Selective Hydrolysis by Engineered Cutinases: Characterization of Aliphatic-Aromatic Homo and Co-Polyesters by LC and LC-MS Methods

The performance, biodegradability, and recyclability of polymers can be tuned during synthesis by adopting monomers with different chemical characteristics. Recent research has shown the aptness of some hydrolases to depolymerize polyesters under mild conditions compared to chemical approaches. Herein, we engineered a cutinase from Thermobifida cellulosilytica (Tc_Cut2NVWCCG) for improved thermostability (up to 91 degrees C) and compared it with previously reported leaf-branch compost cutinase (LCCWCCG) for the hydrolysis of low molar mass substrates, as well as aliphatic and aromatic homo- and co-polyesters. For both enzymes, higher hydrolysis rates were observed for aliphatic compared to aromatic homo-polyesters. SEC-MS analysis revealed that the hydrolysis of aliphatic/aromatic co-polyesters occurred at the aliphatic monomers, significantly reducing the molecular weight and changing the end-group composition. These results underline the importance of co-polymer composition in the biodegradation of co-polymer systems and demonstrate the applicability of enzymes for the analytical characterization of synthetic polymers by selectively reducing their molecular weight. Finally, the discovery and engineering of highly active enzymes that can efficiently hydrolyze a wide variety of synthetic polyesters create new opportunities for their efficient recycling under mild conditions.