A disulfide bridge in the calcium binding site of a polyester hydrolase increases its thermal stability and activity against polyethylene terephthalate

• Main Authors: Then, Johannes, Wei, Ren, Oeser, Thorsten, Gerdts, André, Schmidt, Juliane, Barth, Markus, Zimmermann, Wolfgang
• Other Authors: Universität Leipzig, Fakultät für Biowissenschaften, Pharmazie und Psychologie
• Format: Article
• Language: English
• Published: Universitätsbibliothek Leipzig 2016
• Subjects: Biokatalyse; Kalzium; Disulfidbrücke; Polyethylenterephthalat; Protein-Engineering; Proteinstabilität; biocatalysis; calcium; disulfide bridge; polyethylene terephthalate; protein engineering; protein stability; ddc:540; ddc:570
• Online Access: http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-205582; http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-205582; http://www.qucosa.de/fileadmin/data/qucosa/documents/20558/OAP-2016-090_Zimmermann_feb412053.pdf

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.