scholarly journals Development of a targeted gene disruption system in the PET-degrading bacterium Ideonella sakaiensis and its applications to PETase and MHETase genes

2021 ◽  
Author(s):  
Shin-ichi Hachisuka ◽  
Tarou Nishii ◽  
Shosuke Yoshida
Keyword(s):  
Terephthalic Acid ◽  
Parent Strain ◽  
Gene Disruption ◽  
Ethylene Terephthalate ◽  
Targeted Gene Disruption ◽  
Degrading Bacterium ◽  
Poly Ethylene Terephthalate ◽  
Poly Ethylene ◽  
Pet Hydrolase

Poly(ethylene terephthalate) (PET) is a commonly used synthetic plastic; however its non-biodegradability results in a large amount of waste accumulation that has a negative impact on the environment. Recently, a PET-degrading bacterium Ideonella sakaiensis 201-F6 strain was isolated and the enzymes involved in PET-digestion, PET hydrolase (PETase) and mono(2-hydroxyethyl) terephthalic acid (MHET) hydrolase (MHETase), were identified. Despite the great potentials of I. sakaiensis in bioremediation and biorecycling, approaches to studying this bacterium remain limited. In this study, to enable the functional analysis of PETase and MHETase genes in vivo , we have developed a gene disruption system in I. sakaiensis . The pT18 mobsacB -based disruption vector harboring directly connected 5'- and 3'-flanking regions of the target gene for homologous recombination was introduced into I. sakaiensis cells via conjugation. First, we deleted the orotidine 5'-phosphate decarboxylase gene ( pyrF ) from the genome of the wild-type strain, producing the Δ pyrF strain with 5-fluoroorotic acid (5-FOA) resistance. Next, using the Δ pyrF strain as a parent strain, and pyrF as a counterselection marker, we disrupted the genes for PETase and MHETase. The growth of both Δ petase and Δ mhetase strains on terephthalic acid (TPA, one of the PET hydrolytic products) was comparable to that of the parent strain. However, these mutant strains dramatically decreased the growth level on PET to that on no carbon source. Moreover, the Δ petase strain completely abolished PET degradation capacity. These results demonstrate that PETase and MHETase are essential for I. sakaiensis metabolism of PET. IMPORTANCE The poly(ethylene terephthalate) (PET)-degrading bacterium Ideonella sakaiensis possesses two unique enzymes able to serve in PET hydrolysis. PET hydrolase (PETase) hydrolyzes PET into mono(2-hydroxyethyl) terephthalic acid (MHET) and MHET hydrolase (MHETase) hydrolyzes MHET into terephthalic acid (TPA) and ethylene glycol (EG). These enzymes have attracted global attention as they have potential to be used for bioconversion of PET. Compared to many in vitro studies including the biochemical and crystal structure analyses, few in vivo studies have been reported. Here, we developed a targeted gene disruption system in I. sakaiensis , which was then applied for constructing Δ petase and Δ mhetase strains. Growth of these disruptants revealed that PETase is a sole enzyme responsible for PET degradation in I. sakaiensis , while PETase and MHETase play essential roles in its PET assimilation.

Depolymerization of Poly(ethylene terephthalate) to Terephthalic Acid and Ethylene Glycol in High-temperature Liquid Water

2009 ◽  
Vol 38 (3) ◽  
pp. 268-269 ◽  
Author(s):  
Osamu Sato ◽  
Eiichi Mine ◽  
Mitsumasa Osada ◽  
Norihito Hiyoshi ◽  
Aritomo Yamaguchi ◽  
...  
Keyword(s):  
High Temperature ◽  
Ethylene Glycol ◽  
Liquid Water ◽  
Terephthalic Acid ◽  
Ethylene Terephthalate ◽  
Poly Ethylene Terephthalate ◽  
Temperature Liquid ◽  
Poly Ethylene

Microwave-assisted esterification step of poly(ethylene terephthalate) (PET) synthesis through ethylene glycol and terephthalic acid

2018 ◽  
Vol 76 (6) ◽  
pp. 2931-2944 ◽  
Author(s):  
Ana Christy Espinosa-López ◽  
Carlos Alberto Ávila-Orta ◽  
Francisco Javier Medellín-Rodríguez ◽  
Pablo González-Morones ◽  
Carlos Alberto Gallardo-Vega ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Terephthalic Acid ◽  
Ethylene Terephthalate ◽  
Microwave Assisted ◽  
Poly Ethylene Terephthalate ◽  
Poly Ethylene

Synthesis of ethylene glycol and terephthalic acid from biomass for producing PET

2016 ◽  
Vol 18 (2) ◽  
pp. 342-359 ◽  
Author(s):  
Jifeng Pang ◽  
Mingyuan Zheng ◽  
Ruiyan Sun ◽  
Aiqin Wang ◽  
Xiaodong Wang ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Terephthalic Acid ◽  
Ethylene Terephthalate ◽  
Recent Advances ◽  
Poly Ethylene Terephthalate ◽  
Poly Ethylene

This review presents recent advances in typical routes for drop-in replacement of poly(ethylene terephthalate) monomers from biomass.

Antimicrobial nature and healing behavior of plasma functionalized polyester sutures

2016 ◽  
Vol 32 (3) ◽  
pp. 263-279 ◽  
Author(s):  
Sadiya Anjum ◽  
Amlan Gupta ◽  
Deepika Sharma ◽  
Shanti Kumari ◽  
Plabita Sahariah ◽  
...  
Keyword(s):  
Ethylene Terephthalate ◽  
Antimicrobial Properties ◽  
Silver Ions ◽  
Surgical Infection ◽  
Surgical Suture ◽  
Poly Ethylene Terephthalate ◽  
Poly Ethylene ◽  
Fast Healing ◽  
Carbon Dioxide Plasma

This study deals with the development of bioactive poly(ethylene terephthalate) surgical suture by adopting the immobilization route with bioactive nanogels and chlorhexidine. Carbon dioxide plasma was used for the generation of carboxyl functionality on poly(ethylene terephthalate) surface for the immobilization of the bioactive components. The nanosilver nanogel was prepared using polyethylene glycol which helps in the reduction of silver ions into nanosilver as well as the stabilization of nanoparticles. The particle size of the nanogels, as evaluated by high-resolution transmission electron microscopy, was observed to be in the range of 10–50 nm. Surface functionalization of poly(ethylene terephthalate) filament was observed by attenuated total reflectance measurements and mechanical studies were investigated by Instron. Elemental analysis and surface topography were carried out by energy dispersive X-ray and atomic force microscopy. The cumulative release of silver from the dressing was found to be 68% of the total loading after 72 h. Coated sutures have excellent antimicrobial activity against both Escherichia coli and Staphylococcus aureus. In vivo wound healing and histopathology studies were carried out over a period of 72 h for skin wounds created on Swiss albino mice. Fast healing was observed in nanogel-treated wounds without any inflammatory effects on the newly generated skin. These sutures offer improved healing along with excellent antimicrobial properties and appear to be promising material against surgical infection.

scholarly journals Assessment of the PETase Conformational Changes Induced by Poly(ethylene terephthalate) Binding

2021 ◽  
Author(s):  
Clauber Henrique Costa ◽  
Alberto dos Santos ◽  
Cláudio Nahum Alves ◽  
Sérgio Martí ◽  
Vicente Moliner ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Conformational Changes ◽  
Active Site ◽  
Terephthalic Acid ◽  
Structural Changes ◽  
Ethylene Terephthalate ◽  
Md Simulations ◽  
Poly Ethylene Terephthalate ◽  
Bounded State ◽  
Poly Ethylene

Recently, a bacterium strain of Ideonella sakaiensis was identified with the uncommon ability to degrade the poly(ethylene terephthalate) (PET). The PETase from I. sakaiensis strain 201-F6 catalyzes the hydrolysis of PET converting it to mono(2-hydroxyethyl) terephthalic acid (MHET), bis(2-hydroxyethyl)-TPA (BHET), and terephthalic acid (TPA). Despite the potential of this enzyme for mitigation or elimination of environmental contaminants, one of the limitations of the use of PETase for PET degradation is the fact that it acts only at moderate temperature due to its low thermal stability. Besides, molecular details of the main interaction of PET in the active site of PETase remains unclear. Herein, molecular docking and molecular dynamics (MD) simulations were applied to analyze structural changes of PETase induced by PET binding. Results from the essential dynamics revealed that β1-β2 connecting loop is very flexible. This Loop is located far from the active site of PETase and we suggest that it can be considered for mutagenesis in order to increase the thermal stability of PETase. The free energy landscape (FEL) demonstrates that the main change in the transition between the unbounded to the bounded state is associated with β7-α5 connecting loop, where the catalytic residue Asp206 is located. Overall, the present study provides insights into the molecular binding mechanism of PET into the PETase structure and a computational strategy for mapping flexible regions of this enzyme, which can be useful for the engineering of more efficient enzymes for recycling the plastic polymers using biological systems.

scholarly journals Chemical and Electrochemical Recycling of End-Use Poly(ethylene terephthalate) (PET) Plastics in Batch, Microwave and Electrochemical Reactors

Molecules ◽  
2020 ◽  
Vol 25 (12) ◽  
pp. 2742
Author(s):  
Tessa H. T. Myren ◽  
Taylor A. Stinson ◽  
Zachary J. Mast ◽  
Chloe G. Huntzinger ◽  
Oana R. Luca
Keyword(s):  
Terephthalic Acid ◽  
Ethylene Terephthalate ◽  
Low Cost ◽  
Supporting Electrolyte ◽  
Chemical Recycling ◽  
High Concentration ◽  
Electrochemical Reactors ◽  
Poly Ethylene Terephthalate ◽  
Poly Ethylene ◽  
End Use

This work describes new methods for the chemical recycling of end-use poly(ethylene terephthalate) (PET) in batch, microwave and electrochemical reactors. The reactions are based on basic hydrolysis of the ester moieties in the polymer framework and occur under mild reaction conditions with low-cost reagents. We report end-use PET depolymerization in refluxing methanol with added NaOH with 75% yield of terephthalic acid in batch after 12 h, while yields up to 65% can be observed after only 40 min under microwave irradiation at 85 °C. Using basic conditions produced in the electrochemical reduction of protic solvents, electrolytic experiments have been shown to produce 17% terephthalic acid after 1 h of electrolysis at −2.2 V vs. Ag/AgCl in 50% water/methanol mixtures with NaCl as a supporting electrolyte. The latter method avoids the use of caustic solutions containing high-concentration NaOH at the outset, thus proving the concept for a novel, environmentally benign method for the electrochemical recycling of end-use PET based on low-cost solvents (water and methanol) and reagents (NaCl and electricity).

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