Characterisation and engineering of plastic degrading enzymes Biokatalyse Proteindesign High throughput screening Biosensor High throughput assay PET PET Hydrolases Protein Engineering This thesis deals with the characterisation and engineering of new thermophilic PET hydrolases as potential candidates for an eco friendly biocatalytic recycling approach for the upcycling or downcycling of polyethylene terephthalate (PET) on industrial scale. Furthermore high throughput screening methods are described that detect the products of PET hydrolysis. The high demand of PET in the packaging and textile industries with a global production of 82 million metric tons per year has significantly contributed to the global solid waste stream and environmental plastic pollution after its end of life. Although PET hydrolases have been identified in various microorganisms only a handful of benchmark enzymes have been engineered for industrial applications. Therefore the identification of new PET hydrolases from metagenomes or via protein engineering approaches especially thermophilic PET hydrolases with optimal operating temperatures (i.e. increased thermostability and activity) near the glass transition temperature of the polymer PET is a crucial step towards a bio based circular plastic economy. Article I demonstrates that metagenome derived thermophilic PET hydrolases can be significantly improved using different engineering approaches to achieve a similar activity level as the well established leaf branch compost cutinase (LCC) F243I D238C S283C Y127G variant (LCC ICCG). In Article II thermostable variants of a mesophilic enzyme (PETase from Ideonella sakaiensis) ...