Improving the activity and thermostability of PETase from Ideonella sakaiensis through modulating its post-translational glycan modification

Abstract

AbstractThe large-scale preparation of Polyehylene terephthalate (PET) hydrolysing enzymes in low-cost is critical for the biodegradation of PET in industry. In the present study, we demonstrate that the post-translational glycosylation ofPichia pastorismakes it a remarkable host for the heterologous expression of PETase fromIdeonella sakaiensis201-F6 (IsPETase). Taking advantage of the abundant N- and O-linked glycosylation sites inIsPETase and the efficient post-translational modification in endoplasmic reticulum,IsPETase is heavily glycosylated during secretory expression withP. pastoris, which improves the specific activity and thermostability of the enzyme dramatically. Moreover, the specific activity ofIsPETase increased further after the bulky N-linked polysaccharide chains were eliminated by Endo-β-N-acetylglucosaminidase H (Endo H). Importantly, the partially deglycosylatedIsPETase still maintained high thermostability because of the remaining mono- and oligo-saccharide residues on the protein molecules. Consequently, the partially deglycosylatedIsPETase was able to be applied at 50 °C and depolymerized raw, untreated PET flakes completely in 2 to 3 days. This platform was also applied for the preparation of a famous variant ofIsPETase, Fast-PETase, and the same result was achieved. Partially deglycosylated Fast-PETase demonstrates elevated efficiency in degrading postconsumer-PET trays under 55 °C than 50 °C, the reported optimal temperature of Fast-PETase. The present study provides a strategy to modulate thermostableIsPETase through glycosylation engineering and paves the way for promoting PET biodegradation from laboratories to factories.