Complete Depolymerization of PET Wastes by an Evolved PET Hydrolase from Directed Evolution

Abstract

PETase displays great potential in PET depolymerization. Directed evolution has been limited to engineer PETase due to the lack of high-throughput screening assay. In this study, a novel fluorescence-based high-throughput screening assay employing a newly designed substrate, bis (2-hydroxyethyl) 2-hydroxyterephthalate (termed BHET-OH), was developed for PET hydrolases. The best variant DepoPETase produced 1407-fold more products towards amorphous PET film at 50 °C and showed a 23.3 °C higher T m value than the PETase WT. DepoPETase enabled complete depolymerization of seven untreated PET wastes and 19.1 g PET waste (0.4 % W enzyme /W PET ) in liter-scale reactor, suggesting that it is a potential candidate for industrial PET depolymerization processes. The molecular dynamic simulations revealed that the distal substitutions stabilized the loops around the active sites and transmitted the stabilization effect to the active sites through enhancing inter-loop interactions network.

De novo design of a polycarbonate hydrolase

“…The results indicate that PETases are strongly bound in sequence and topology, and as a consequence may be restricted in terms of achievable stability and catalytic efficiency, potentially limiting optimization. Indeed, directed evolution and rational design of PETases to increase melting temperature show diminishing returns in terms of activity [24,28,30]. It is thus of interest to design enzymes with activity towards plastics from highly thermostable scaffolds with a low homology to natural PETases as to escape the sequence space by which they appear to be confined.…”

“…IsPETase has undergone several iterations of protein engineering to improve thermostability and thereby appararent activity. The wildtype T M of 45.1 • C has been increased through rational design or structural comparison with other PET hydrolases [20,21,22,23,24,25,26], machine learning [27], and directed evolution [24,28,29,30].…”

Advancements in the Bio-degradation of Plastic Waste into Value-added Chemicals: A Recent Perspective

“…Directed evolution was conducted on PETase using a novel high-throughput fluorescence screening assay, which utilized a novel substrate bis(2-hydroxyethyl)2hydroxyterephthalate (BHET-OH). The generated DepoPETase enabled complete depolymerization of untreated PET wastes at mild temperature [67]. The novel PET hydrolase CaPETase, which combined the advantages of PETase and LCC, exhibited high catalytic activity and high thermal stability at ambient temperature.…”