimide 1-ethyl-3-(dimethylaminopropyl) carbodiimide produce long-lasting viable biocatalysts as their catalytic activities will (Kumar et al., 2020; Samak et al., 2020, 2018b; Xia et al., 2016) could be be well-preserved. used as an effective support and linkers for single or double enzyme Whole-cell immobilization has many advantages such as long-term immobilization for complete PET bioconversion into value-added metabolic activity, high cell mass loading capacity, economical viable products such as TPA. cells re-usability, genetic stability improvement, cost-effective, sustain­ Table 4 indicates the enhancement of PET degrading enzyme’s cat­ ability, easy separation of immobilized cells from degradation medium, alytic activity, thermal stability, storage stability, and re-usability via resistance to high pH, temperatures, and solvents and high operational enzyme immobilization. Glutaraldehyde-activated chitosan beads were stability (Gardin and Pauss 2001). used to covalently immobilize T. fusca cutinases with 74% adsorption Effective immobilization of live cells depends on the inner structure ◦ capacity and showed optimal activity at 55 C, pH 8. The immobilized of the matrix; the spherical matrix, for example, is more favorable or ◦ ◦ enzyme showed operational stability in the range of 45 C to 70 C, re- rapid production with control of particle diameter. Adsorption, used for ten consecutive cycles with 80% efficiency, and the catalytic Entrapment inside different polymeric supports, encapsulation within activity was decreased after 13 days storage to reach 50% (Hegde and spherical permeable membranes, and covalent binding are different Veeranki 2014). Thielavia terrestris Cutinase was physically immobilized methods for whole-cell immobilization (Bayat et al. 2015). Adsorption on the Lewatit VP OC 1600 cross-linked polymer, showed high thermal method depends on binding the viable cells with water-insoluble par­ ◦ ◦ stability at 80 C and retained about 64% of its catalytic activity at 90 C ticles, this produces electrostatic interaction between the particles and ˙ (Su et al. 2018). T. cellulosilytica cutinase was covalently immobilized on microorganisms’ cellular surface (Zur et al. 2016). Entrapment is the Amber chelated with ferric ions via His-tag binding. The immobilized most common method used for whole viable cell immobilization, and enzyme showed high catalytic activity toward the cleavage of C6 – C4 polymeric hydrogels, such as polyacrylamide gel (PAA) and calcium ester-diol bond with a conversion rate up to 78% and the re- alginate, are the widely used carriers so far (Trelles and Rivero 2013). usability reached to 24 h cycles with more than 94% residual activity PAA polymeric hydrogels protect the immobilized cells from any haz­ (Pellis et al., 2017). Dual enzymes, TfCut2 from Thermobifidafusca KW3 ardous shocks produced by chemical or mechanical environmental immobilized on SulfoLink coupling resin, and free LC-cutinase were factors, so they prevent the loss of cellular activity and facilitate the used for complete PET biodegradation. The dual enzymes showed a 2.4 diffusion of substrate and product (Trevors 1992). Fungi spores also ◦ fold higher degradation rate of PET films at 60 C compared to the could be entrapped using PAA to induce germination in situ in the degradation rate using the free TfCut2 enzyme. The immobilized presence of cultural nutrients (Fravel et al., 1985; Wang et al., 2018). enzyme enhanced further degradation of the produced MHET to produce Entrapment using calcium alginate may be unfavorable even though it is TPA and EG (Barth et al. 2016). a mild process with no toxic and heating requirements, but the enzy­ matic cellular activity usually depleted through its re-usability (Busscher et al. 1995). In this regards, using the whole-cell immobilization method 5.10. Whole-cell immobilization for PET biocatalyst will open the field for more dynamic and robust enzymes that could enrich the industrial applications of these enzymes Whole-cell viable microorganism immobilization is another method and fill the gap to move forward into reality. to enhance plastic biodegradation. Viable bacterial or fungal cells could be immobilized in or on the surface of the polymerized matrix via 6. Economic analysis physical confinement or chemical binding with cellular adherence mechanisms for a more effective plastic degradation process. Immobi­ The economic feasibility of PET waste green recycling depends on lization here will control microorganisms’ growth by controlling the cost-effective large-scale productivity of PET hydrolases, the required porosity degree, reticulation size, hydration rate in organic and inor­ time for PET bioconversion, and the useful applications of degradation ganic solutions (Huang et al., 2012; Li et al., 2011; Mu et al., 2017; Shan byproducts. As noted earlier, the mechanical and chemical degradation et al., 2005). Herein, the immobilization of pure strains is favorable so,

Table 4 Examples of different immobilization methods and supports to improve the catalytic performance and re-usability of PET hydrolases.

Name of Enzyme Immobilization Immobilization Thermal stability Optimum Re-usability Storage Ref Support method pH stability

T. fusca Cut1 glutaraldehyde Covalent Optimum temperature 8 80% after 10 reuse 50% after (Hegde and activated chitosan 55 ℃ cycles 13 days Veeranki beads 2014) TfCa from SulfoLink resin covalent Retained 94% of 8 ND ND (Barth et al. ◦ T. fusca used with LCC activity at 60 C 2016) enzyme Aspergillus oryzae Cutinase Lewatit VP OC 1600 Physical Retained full activity 5–9 ND ND (Su et al. ◦ for 1 h at 80 C 2018) Humicola insolens Cutinase Lewatit VP OC 1600 Physical activity decreased 5–9 ND ND (Su et al. after 2 h by 50% at 2018) ◦ 80 C Thielavia terrestris Cutinase Lewatit VP OC 1600 Physical Retained 77% of 5–9 ND ND (Su et al. enzyme activity after 2018) ◦ 2 h at 80 C Thermobifida cellulosilytica Opal covalent 57% monomer 7 Conversion decrease ND (Pellis et al., ◦ cutinase 1 conversions at 21 C 42% after 24 h 2017) reaction (3 cycles) Thermobifida cellulosilytica Coral covalent 76% monomer 7 decrease 58% after 24 ND (Pellis et al., ◦ cutinase 1 conversions at 21 C h reaction (3 cycles) 2017) Thermobifida cellulosilytica Amber covalent 78% monomer 7 decrease 58% after 24 ND (Pellis et al., ◦ cutinase 1 conversions at 21 C h reaction (3 cycles) 2017)

ND, not detected.

14 N.A. Samak et al. Environment International 145 (2020) 106144 can cost ~250 and 500 €/ton plastic, respectively. In comparison, Declaration of Competing Interest Tournier et al (Tournier et al. 2020) stated that the production cost of 1 kg enzymes required to recycle PET waste is 21 €. The recycling of 1-ton The authors declare that they have no known competing financial PET waste needs ~3 kg protein which costs ~63 €, and this process interests or personal relationships that could have appeared to influence produces sodium sulfate by 0.6 kg/kg-recycled PET and 863 kg ter­ the work reported in this paper. ephthalic acid, which was purifiedafter that to 99.8%, during the whole process. In addition to the produced amounts of phosphorus, antimony, Acknowledgements and ethylene glycol. Thus, it is estimated that the recycling process of 100,000 tons of PET/year will yield 60,000 tons of sodium sulfate, This article acknowledges the funding support provided by the Na­ which represents 0.28% of the global market need. Moreover, the pro­ tional Natural Science Foundation of China (grant numbers duced terephthalic acid could be used for the manufacturing of PET 31961133017, 31961133018, 31961133019). These grants are part of bottles to close the gap of the circular economy. By contrast, given that “MIXed plastics biodegradation and using microbial com­ all the available bioengineering strategies for PET biocatalysts that were munities” MIX-UP research project, which is a joint NSFC and EU H2020 mentioned during this review to enhance their productivity and cata­ collaboration. In Europe, MIX-UP has received funding from the Euro­ lytic activity and followed by immobilization on suitable support, this pean Union’s Horizon 2020 research and innovation programme under will enable the biocatalysts re-usability for at least 10 cycles. This sup­ grant agreement No 870294. posed to reduce the estimated actual production cost of PET biocatalysts and PET waste green recycling by 10 fold. Further studies of genetic References improvement, large-scale production, and immobilization of PET bio­ catalysts will bring the process of PET waste green recycling closer to Abdel-Shafy, H.I., Mansour, M.S., 2018. Solid waste issue: Sources, composition, disposal, recycling, and valorization. Egypt. J. Pet. 27, 1275–1290. practical and commercial reality. Besides, it will fill the global need to Al-Sabagh, A., Yehia, F., Eshaq, G., Rabie, A., ElMetwally, A., 2016. Greener routes for solve the hazardous problem of plastic waste. recycling of polyethylene terephthalate. Egypt. J. Pet. 25, 53–64. Alisch-Mark, M., Herrmann, A., Zimmermann, W., 2006. Increase of the hydrophilicity of polyethylene terephthalate fibres by hydrolases from Thermomonospora fusca and 7. Concluding remarks and future perspectives Fusarium solani f. sp. pisi. Biotechnol. Lett. 28, 681–685. Almeida, E.L., Carrillo Rincon, A.F., Jackson, S.A., Dobson, A.D., 2019. In silico screening The continuous and rapid development of the plastic industry has and heterologous expression of a Polyethylene Terephthalate hydrolase (PETase)- like enzyme (SM14est) with Polycaprolactone (PCL)-degrading activity, from the risen a global warning because of their environmental impact, which is marine sponge-derived strain Streptomyces sp. SM14. Front. Microbiol. 10, 2187. represented in the massive PET waste accumulations in the landfills, Alves, N., Mano, J.F., Balaguer, E., Duenas,˜ J.M., Ribelles, J.G., 2002. Glass transition seas, and oceans. Traditional methods for PET waste recycling still and structural relaxation in semi-crystalline poly (ethylene terephthalate): a DSC study. Polymer 43, 4111–4122. + problematic because of the lethal effect on marine animals and humans. Amada, K., Kwon, H.-J., Haruki, M., Morikawa, M., Kanaya, S., 2001. Ca2 -induced + Finding an effective and environment-friendly strategy for PET waste folding of a family I. 3 lipase with repetitive Ca2 binding motifs at the C-terminus. green recycling is in high demand. The discovery of new PET bio­ FEBS Lett. 509, 17–21. catalysts and degrading microorganisms is an excellent movement to­ Ammala, A., Bateman, S., Dean, K., Petinakis, E., Sangwan, P., Wong, S., Yuan, Q., Yu, L., Patrick, C., Leong, K., 2011. An overview of degradable and biodegradable ward a green recycling scheme for PET waste. Besides, studying their polyolefins. Prog. Polym. Sci. 36, 1015–1049. molecular mechanism extensively via solving their crystal structure will Andrady, A.L., 2015. Plastics and environmental sustainability ed^eds. John Wiley & widen this research area to move forward the industrial applications. Sons. Andri´c, P., Meyer, A.S., Jensen, P.A., Dam-Johansen, K., 2010. Reactor design for This will require further assistance from computational and structural minimizing product inhibition during enzymatic lignocellulose hydrolysis: II. biology, biochemistry scientists, and expertise of material sciences. Quantification of inhibition and suitability of membrane reactors. Biotechnol. Adv. Utilization of alternative and more dynamic chassis for enhancing PET 28, 407–425. Araújo, R., Silva, C., O’Neill, A., Micaelo, N., Guebitz, G., Soares, C.M., Casal, M., biocatalysts production needs further investigations due to the severe Cavaco-Paulo, A., 2007. Tailoring cutinase activity towards polyethylene shortage in this field. Utilization of the reported bioengineering strate­ terephthalate and polyamide 6, 6 fibers. J. Biotechnol. 128, 849–857. gies during this review can potentially dramatically boost the produc­ Austin, H.P., Allen, M.D., Donohoe, B.S., Rorrer, N.A., Kearns, F.L., Silveira, R.L., Pollard, B.C., Dominick, G., Duman, R., El Omari, K., 2018. Characterization and tivity in large scale industrial processes, the enhancement of catalytic engineering of a plastic-degrading aromatic polyesterase. Proc. Natl. Acad. Sci. 115, performance, and the improvement in thermostability and re-usability E4350–E4357. characteristics for sustainable applications. More potentially, it may Azapagic, A., Emsley, A., Hamerton, I., 2003. Polymers: the environment and sustainable development ed^eds. John Wiley & Sons. lead to the reduction of PET crystallinity. It can be concluded that Barth, M., Honak, A., Oeser, T., Wei, R., Belisario-Ferrari,´ M.R., Then, J., Schmidt, J., protein engineering strategies performed by (Tournier et al. 2020) were Zimmermann, W., 2016. A dual enzyme system composed of a polyester hydrolase highly effective to make the engineered LCC enzyme the best candidate and a carboxylesterase enhances the biocatalytic degradation of polyethylene – for petrochemical PET hydrolysis so far as 90% depolymerization of PET terephthalate films. Biotechnol. J. 11, 1082 1087. Barth, M., Wei, R., Oeser, T., Then, J., Schmidt, J., Wohlgemuth, F., Zimmermann, W., was achieved within 10 h with 16.7 g TPA productivity/L/h. However, it 2015. Enzymatic hydrolysis of polyethylene terephthalate films in an ultrafiltration is recommended to find alternative biodegradable plastics such as pol­ membrane reactor. J. Membr. Sci. 494, 182–187. yhydroxyalkanoates (PHAs) that are safe and non-toxic bioplastics Bayat, Z., Hassanshahian, M., Cappello, S., 2015. Immobilization of microbes for bioremediation of crude oil polluted environments: a mini review. The open produced by different microorganisms to facilitate the biodegradation microbiology journal 9, 48. and recycling processes. PHAs are similar to the petrochemical-based Bermúdez-García, E., Pena-Montes,˜ C., Castro-Rodríguez, J.A., Gonzalez-Canto,´ A., ˜ ´ plastics, but they are completely biocompatible and biodegradable Navarro-Ocana, A., Farres, A., 2017. ANCUT2, a thermo-alkaline cutinase from Aspergillus nidulans and its potential applications. Appl. Biochem. Biotechnol. 182, (Ong et al. 2017). PHAs are easily degradable by microorganisms under 1014–1036. limited carbon and energy sources (Chen and Patel 2012). Bacteria such Biundo, A., Reich, J., Ribitsch, D., Guebitz, G.M., 2018. Synergistic effect of mutagenesis as Bacillus, Nocardiopsis, Burkholderia, and Cupriavidus and fungi like and truncation to improve a polyesterase from Clostridium botulinum for polyester hydrolysis. Sci. Rep. 8. Micromycetes and Mycobacterium have been reported for their PHA Bollinger, A., Thies, S., Knieps-Grünhagen, E., Gertzen, C., Kobus, S., Hoppner,¨ A., assimilation under aerobic and anaerobic conditions (Boyandin et al. Ferrer, M., Gohlke, H., Smits, S.H., Jaeger, K.-E., 2020. A novel polyester hydrolase 2013). Other examples of the bio-based and biodegradable alternatives from the marine bacterium Pseudomonas aestusnigri–structural and functional insights. Front. Microbiol. 11, 114. of PET are aromatic-aliphatic polyesters based on diethyl-2,5- Bombelli, P., Howe, C.J., Bertocchini, F., 2017. Polyethylene bio-degradation by furandicarboxylate, the petroleum-based diethyl terephthalate, and caterpillars of the wax moth Galleria mellonella. Curr. Biol. 27, R292–R293. diethyl isophthalate which were produced via enzymatic catalysis and Boyandin, A.N., Prudnikova, S.V., Karpov, V.A., Ivonin, V.N., Đỗ, N.L., Nguyễn, T.H., ˆ the furan-aromatic polyesters prepared from biomass-based HMF (Pellis Le, T.M.H., Filichev, N.L., Levin, A.L., Filipenko, M.L., 2013. Microbial degradation of polyhydroxyalkanoates in tropical soils. Int. Biodeterior. Biodegrad. 83, 77–84. et al., 2019; Zhang et al., 2019).

15 N.A. Samak et al. Environment International 145 (2020) 106144

Brandon, A.M., Gao, S.-H., Tian, R., Ning, D., Yang, S.-S., Zhou, J., Wu, W.-M., Criddle, C. Gong, J., Kong, T., Li, Y., Li, Q., Li, Z., Zhang, J., 2018. Biodegradation of microplastic S., 2018. Biodegradation of polyethylene and plastic mixtures in mealworms (larvae derived from poly (ethylene terephthalate) with bacterial whole-cell biocatalysts. of Tenebrio molitor) and effects on the gut microbiome. Environ. Sci. Technol. 52, Polymers 10, 1326. 6526–6533. Gong, Y., Hu, H., Gao, Y., Xu, X., Gao, H., 2011. Microalgae as platforms for production Brueckner, T., Eberl, A., Heumann, S., Rabe, M., Guebitz, G.M., 2008. Enzymatic and of recombinant proteins and valuable compounds: progress and prospects. J. Ind. chemical hydrolysis of poly (ethylene terephthalate) fabrics. J. Polym. Sci., Part A: Microbiol. Biotechnol. 38, 1879–1890. Polym. Chem. 46, 6435–6443. Grigore, M.E., 2017. Methods of recycling, properties and applications of recycled Burnley, S., Coleman, T., Peirce, A., 2015. Factors influencing the life cycle burdens of thermoplastic polymers. Recycling 2, 24. the recovery of energy from residual municipal waste. Waste Manage. 39, 295–304. Gupta, D., Chaudhary, H., Gupta, C., 2015. Topographical changes in polyester after Busscher, H., Bos, R., Van der Mei, H., 1995. Initial microbial adhesion is a determinant chemical, physical and enzymatic hydrolysis. J. Textile Institute 106, 690–698. for the strength of biofilm adhesion. FEMS Microbiol. Lett. 128, 229–234. Han, X., Liu, W., Huang, J.-W., Ma, J., Zheng, Y., Ko, T.-P., Xu, L., Cheng, Y.-S., Chen, C.- Calleja, F.B., Cagiao, M., Zachmann, H., Vanderdonckt, C., 1994. Hydrolysis etching of C., Guo, R.-T., 2017. Structural insight into catalytic mechanism of PET hydrolase. crystalline and amorphous poly (ethylene terephthalate): Influence of molecular Nat. Commun. 8, 1–6. weight and microstructure. J. Macromol. Sci.—Phys. 33, 333–346. Hegde, K., Veeranki, V.D., 2014. Studies on immobilization of cutinases from Carniel, A., Valoni, E.,´ Junior, J.N., da Conceiçao˜ Gomes, A., de Castro, A.M., 2017. Thermobifida fusca on glutaraldehyde activated chitosan beads. Biotechnol. J. Int. Lipase from Candida antarctica (CALB) and cutinase from Humicola insolens act 1049–1063. synergistically for PET hydrolysis to terephthalic acid. Process Biochem. 59, 84–90. Herrero Acero, E., Ribitsch, D., Dellacher, A., Zitzenbacher, S., Marold, A., Carta, D., Cao, G., D’Angeli, C., 2003. Chemical recycling of poly (ethylene Steinkellner, G., Gruber, K., Schwab, H., Guebitz, G.M., 2013. Surface engineering of terephthalate)(PET) by hydrolysis and glycolysis. Environ. Sci. Pollut. Res. 10, a cutinase from Thermobifida cellulosilytica for improved polyester hydrolysis. 390–394. Biotechnol. Bioeng. 110, 2581–2590. Cedrone, F., Menez,´ A., Quem´ eneur,´ E., 2000. Tailoring new enzyme functions by Herrero Acero, E., Ribitsch, D., Steinkellner, G.T., Gruber, K., Greimel, K., Eiteljoerg, I., rational redesign. Curr. Opin. Struct. Biol. 10, 405–410. Trotscha, E., Wei, R., Zimmermann, W., Zinn, M., Cavaco-Paulo, A., Freddi, G., Cerda-Cu` ellar,´ M., Kint, D.P., Munoz-Guerra,˜ S., Marques-Calvo,´ M.S., 2004. Schwab, H., Guebitz, G.M., 2011;44.. Enzymatic surface hydrolysis of PET: effect of Biodegradability of aromatic building blocks for poly (ethylene terephthalate) structural diversity on kinetic properties of cutinases from Thermobifida. copolyesters. Polym. Degrad. Stab. 85, 865–871. Macromolecules. Chae, Y., Kim, D., Choi, M.-J., Cho, Y., An, Y.-J., 2019. Impact of nano-sized plastic on Hopewell, J., Dvorak, R., Kosior, E., 2009. Plastics recycling: challenges and the nutritional value and gut microbiota of whiteleg shrimp Litopenaeus vannamei via opportunities. Philos Trans. Roy. Soc. B: Biol. Sci. 364, 2115–2126. dietary exposure. Environ. Int. 130, 104848. Huang, T., Qiang, L., Zelong, W., Daojiang, Y., Jianmin, X., 2012. Simultaneous removal Chen, C.C., Han, X., Ko, T.P., Liu, W., Guo, R.T., 2018. Structural studies reveal the of thiophene and dibenzothiophene by immobilized Pseudomonas delafieldiiR-8 cells. molecular mechanism of PETase. FEBS J. 285, 3717–3723. Chin. J. Chem. Eng. 20, 47–51. Chen, G.-Q., Patel, M.K., 2012. Plastics derived from biological sources: present and Joo, S., Cho, I.J., Seo, H., Son, H.F., Sagong, H.-Y., Shin, T.J., Choi, S.Y., Lee, S.Y., future: a technical and environmental review. Chem. Rev. 112, 2082–2099. Kim, K.-J., 2018. Structural insight into molecular mechanism of poly (ethylene Chen, M.-L., Chen, J.-S., Tang, C.-L., Mao, I.-F., 2008. The internal exposure of Taiwanese terephthalate) degradation. Nat. Commun. 9, 382. to phthalate—an evidence of intensive use of plastic materials. Environ. Int. 34, Kale, S.K., Deshmukh, A.G., Dudhare, M.S., Patil, V.B., 2015. Microbial degradation of 79–85. plastic: a review. J. Biochem. Technol. 6, 952–961. Choi, K.Y., Kim, D., Sul, W.J., Chae, J.-C., Zylstra, G.J., Kim, Y.M., Kim, E., 2005. Karayannidis, G.P., Achilias, D.S., 2007. Chemical recycling of poly (ethylene Molecular and biochemical analysis of phthalate and terephthalate degradation by terephthalate). Macromol. Mater. Eng. 292, 128–146. Rhodococcus sp. strain DK17. FEMS Microbiol. Lett. 252, 207–213. Kawabata, T., Oda, M., Kawai, F., 2017. Mutational analysis of cutinase-like enzyme, Cleary, J., 2014. A life cycle assessment of residential waste management and Cut190, based on the 3D docking structure with model compounds of polyethylene prevention. Int. J. Life Cycle Assess. 19, 1607–1622. terephthalate. J. Biosci. Bioeng. 124, 28–35. Cui, Y., Chen, Y., Liu, X., Dong, S., Qiao, Y., Han, J., Li, C., Han, X., Liu, W., Chen, Q., Kawai, F., Kawabata, T., Oda, M., 2019. Current knowledge on enzymatic PET 2019. Computational redesign of PETase for plastic biodegradation by GRAPE degradation and its possible application to waste stream management and other strategy. BioRxiv 787069. fields. Appl. Microbiol. Biotechnol. 103, 4253–4268. d’Ambrieres,` W., 2019. Plastics recycling worldwide: current overview and desirable Kawai, F., Oda, M., Tamashiro, T., Waku, T., Tanaka, N., Yamamoto, M., Mizushima, H., + changes. Field Actions Science Reports J. Field Actions 12–21. Miyakawa, T., Tanokura, M., 2014. A novel Ca2 -activated, thermostabilized de Carvalho, C.C., 2017. Whole cell biocatalysts: essential workers from Nature to the polyesterase capable of hydrolyzing polyethylene terephthalate from industry. Microb. Biotechnol. 10, 250–263. Saccharomonospora viridis AHK 190. Appl. Microbiol. Biotechnol. 98. Donelli, I., Taddei, P., Smet, P.F., Poelman, D., Nierstrasz, V.A., Freddi, G., 2009. Kaza, S., Yao, L., Bhada-Tata, P., Van Woerden, F., 2018. What a waste 2.0: a global Enzymatic surface modificationand functionalization of PET: a water contact angle, snapshot of solid waste management to 2050 ed^eds. The World Bank. FTIR, and fluorescence spectroscopy study. Biotechnol. Bioeng. 103, 845–856. Kikkawa, Y., Fujita, M., Abe, H., Doi, Y., 2004. Effect of water on the surface molecular Eberl, A., Heumann, S., Brückner, T., Araujo, R., Cavaco-Paulo, A., Kaufmann, F., mobility of poly (lactide) thin films: an atomic force microscopy study. Kroutil, W., Guebitz, G.M., 2009. Enzymatic surface hydrolysis of poly (ethylene Biomacromolecules 5, 1187–1193. terephthalate) and bis (benzoyloxyethyl) terephthalate by lipase and cutinase in the Kim, H.R., Song, W.S., 2006. Lipase treatment of polyester fabrics. Fibers Polym. 7, presence of surface active molecules. J. Biotechnol. 143, 207–212. 339–343. Eriksen, M., Lebreton, L.C., Carson, H.S., Thiel, M., Moore, C.J., Borerro, J.C., Galgani, F., Kim, J.W., Park, S.-B., Tran, Q.-G., Cho, D.-H., Choi, D.-Y., Lee, Y.J., Kim, H.-S., 2020. Ryan, P.G., Reisser, J., 2014. Plastic pollution in the world’s oceans: more than 5 Functional expression of polyethylene terephthalate-degrading enzyme (PETase) in trillion plastic pieces weighing over 250,000 tons afloat at sea. PLoS ONE 9, green microalgae. Microb. Cell Fact. 19, 1–9. e111913. Kim, M.-H., Kim, H.-K., Lee, J.-K., Park, S.-Y., Oh, T.-K., 2000. Thermostable lipase of Facts, P.E.P.T. An analysis of European plastics production, demand and waste data. Bacillus stearothermophilus: high-level production, purification, and calcium- 2019. dependent thermostability. Biosci. Biotechnol. Biochem. 64, 280–286. Fatima, S., 2014. Chemical recycle of plastic. Am. J. Eng. Res. 3, 93–108. Kitadokoro, K., Thumarat, U., Nakamura, R., Nishimura, K., Karatani, H., Suzuki, H., Fecker, T., Galaz-Davison, P., Engelberger, F., Narui, Y., Sotomayor, M., Parra, L.P., Kawai, F., 2012. Crystal structure of cutinase Est119 from Thermobifidaalba AHK119 Ramírez-Sarmiento, C.A., 2018. Active site flexibilityas a hallmark for efficientPET that can degrade modified polyethylene terephthalate at 1.76 Å resolution. Polym. degradation by I. sakaiensis PETase. Biophys. J. 114, 1302–1312. Degrad. Stability 97, 771–775. Fravel, D., Marois, J., Lumsden, R., Connick Jr, W., 1985. Encapsulation of potential Korf, U., Kohl, T., Van Der Zandt, H., Zahn, R., Schleeger, S., Ueberle, B., biocontrol agents in an alginate-clay matrix. Phytopathology 75, 774–777. Wandschneider, S., Bechtel, S., Schnolzer,¨ M., Ottleben, H., 2005. Large-scale Furukawa, M., Kawakami, N., Oda, K., Miyamoto, K., 2018. Acceleration of enzymatic protein expression for proteome research. Proteomics 5, 3571–3580. degradation of poly (ethylene terephthalate) by surface coating with anionic Kubowicz, S., Booth, A.M., 2017. Biodegradability of plastics: challenges and surfactants. ChemSusChem 11, 4018–4025. misconceptions. ACS Publications. Gamerith, C., Vastano, M., Ghorbanpour, S.M., Zitzenbacher, S., Ribitsch, D., Kumar, V., Lee, Y.-S., Shin, J.-W., Kim, K.-H., Kukkar, D., Tsang, Y.F., 2020. Potential Zumstein, M.T., Sander, M., Herrero Acero, E., Pellis, A., Guebitz, G.M., 2017. applications of graphene-based nanomaterials as adsorbent for removal of volatile Enzymatic degradation of aromatic and aliphatic polyesters by P. pastoris expressed organic compounds. Environ. Int. 135, 105356. cutinase 1 from Thermobifida cellulosilytica. Front. Microbiol. 8, 938. Lauersen, K.J., Berger, H., Mussgnug, J.H., Kruse, O., 2013a. Efficient recombinant Gangl, D., Zedler, J.A., Rajakumar, P.D., Martinez, E.M.R., Riseley, A., Włodarczyk, A., protein production and secretion from nuclear transgenes in Chlamydomonas Purton, S., Sakuragi, Y., Howe, C.J., Jensen, P.E., 2015. Biotechnological reinhardtii. J. Biotechnol. 167, 101–110. exploitation of microalgae. J. Exp. Bot. 66, 6975–6990. Lauersen, K.J., Vanderveer, T.L., Berger, H., Kaluza, I., Mussgnug, J.H., Walker, V.K., Garcia, J.M., Robertson, M.L., 2017. The future of plastics recycling. Science 358, Kruse, O., 2013b. Ice recrystallization inhibition mediated by a nuclear-expressed 870–872. and-secreted recombinant ice-binding protein in the microalga Chlamydomonas Gardin, H., Pauss, A., 2001. κ-carrageenan/gelatin gel beads for the co-immobilization of reinhardtii. Appl. Microbiol. Biotechnol. 97, 9763–9772. aerobic and anaerobic microbial communities degrading 2, 4, 6-trichlorophenol Lebreton, L., Andrady, A., 2019. Future scenarios of global plastic waste generation and under air-limited conditions. Appl. Microbiol. Biotechnol. 56, 517–523. disposal. Palgrave Commun. 5, 1–11. Gewert, B., Plassmann, M.M., MacLeod, M., 2015. Pathways for degradation of plastic Lechner, A., Keckeis, H., Lumesberger-Loisl, F., Zens, B., Krusch, R., Tritthart, M., polymers floating in the marine environment. Environ. Sci. Processes Impacts 17, Glas, M., Schludermann, E., 2014. The Danube so colourful: a potpourri of plastic 1513–1521. litter outnumbers fish larvae in Europe’s second largest river. Environ. Pollut. 188, Geyer, R., Jambeck, J.R., Law, K.L., 2017. Production, use, and fate of all plastics ever 177–181. made. Sci. Adv. 3, e1700782.

16 N.A. Samak et al. Environment International 145 (2020) 106144

Leon-Zayas,´ R., Roberts, C., Vague, M., Mellies, J.L., 2019. Draft Genome Sequences of Perz, V., Baumschlager, A., Bleymaier, K., Zitzenbacher, S., Hromic, A., Steinkellner, G., Five Environmental Bacterial Isolates That Degrade Polyethylene Terephthalate Pairitsch, A., Łyskowski, A., Gruber, K., Sinkel, C., 2016. Hydrolysis of synthetic Plastic. Microbiol. Resource Announcements 8, e00237–00219. polyesters by Clostridium botulinum esterases. Biotechnol. Bioeng. 113, 1024–1034. Levchik, S.V., Weil, E.D., 2004. A review on thermal decomposition and combustion of Qasaimeh, A., Abdallah-Qasaimeh, M., Hani, F.B., 2016. A review on biogas interception thermoplastic polyesters. Polym. Adv. Technol. 15, 691–700. processes in municipal landfill. J. Environ. Sci. Technol. 9, 1–25. Li, C.-T., Zhuang, H.-K., Hsieh, L.-T., Lee, W.-J., Tsao, M.-C., 2001. PAH emission from Quartinello, F., Vajnhandl, S., Volmajer Valh, J., Farmer, T.J., Vonˇcina, B., Lobnik, A., the incineration of three plastic wastes. Environ. Int. 27, 61–67. Herrero Acero, E., Pellis, A., Guebitz, G.M., 2017. Synergistic chemo-enzymatic Li, Y., Li, W., Gao, H., Xing, J., Liu, H., 2011. Integration of flocculation and adsorptive hydrolysis of poly (ethylene terephthalate) from textile waste. Microb. Biotechnol. immobilization of Pseudomonas delafieldii R-8 for diesel oil biodesulfurization. 10, 1376–1383. J. Chem. Technol. Biotechnol. 86, 246–250. Ragaert, K., Delva, L., Van Geem, K., 2017. Mechanical and chemical recycling of solid Liebminger, S., Eberl, A., Sousa, F., Heumann, S., Fischer-Colbrie, G., Cavaco-Paulo, A., plastic waste. Waste Manage. 69, 24–58. Guebitz, G.M., 2007. Hydrolysis of PET and bis-(benzoyloxyethyl) terephthalate Restrepo-Florez,´ J.-M., Bassi, A., Thompson, M.R., 2014. Microbial degradation and with a new polyesterase from Penicillium citrinum. Biocatal. Biotransform. 25, deterioration of polyethylene–A review. Int. Biodeterior. Biodegrad. 88, 83–90. 171–177. Ribitsch, D., Acero, E.H., Greimel, K., Dellacher, A., Zitzenbacher, S., Marold, A., Liu, B., He, L., Wang, L., Li, T., Li, C., Liu, H., Luo, Y., Bao, R., 2018. Protein Rodriguez, R.D., Steinkellner, G., Gruber, K., Schwab, H., Guebitz, G.M., 2012a. Crystallography and Site-Direct Mutagenesis Analysis of the Poly (ethylene A new esterase from Thermobifidahalotolerans hydrolyses polyethylene terephthalate terephthalate) Hydrolase PETase from Ideonella sakaiensis. ChemBioChem 19, (PET) and (PLA). Polymers 4, 617–629. 1471–1475. Ribitsch, D., Herrero Acero, E., Greimel, K., Eiteljoerg, I., Trotscha, E., Freddi, G., Longhi, S., Cambillau, C., 1999. Structure-activity of cutinase, a small lipolytic enzyme. Schwab, H., Guebitz, G.M., 2012b. Characterization of a new cutinase from Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of. Lipids 1441, Thermobifida alba for PET-surface hydrolysis. Biocatal. Biotransform. 30. 185–196. Ribitsch, D., Heumann, S., Trotscha, E., Herrero Acero, E., Greimel, K., Leber, R., Birner- Luo, Z.W., Lee, S.Y., 2017. Biotransformation of p-xylene into terephthalic acid by Gruenberger, R., Deller, S., Eiteljoerg, I., Remler, P., 2011. Hydrolysis of engineered Escherichia coli. Nat. Commun. 8, 1–8. polyethyleneterephthalate by p-nitrobenzylesterase from Bacillus subtilis. Biotechnol. Ma, Y., Yao, M., Li, B., Ding, M., He, B., Chen, S., Zhou, X., Yuan, Y., 2018. Enhanced Prog. 27, 951–960. poly (ethylene terephthalate) hydrolase activity by protein engineering. Engineering Ricca, E., Brucher, B., Schrittwieser, J.H., 2011. Multi-enzymatic cascade reactions: 4, 888–893. overview and perspectives. Adv. Synth. Catal. 353, 2239–2262. Mayfield, S.P., Manuell, A.L., Chen, S., Wu, J., Tran, M., Siefker, D., Muto, M., Marin- Ronkvist, Å.M., Xie, W., Lu, W., Gross, R.A., 2009. Cutinase-catalyzed hydrolysis of poly Navarro, J., 2007. Chlamydomonas reinhardtii chloroplasts as protein factories. Curr. (ethylene terephthalate). Macromolecules 42, 5128–5138. Opin. Biotechnol. 18, 126–133. Rosano, G.L., Ceccarelli, E.A., 2014. Recombinant protein expression in Escherichia coli: Mierzwa-Hersztek, M., Gondek, K., Kope´c, M., 2019. Degradation of polyethylene and advances and challenges. Front. Microbiol. 5, 172. biocomponent-derived polymer materials: An overview. J. Polym. Environ. 27, Roth, C., Wei, R., Oeser, T., Then, J., Foellner, C., Zimmermann, W., Straeter, N., 2014. 600–611. Structural and functional studies on a thermostable polyethylene terephthalate Miyakawa, T., Mizushima, H., Ohtsuka, J., Oda, M., Kawai, F., Tanokura, M., 2015. degrading hydrolase from Thermobifida fusca. Appl. Microbiol. Biotechnol. 98. + Structural basis for the Ca2 -enhanced thermostability and activity of PET-degrading Ruan, J., Qin, B., Huang, J., 2018. Controlling measures of micro-plastic and nano cutinase-like enzyme from Saccharomonospora viridis AHK190. Appl. Microbiol. pollutants: a short review of disposing waste toners. Environ. Int. 118, 92–96. Biotechnol. 99, 4297–4307. Sagong, H.-Y., Seo, H., Kim, T., Son, H.F., Joo, S., Lee, S.H., Kim, S., Woo, J.-S., Hwang, S. Moog, D., Schmitt, J., Senger, J., Zarzycki, J., Rexer, K.-H., Linne, U., Erb, T., Maier, U. Y., Kim, K.-J., 2020. Decomposition of the PET Film by MHETase Using Exo-PETase G., 2019. Using a marine microalga as a chassis for polyethylene terephthalate (PET) Function. ACS Catal. 10, 4805–4812. degradation. Microb. Cell Fact. 18, 171. Salvador, M., Abdulmutalib, U., Gonzalez, J., Kim, J., Smith, A.A., Faulon, J.-L., Wei, R., Mu, T., Zhao, J., Guan, Y., Tian, J., Yang, M., Guo, C., Xing, J., 2017. Desulfurization Zimmermann, W., Jimenez, J.I., 2019. Genes for a Circular and Sustainable Bio-PET with Thialkalivibrio versutus immobilized on magnetic nanoparticles modifiedwith 3- Economy. aminopropyltriethoxysilane. Biotechnol. Lett. 39, 865–871. Samak, N.A., Hu, J., Wang, K., Guo, C., Liu, C., 2018a. Development of a novel micro- Mückschel, B., Simon, O., Klebensberger, J., Graf, N., Rosche, B., Altenbuchner, J., aerobic cultivation strategy for high potential CotA laccase production. Waste Pfannstiel, J., Huber, A., Hauer, B., 2012. Ethylene glycol metabolism by Biomass Valorization 9, 369–377. Pseudomonas putida. Appl. Environ. Microbiol. 78, 8531–8539. Samak, N.A., Selim, M.S., Hao, Z., Xing, J., 2020. Controlled-synthesis of alumina- Müller, R.J., Schrader, H., Profe, J., Dresler, K., Deckwer, W.D., 2005. Enzymatic graphene oxide nanocomposite coupled with DNA/sulfide fluorophore for eco- degradation of poly (ethylene terephthalate): rapid hydrolyse using a hydrolase from friendly “Turn off/on” H2S nanobiosensor. Talanta 211, 120655. T. fusca. Macromol. Rapid Commun. 26, 1400–1405. Samak, N.A., Tan, Y., Sui, K., Xia, T.-T., Wang, K., Guo, C., Liu, C., 2018b. CotA laccase Nechwatal, A., Blokesch, A., Nicolai, M., Krieg, M., Kolbe, A., Wolf, M., Gerhardt, M., immobilized on functionalized magnetic graphene oxide nano-sheets for efficient 2006. A Contribution to the Investigation of Enzyme-Catalysed Hydrolysis of Poly biocatalysis. Mol. Cataly. 445, 269–278. (ethylene terephthalate) Oligomers. Macromol. Mater. Eng. 291, 1486–1494. Sasoh, M., Masai, E., Ishibashi, S., Hara, H., Kamimura, N., Miyauchi, K., Fukuda, M., Nelms, S.E., Duncan, E.M., Broderick, A.C., Galloway, T.S., Godfrey, M.H., Hamann, M., 2006. Characterization of the terephthalate degradation genes of Comamonas sp. Lindeque, P.K., Godley, B.J., 2016. Plastic and marine turtles: a review and call for strain E6. Appl. Environ. Microbiol. 72, 1825–1832. research. ICES J. Mar. Sci. 73, 165–181. Scalenghe, R., 2018. Resource or waste? A perspective of plastics degradation in soil with Numoto, N., Kamiya, N., Bekker, G.-J., Yamagami, Y., Inaba, S., Ishii, K., Uchiyama, S., a focus on end-of-life options. Heliyon 4, e00941. Kawai, F., Ito, N., Oda, M., 2018. Structural dynamics of the PET-degrading cutinase- Schilling, M., Patett, F., Schwab, W., Schrader, J., 2007. Influenceof solubility-enhancing like enzyme from Saccharomonospora viridis AHK190 in substrate-bound states fusion proteins and organic solvents on the in vitro biocatalytic performance of the + elucidates the Ca2 -driven catalytic cycle. Biochemistry 57, 5289–5300. carotenoid cleavage dioxygenase AtCCD1 in a micellar reaction system. Appl. Oda, M., Yamagami, Y., Inaba, S., Oida, T., Yamamoto, M., Kitajima, S., Kawai, F., 2018. Microbiol. Biotechnol. 75, 829. + Enzymatic hydrolysis of PET: functional roles of three Ca2 ions bound to a cutinase- Schoffelen, S., van Hest, J.C., 2013. Chemical approaches for the construction of multi- like enzyme, Cut190*, and its engineering for improved activity. Appl. Microbiol. enzyme reaction systems. Curr. Opin. Struct. Biol. 23, 613–621. Biotechnol. 102, 10067–10077. Seo, H., Kim, S., Son, H.F., Sagong, H.-Y., Joo, S., Kim, K.-J., 2019. Production of Ohlson, I., Tragårdh,¨ G., Hahn-Hagerdal,¨ B., 1984. Enzymatic hydrolysis of sodium- extracellular PETase from Ideonella sakaiensis using sec-dependent signal peptides in hydroxide-pretreated sallow in an ultrafiltration membrane reactor. Biotechnol. E. coli. Biochem. Biophys. Res. Commun. 508, 250–255. Bioeng. 26, 647–653. Seo, S., Park, Y.-G., 2020. Destination of floating plastic debris released from ten major Olajuyin, A.M., Yang, M., Liu, Y., Mu, T., Tian, J., Adaramoye, O.A., Xing, J., 2016. rivers around the Korean Peninsula. Environ. Int. 138, 105655. Efficient production of succinic acid from Palmaria palmata hydrolysate by Shan, G., Xing, J., Zhang, H., Liu, H., 2005. Biodesulfurization of dibenzothiophene by metabolically engineered Escherichia coli. Bioresour. Technol. 214, 653–659. microbial cells coated with magnetite nanoparticles. Appl. Environ. Microbiol. 71, Olson, D.G., Lynd, L.R., 2012. Transformation of Clostridium thermocellum by 4497–4502. electroporation. Methods in Enzymology: Elsevier. Sharon, C., Sharon, M., 2012. Studies on biodegradation of polyethylene terephthalate: A Ong, S.Y., Chee, J.Y., Sudesh, K., 2017. Degradation of polyhydroxyalkanoate (PHA): a synthetic polymer. J. Microbiol. Biotechnol. Res. 2, 248–257. review. Sharshar, M.M., Samak, N.A., Hao, X., Mu, T., Zhong, W., Yang, M., Peh, S., Ambreen, S., Palm, G.J., Reisky, L., Bottcher,¨ D., Müller, H., Michels, E.A., Walczak, M.C., Berndt, L., Xing, J., 2019. Enhanced growth-driven stepwise inducible expression system Weiss, M.S., Bornscheuer, U.T., Weber, G., 2019. Structure of the plastic-degrading development in haloalkaliphilic desulfurizing Thioalkalivibrio versutus. Bioresour. Ideonella sakaiensis MHETase bound to a substrate. Nat. Commun. 10, 1717. Technol. 288, 121486. Paszun, D., Spychaj, T., 1997. Chemical recycling of poly (ethylene terephthalate). Ind. Shen, Y., 2011. Isolation of intestinal bacteria from T. molitor L. and study on the Eng. Chem. Res. 36, 1373–1383. phenomenon of plastic degradation. Shanghai: Master’s Thesis of East. China Normal Pellis, A., Comerford, J.W., Weinberger, S., Guebitz, G.M., Clark, J.H., Farmer, T.J., University. 2019. Enzymatic synthesis of lignin derivable pyridine based polyesters for the Shih, Y.P., Kung, W.M., Chen, J.C., Yeh, C.H., Wang, A.H.J., Wang, T.F., 2002. High- substitution of petroleum derived plastics. Nat. Commun. 10, 1–9. throughput screening of soluble recombinant proteins. Protein Sci. 11, 1714–1719. Pellis, A., Vastano, M., Quartinello, F., Herrero Acero, E., Guebitz, G.M., 2017. His-tag Shirke, A.N., White, C., Englaender, J.A., Zwarycz, A., Butterfoss, G.L., Linhardt, R.J., immobilization of cutinase 1 from Thermobifida cellulosilytica for solvent-free Gross, R.A., 2018. Stabilizing leaf and branch compost cutinase (LCC) with synthesis of polyesters. Biotechnol. J. 12, 1700322. glycosylation: Mechanism and effect on PET hydrolysis. Biochemistry 57, Peng, C., Shi, C., Cao, X., Li, Y., Liu, F., Lu, F., 2019. Factors influencing recombinant 1190–1200. protein secretion efficiency in gram-positive bacteria: signal peptide and beyond. Front. Bioeng. Biotechnol. 7, 139.

17 N.A. Samak et al. Environment International 145 (2020) 106144

Silva, C., Da, S., Silva, N., Matama,´ T., Araújo, R., Martins, M., Chen, S., Chen, J., Wu, J., Wei, R., Oeser, T., Schmidt, J., Meier, R., Barth, M., Then, J., Zimmermann, W., 2016. Casal, M., 2011. Engineered Thermobifida fusca cutinase with increased activity on Engineered bacterial polyester hydrolases efficiently degrade polyethylene polyester substrates. Biotechnol. J. 6, 1230–1239. terephthalate due to relieved product inhibition. Biotechnol. Bioeng. 113, Simons, J.-W.F., van Kampen, M.D., Ubarretxena-Belandia, I., Cox, R.C., Alves dos 1658–1665. Santos, C.M., Egmond, M.R., Verheij, H.M., 1999. Identificationof a calcium binding Wei, R., Oeser, T., Then, J., Kühn, N., Barth, M., Schmidt, J., Zimmermann, W., 2014. site in Staphylococcus hyicus lipase: generation of calcium-independent variants. Functional characterization and structural modeling of synthetic polyester- Biochemistry 38, 2–10. degrading hydrolases from Thermomonospora curvata. AMB express 4, 44. Son, H.F., Cho, I.J., Joo, S., Seo, H., Sagong, H.-Y., Choi, S.Y., Lee, S.Y., Kim, K.-J., 2019. Wei, R., Zimmermann, W., 2017. Biocatalysis as a green route for recycling the Rational protein engineering of thermo-stable PETase from Ideonella sakaiensis for recalcitrant plastic polyethylene terephthalate. Microb. Biotechnol. 10, 1302–1307. highly efficient PET degradation. ACS Catal. 9, 3519–3526. Weinberger, S., Haernvall, K., Scaini, D., Ghazaryan, G., Zumstein, M.T., Sander, M., Sowdhamini, R., Srinivasan, N., Shoichet, B., Santi, D.V., Ramakrishnan, C., Balaram, P., Pellis, A., Guebitz, G.M., 2017. Enzymatic surface hydrolysis of poly (ethylene 1989. Stereochemical modeling of disulfide bridges. Criteria for introduction into furanoate) thin films of various crystallinities. Green Chem. 19, 5381–5384. proteins by site-directed mutagenesis. Protein Eng. Des. Sel. 3, 95–103. Welzel, K., Müller, R.J., Deckwer, W.D., 2002. Enzymatischer Abbau von Polyester- Su, A., Shirke, A., Baik, J., Zou, Y., Gross, R., 2018. Immobilized cutinases: Preparation, Nanopartikeln. Chem. Ing. Tech. 74, 1496–1500. solvent tolerance and thermal stability. Enzyme Microb. Technol. 116, 33–40. Wierckx, N., Prieto, M.A., Pomposiello, P., de Lorenzo, V., O’Connor, K., Blank, L.M., Sulaiman, S., Yamato, S., Kanaya, E., Kim, J.J., Koga, Y., Takano, K., Kanaya, S., 2012. 2015. Plastic waste as a novel substrate for industrial biotechnology. Microb. Isolation of a novel cutinase homolog with polyethylene terephthalate-degrading Biotechnol. 8, 900. activity from leaf-branch compost by using a metagenomic approach. Appl. Environ. Wilcox, C., Van Sebille, E., Hardesty, B.D., 2015. Threat of plastic pollution to seabirds is Microbiol. 78. global, pervasive, and increasing. Proc. Natl. Acad. Sci. 112, 11899–11904. Sulaiman, S., You, D.-J., Kanaya, E., Koga, Y., Kanaya, S., 2014. Crystal structure and Xia, T.-T., Liu, C.-Z., Hu, J.-H., Guo, C., 2016. Improved performance of immobilized thermodynamic and kinetic stability of metagenome-derived LC-cutinase. laccase on amine-functioned magnetic Fe3O4 nanoparticles modified with Biochemistry 53, 1858–1869. polyethylenimine. Chem. Eng. J. 295, 201–206. Then, J., Wei, R., Oeser, T., Barth, M., Belisario-Ferrari,´ M.R., Schmidt, J., Yan, F., Wei, R., Cui, Q., Bornscheuer, U.T., Liu, Y.J., 2020. Thermophilic whole-cell + + Zimmermann, W., 2015. Ca2 and Mg2 binding site engineering increases the degradation of polyethylene terephthalate using engineered Clostridium degradation of polyethylene terephthalate films by polyester hydrolases from thermocellum. Microb. Biotechnol. Thermobifida fusca. Biotechnol. J. 10, 592–598. Yang, J., Yang, Y., Wu, W.-M., Zhao, J., Jiang, L., 2014. Evidence of polyethylene Thompson, R.C., Moore, C.J., Vom Saal, F.S., Swan, S.H., 2009. Plastics, the environment biodegradation by bacterial strains from the guts of plastic-eating waxworms. and human health: current consensus and future trends. Philos. Trans. Roy. Soc. B: Environ. Sci. Technol. 48, 13776–13784. Biol. Sci. 364, 2153–2166. Yang, S.-S., Wu, W.-M., Brandon, A.M., Fan, H.-Q., Receveur, J.P., Li, Y., Wang, Z.-Y., Thumarat, U., Nakamura, R., Kawabata, T., Suzuki, H., Kawai, F., 2012. Biochemical and Fan, R., McClellan, R.L., Gao, S.-H., 2018. Ubiquity of polystyrene digestion and genetic analysis of a cutinase-type polyesterase from a thermophilic Thermobifida biodegradation within yellow mealworms, larvae of Tenebrio molitor Linnaeus alba AHK 119. Appl. Microbiol. Biotechnol. 95. (Coleoptera: Tenebrionidae). Chemosphere 212, 262–271. Tokiwa, Y., Calabia, B.P., Ugwu, C.U., Aiba, S., 2009. Biodegradability of plastics. Int. J. Yang, S., Xu, H., Yan, Q., Liu, Y., Zhou, P., Jiang, Z., 2013. A low molecular mass cutinase Mol. Sci. 10, 3722–3742. of Thielavia terrestris efficiently hydrolyzes poly (esters). J. Ind. Microbiol. Tournier, V., Topham, C., Gilles, A., David, B., Folgoas, C., Moya-Leclair, E., Biotechnol. 40, 217–226. Kamionka, E., Desrousseaux, M.-L., Texier, H., Gavalda, S., 2020. An engineered PET Yang, Y., Chen, J., Wu, W.-M., Zhao, J., Yang, J., 2015a. Complete genome sequence of depolymerase to break down and recycle plastic bottles. Nature 580, 216–219. Bacillus sp. YP1, a polyethylene-degrading bacterium from waxworm’s gut. Trelles, J.A., Rivero, C.W., 2013. Whole cell entrapment techniques. Immobilization of J. Biotechnol. 200, 77–78. enzymes and cells. Springer. Yang, Y., Yang, J., Wu, W.-M., Zhao, J., Song, Y., Gao, L., Yang, R., Jiang, L., 2015b. Trevors, J., 1992. Use of alginate and other carries for encapsulation of microbial cells Biodegradation and mineralization of polystyrene by plastic-eating mealworms: Part for use in soil. Microvial Rel 1, 61–69. 1. Chemical and physical characterization and isotopic tests. Environ. Sci. Technol. Trifunovi´c, D., Schuchmann, K., Müller, V., 2016. Ethylene glycol metabolism in the 49, 12080–12086. acetogen Acetobacterium woodii. J. Bacteriol. 198, 1058–1065. Yoshida, S., Hiraga, K., Takehana, T., Taniguchi, I., Yamaji, H., Maeda, Y., Toyohara, K., Vertommen, M., Nierstrasz, V., Van Der Veer, M., Warmoeskerken, M., 2005. Enzymatic Miyamoto, K., Kimura, Y., Oda, K., 2016. A bacterium that degrades and assimilates surface modification of poly (ethylene terephthalate). J. Biotechnol. 120, 376–386. poly (ethylene terephthalate). Science 351, 1196–1199. Wang, K.-F., Guo, C., Ju, F., Samak, N.A., Zhuang, G.-Q., Liu, C.-Z., 2018. Farnesol- Zhang, J., Liang, Q., Xie, W., Peng, L., He, L., He, Z., Chowdhury, S.P., Christensen, R., induced hyperbranched morphology with short hyphae and bulbous tips of Coriolus Ni, Y., 2019. An eco-friendly method to get a bio-based dicarboxylic acid monomer versicolor. Sci. Rep. 8, 1–12. 2, 5-furandicarboxylic acid and its application in the synthesis of poly (hexylene 2, 5- Wang, Y.Z., Zhou, Y., Zylstra, G.J., 1995. Molecular analysis of isophthalate and furandicarboxylate)(PHF). Polymers 11, 197. terephthalate degradation by Comamonas testosteroni YZW-D. Environ. Health Zur,˙ J., Wojcieszynska,´ D., Guzik, U., 2016. Metabolic responses of bacterial cells to Perspect. 103, 9–12. immobilization. Molecules 21, 958.

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