title: A disulfide bridge in the calcium binding site of a polyester hydrolase increases its thermal stability and activity against polyethylene terephthalate.
abstract: Elevated reaction temperatures are crucial for the efficient enzymatic degradation of polyethylene terephthalate (PET). A disulfide bridge was introduced to the polyester hydrolase TfCut2 to substitute its calcium binding site. The melting point of the resulting variant increased to 94.7 °C (wild-type TfCut2: 69.8 °C) and its half-inactivation temperature to 84.6 °C (TfCut2: 67.3 °C). The variant D204C-E253C-D174R obtained by introducing further mutations at vicinal residues showed a temperature optimum between 75 and 80 °C compared to 65 and 70 °C of the wild-type enzyme. The variant caused a weight loss of PET films of 25.0 ± 0.8% (TfCut2: 0.3 ± 0.1%) at 70 °C after a reaction time of 48 h. The results demonstrate that a highly efficient and calcium-independent thermostable polyester hydrolase can be obtained by replacing its calcium binding site with a disulfide bridge.
authors.affiliation: Department of Microbiology and Bioprocess Technology Institute of Biochemistry Leipzig University Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Then
authors.affiliation: Department of Microbiology and Bioprocess Technology Institute of Biochemistry Leipzig University Germany.
authors.forename: Ren
authors.initials: R
authors.lastname: Wei
authors.affiliation: Department of Microbiology and Bioprocess Technology Institute of Biochemistry Leipzig University Germany.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Oeser
authors.affiliation: Department of Microbiology and Bioprocess Technology Institute of Biochemistry Leipzig University Germany.
authors.forename: André
authors.initials: A
authors.lastname: Gerdts
authors.affiliation: Department of Microbiology and Bioprocess Technology Institute of Biochemistry Leipzig University Germany.
authors.forename: Juliane
authors.initials: J
authors.lastname: Schmidt
authors.affiliation: Department of Microbiology and Bioprocess Technology Institute of Biochemistry Leipzig University Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Barth
authors.affiliation: Department of Microbiology and Bioprocess Technology Institute of Biochemistry Leipzig University Germany.
authors.forename: Wolfgang
authors.identifier: 0000-0002-5730-6663
authors.initials: W
authors.lastname: Zimmermann
keywords: biocatalysis; calcium; disulfide bridge; polyethylene terephthalate; protein engineering; protein stability