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

Abstract

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 SbPETase from Schlegelella brevitalea sp. nov. was biochemically characterized, and rational design was performed based on its sequence similarity with the previously reported IsPETase from Ideonella sakaiensis, 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 Escherichia coli 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 IsPETase and SbPETase through DNA shuffling. Finally, we generated a mutant IsPETaseS139T with increased activity from the mutant library.

Keywords

• PETase
• Protein secretion
• Directed evolution
• DNA shuffling
• High-throughput screening