Construction And Application Of Whole-cell Biocatalyst System For Efficient Biodegradation Of Highly Crystallized Polyethylene Terephthalate(PET)

Polyethylene terephthalate(PET)is one of the most widely used plastics in the world.Accumulation of the discarded PET in the environment has caused a global environmental pollution problem,and the enzymatic degradation of high crystallinity PET(hcPET)is facing great challenges.In recent years,a bacterial enzyme named PETase has been found to have the ability to degrade hcPET.However,the enzyme activity of wild-type PETase is still low,which limits its application in PET recycling.In this study,we developed a whole-cell biocatalyst by displaying PETase on the surface of yeast(Pichia pastoris)cells to improve its degradation efficiency.Our data shows that PETase could be functionally displayed on the yeast cells,and the anchoring protein and induction time have an important effect on degradation efficiency.Next,through a series of tests to verify the improvement of pH and thermal stability.The turnover rate of whole-cell biocatalysts displaying PETase towards hcPET dramatically increased about 36-fold compared with that of purified PETase.Furthermore,the whole-cell biocatalyst showed stable turnover rate after 7 repeated use and under some chemical/solvent conditions,and its ability to degrade different commercial hcPET bottles.To further optimize the surface display system and increase enzyme activity,we introduced hydrophobin to construct a co-display system to simulate the adsorption and degradation steps in the enzymatic degradation process.Our data indicate that the hydrophobin HFBI and PETase are successfully displayed on the surface of yeast cells.In addition,it was confirmed that the displayed HFBI can enhance the adsorption of codisplayed cells on the surface of hydrophobic hcPET by increasing the hydrophobicity of yeast cells.Compared with PETase,the turnover rate of co-display cells to hcPET increased significantly by about 332-fold.In addition,co-display cells can also be reused and have a certain resistance.Finally,using the co-displayed whole-cell catalyst to degrade several commercial hcPET bottles,the decomposition rate was increased from 0.01 % to 3.12 %.This research is of breakthrough significance for the decomposition of hcPET,and also provides a strategy for the development of other high-performance co-display systems.