Sequentially co-immobilized PET and MHET hydrolases via Spy chemistry in calcium phosphate nanocrystals present high-performance PET degradation.

Accumulation of polyethylene terephthalate (PET) has brought an enormous threat to the ecosystem. The recently reported PET hydrolase (DuraPETase) and MHET hydrolase (MHETase) can synergistically catalyze the complete PET degradation. Hence, this work was designed to develop a bienzymatic cascade catalysis by co-immobilizing the two enzymes for PET biodegradation. DuraPETase and MHETase were sequentially co-immobilized in calcium phosphate nanocrystals (CaP) through SpyTag/SpyCatcher system. MHETase-SpyCatcher was first embedded inside the nanocrystals via biomimetic mineralization, and DuraPETase-SpyTag was then conjugated on the outlayer (~1.5 µm). The bienzyme compartmentalization facilitated DuraPETase interaction with the solid substrate, and the layered structures of the nanocrystals protected the enzymes, thus enhancing their stability. The high specific surface area of the nanocrystals and the proximity effects from the bienzymatic cascade were beneficial to the improved enzyme activity. Experimental data and molecular dynamics simulations revealed the activation effect of Ca on DuraPETase. Taken together, the final results indicate that the PET degradation efficiency of DuraPETase-MHETase@CaP increased by 6.1 and 1.5 times over the free bienzyme system within 10 d at 40 °C and 50 °C, with weight losses at 32.2% and 50.3%, respectively. The bienzymatic cascade with DuraPETase-MHETase@CaP can completely degrade PET, contributing to the recycling of PET.