Enzymatic Depolymerization of Synthetic Polyesters by Microbial Carboxylesterases

Several types of biodegradable polyesters with different physical properties have emerged to replace traditional petroleum-based polymers, including polylactic acid (PLA). However, the lack of a sustainable recycling strategy for most biodegradable plastics and their accumulation in aquatic environments necessitate the design of ecologically benign polymers with improved life cycles. Enzymatic degradation of polyesters may not only serve as an energy-efficient approach to convert and re-use post-consumer plastics, but will also pave the way to design plastics with improved biodegradability. Although a number of polyesterase enzymes have been identified, the growing global demand for novel plastics and polymers has stimulated an interest in biocatalytic approaches for plastics recycling technologies. The discovery of novel polyesterases with high activity against a broad range of complex synthetic polyesters remains a key challenge for the development of enzyme-based plastic recycling. In this work, over 200 purified uncharacterized hydrolases from different microbial genomes and metagenomes were screened for esterase activity against polylactic acid, polycaprolactone (PCL), and the model polyethylene terephthalate substrate 3PET producing 39 active enzymes. Direct, activity-based screening of purified uncharacterized hydrolases has revealed the presence of polyesterase activity in several uncharacterized hydrolases from diverse esterase families, whose activity could not have been predicted using current bioinformatics tools. In this work, 17 novel polyester-degrading enzymes were biochemically characterized using both mono-ester and polyester substrates. Some of the identified polyesterases catalysed complete or extensive hydrolysis of solid PLA with the production of soluble lactic acid oligomers and monomers. Agarose-based assays with 24 different synthetic polyesters demonstrated a broad substrate range of the six identified polyesterases. Moreover, the crystal structures of two polyester- hydrolases were determined and the active site residues critical for the enzymatic activity were identified using site-directed mutagenesis. The crystal structure of the RPA1511 polyesterase from Rhodopseudomonas palustris revealed the presence of a molecule of polyethylene glycol bound close to the catalytic Ser residue, which represents the first structure of the polyesterase-substrate mimic complex. The results presented in this thesis indicate that microbial carboxylesterases can hydrolyze a broad range of polyesters with high rates, making them attractive biocatalysts for plastics depolymerization and recycling.Ph.D.2019-02-09 00:00:0