Development and Production of Moderate-Thermophilic PET Hydrolase for PET Bottle and Fiber Recycling

Polyethylene terephthalate (PET) is a widely utilized synthetic polymer found in bottles, textiles, and packaging materials. To enhance the activity of PET hydrolase PET2, seven new mutations were identified through random screening and combined with a previously reported variant, PET2-7M, to generate PET2-14M. In addition, two mutations that enhanced the positive charge on the binding surface of PET were identified. Concurrently, the substrate binding site of PET2-14M was modified using HotPETase as a structural reference, yielding PET2-14M-6Hot. Subsequently, the two mutations were introduced into PET2-14M-6Hot, considering the optimization screening results, and positively charged mutations were also combined. The final variant, PET2-21M, demonstrated a 28.6-fold increase in the concentration of total product compared with the PET2 wild-type. Expression systems for PET2-14M-6Hot and PET2-21M were established inKomagataella phaffii, producing up to 691 mg L–1 of PET2-14M-6Hot after 137 h of cultivation. PET2-21M (5 mg L–1) completely degraded 20 g L–1 powder of commercial PET bottles within 24 h at 60 °C. Furthermore, PET2-14M-6Hot showed twice the degradation rate of LCC-ICCG on PET fiber and PET/cotton-blended fiber and achieved 2.2 times higher product yield than LCC-ICCG on PET/PU-blended fiber at 50 °C. These engineered PET2 variants could be candidates for recycling both pure and blended PET fibers under moderate-temperature conditions.