Engineering And Immobilizing PET Hydrolytic Enzymes For Improving Biocatalytic PET Degradation

Polyethylene terephthalate（PET）is one of the most widespread synthetic polyesters,and its accumulation has brought an enormous threat to the global ecosystem.PET biodegradation is an environmentally friendly method for PET waste disposal.The recently reported PET hydrolase（PETase）and MHET hydrolase（MHETase）can synergistically catalyze the complete degradation of PET to its initial synthetic molecules.Hence,this work was designed to promote the application of bio-catalysis in PET biodegradation by the methods of engineering PETase through enzyme fusion and protein design and immobilizing the two enzymes for cascade catalysis.First,a fusion enzyme modification strategy was introduced by fusing a zwitterionic polypeptide（5-30 k Da）consisting of glutamate（E）and lysine（K）residues with repeating alternating charges to the C-terminus of PETase.It is found that the catalytic performance of the fusion enzymes was higher than that of PETase and increased with the length of the fusion peptide.The degradation ability of highly crystallized PET films（45.2%in crystallinity）by PETase-EK30 was 11.5 times higher than that of PETase.The molecular mechanism of the improved enzyme performance was analyzed through structural analysis,substrate binding,and molecular simulations.Circular dichroism and fluorescence spectroscopic characteristics indicate that the fusion enzyme enhanced its structural stability.Synchronous fluorescence spectroscopy showed that the substrate-binding pocket of the fusion enzyme was more open.Molecular dynamics（MD）simulations of the enzyme-substrate complexes show that polypeptide fusion induces the exposure of hydrophobic amino acids（W185,I208,and W159）in the substrate-binding pocket and rotation of the phenyl ring of Y87,promoting the substrate-binding kinetics,thereby enhancing the substrate affinity.In addition,the MD simulation results show that the fusion enzyme shortens the catalytic distance with the substrate,which also helps to improve the PET degradation of the enzyme.Then,the rational design based on computer simulations was used to improve the stability of the protein by introducing salt bridge interactions in the PETase structure.Among the mutants,I168R/S188D and I168R/S188E exhibited significantly enhanced thermostability,and the Tm value was increased by 7.4 and 8.7°C,respectively.The PET degradation ability at 40°C was 3.8 and 4.3 times higher than that of wild-type PETase.It is found that the maximum fluorescence emissions of the two mutants were blue-shifted,indicating the changed three-dimensional conformations of the enzymes.The MD simulations prove that a stable salt bridge interaction is formed in the protein structure of the mutant,thus increasing the rigidity of the protein structure and enhancing the stability.Finally,Dura PETase and MHETase were sequentially co-immobilized in calcium phosphate nanocrystals（～1.5μm）via biomimetic mineralization through Spy Tag/Spy Catcher system.Dura PETase was located on the outlayer of the nanocrystals,facilitating the interaction with the substrate.The layered structures of the nanocrystals could protect the active center conformation of the enzyme,hence enhancing the stability of the enzyme.The high specific surface area of the nanocrystals and the substrate channeling from the dual-enzymatic cascade were beneficial to the improved enzyme activity.MD simulations showed that the Ca²⁺-induced enzyme conformational changes increased the substrate affinity.In a word,the synergistic effects of these factors promoted Dura PETase@MHETase to exhibit an excellent PET degradation performance.The dual-enzymatic cascade with Dura PETase@MHETase can completely degrade PET,contributing to the recycling of PET.