Biochemical characterization of a polyethylene terephthalate hydrolase and design of high-throughput screening for its directed evolution.

Polyethylene terephthalate (PET), one of the most widely used plastics in the world, causes serious environmental pollution. Recently, researchers have focused their efforts on enzymatic degradation of PET, which is an attractive way of degrading and recycling PET. In this work, PET hydrolase PETase from sp. nov. was biochemically characterized, and rational design was performed based on its sequence similarity with the previously reported PETase from , resulting in a triple mutant with increased activity. Furthermore, using a sec-dependent signal peptide PeIB and colicin release protein Kil, we set up a high-efficiency secretion system of PETase in BL21(DE3), enabling higher PETase secretion. Utilizing this secretion system, we established a high-throughput screening method named SecHTS (secretion-based high-throughput screening) and performed directed evolution of PETase and PETase through DNA shuffling. Finally, we generated a mutant PETase with increased activity from the mutant library.