Engineered bacterial polyester hydrolases efficiently degrade polyethylene terephthalate due to relieved product inhibition

Abstract

Recent studies on the enzymatic degradation of synthetic polyesters have shown the potential of polyester hydrolases from thermophilic actinomycetes for modifying or degrading polyethylene terephthalate (PET). TfCut2 from Thermobifida fusca KW3 and LC-cutinase (LCC) isolated from a compost metagenome are remarkably active polyester hydrolases with high sequence and structural similarity. Both enzymes exhibit an exposed active site in a substrate binding groove located at the protein surface. By exchanging selected amino acid residues of TfCut2 involved in substrate binding with those present in LCC, enzyme variants with increased PET hydrolytic activity at 65°C were obtained. The highest activity in hydrolyzing PET films and fibers were detected with the single variant G62A and the double variant G62A/I213S. Both variants caused a weight loss of PET films of more than 42% after 50 h of hydrolysis, corresponding to a 2.7-fold increase compared to the wild type enzyme. Kinetic analysis based on the released PET hydrolysis products confirmed the superior hydrolytic activity of G62A with a fourfold higher hydrolysis rate constant and a 1.5-fold lower substrate binding constant than those of the wild type enzyme. Mono-(2-hydroxyethyl) terephthalate is a strong inhibitor of TfCut2. A determination of the Rosetta binding energy suggested a reduced interaction of G62A with 2PET, a dimer of the PET monomer ethylene terephthalate. Indeed, G62A revealed a 5.5-fold lower binding constant to the inhibitor than the wild type enzyme indicating that its increased PET hydrolysis activity is the result of a relieved product inhibition by mono-(2-hydroxyethyl) terephthalate. Biotechnol. Bioeng. 2016;113: 1658-1665. © 2016 Wiley Periodicals, Inc.

Screening of commercial enzymes for poly(ethylene terephthalate) (PET) hydrolysis and synergy studies on different substrate sources

“…The best variant of the engineered enzyme yielded a conversion of 42.6%, considerably higher than that found with the wild-type cutinase (15.9%), and the increase was attributed to relieved product inhibition [29]. The best variant of the engineered enzyme yielded a conversion of 42.6%, considerably higher than that found with the wild-type cutinase (15.9%), and the increase was attributed to relieved product inhibition [29].…”

Biocatalytic Degradation Efficiency of Postconsumer Polyethylene Terephthalate Packaging Determined by Their Polymer Microstructures

“…[38] DSC Analysis of the PET Materials: Differential scanning calorimetry was performed using a DSC 8500 calorimeter (PerkinElmer, Waltham, USA) using ≈4-5 mg of a dry PET sample. [38] DSC Analysis of the PET Materials: Differential scanning calorimetry was performed using a DSC 8500 calorimeter (PerkinElmer, Waltham, USA) using ≈4-5 mg of a dry PET sample.…”

Biodegradation of diethyl terephthalate and polyethylene terephthalate by a novel identified degraderDelftiasp. WL-3 and its proposed metabolic pathway

“…; Wei et al . ). However, the micro‐organisms reported to date can only transform PET to TPA, and its further degradation process is not yet clear, which may bring more serious ecotoxicity compared to PET materials themselves (Barth et al .…”

“…However, 72% of plastic packaging is not recovered with 32% estimated to completely escape the collection system and 40% being landfilled (Wei et al . ), which released into the local ecosystems causing serious environmental pollution (Barth et al . 2015a).…”