Directed Evolution of an Efficient Polycarbonate Depolymerase With Exceptional Operational Stability

We recently developed a high-throughput directed evolution platform for engineering polymer degrading enzymes, and showcased its utility through the development of an efficient and thermostable variant of IsPETase, termed HotPETase. Here, we show that this platform can be used to re-engineer PET degrading enzymes for the recycling of other aromatic-containing commodity polymers. Promiscuous poly(bisphenol-A carbonate) (PC) depolymerase activity of LCC^ICCG was enhanced by directed evolution to afford an engineered polycarbonate hydrolase, that also benefits from improved solvent tolerance and operational stability at elevated temperatures. Interestingly, the enzyme-concentration dependent inhibition observed with the parent enzyme is also alleviated through evolution, improving practical utility. PC-2 can achieve rapid and complete depolymerization of a PC film to bisphenol-A (BPA) in just 6 h at 75°C. This study shows how plastic degrading enzymes can be readily adapted through evolution to operate on new and valuable polymer classes.