title: 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.
authors.affiliation: Department of Microbiology and Bioprocess Technology, Institute of Biochemistry, Leipzig University, Johannisallee 21-23, 04103 Leipzig, Germany.
authors.forename: Ren
authors.initials: R
authors.lastname: Wei
authors.affiliation: Department of Microbiology and Bioprocess Technology, Institute of Biochemistry, Leipzig University, Johannisallee 21-23, 04103 Leipzig, Germany.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Oeser
authors.affiliation: Department of Microbiology and Bioprocess Technology, Institute of Biochemistry, Leipzig University, Johannisallee 21-23, 04103 Leipzig, Germany.
authors.forename: Juliane
authors.initials: J
authors.lastname: Schmidt
authors.affiliation: Department of Biochemistry and Bioorganic Chemistry, Institute of Biochemistry, Leipzig University, Leipzig, Germany.
authors.forename: René
authors.initials: R
authors.lastname: Meier
authors.affiliation: Department of Microbiology and Bioprocess Technology, Institute of Biochemistry, Leipzig University, Johannisallee 21-23, 04103 Leipzig, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Barth
authors.affiliation: Department of Microbiology and Bioprocess Technology, Institute of Biochemistry, Leipzig University, Johannisallee 21-23, 04103 Leipzig, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Then
authors.affiliation: Department of Microbiology and Bioprocess Technology, Institute of Biochemistry, Leipzig University, Johannisallee 21-23, 04103 Leipzig, Germany. wolfgang.zimmermann@uni-leipzig.de.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Zimmermann
mesh_terms: D039903:Actinobacteria / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D004795:Enzyme Stability; D004926:Escherichia coli; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D011091:Polyesters / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D011093:Polyethylene Terephthalates / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011091:Polyesters; D011093:Polyethylene Terephthalates; D011994:Recombinant Proteins; D006867:Hydrolases
keywords: biocatalysis; hydrolase; polyester; polyethylene terephthalate; product inhibition