Modification Of PETase:Effect Of Monomer Property On The Modified Enzyme Performance

Polyethylene terephthalate（PET）,which is a common plastic widely used in modern society.Because of the chemical properties of ester bond,structure of PET is very stable and not easy to be degraded.So,the massive accumulation of PET waste in the environment poses serious threats to human health and the ecosystem.Compared with thermophilic PET hydrolases,PETase,a recently reported PET hydrolases,can degrade PET at mild temperature efficiently,but its poor stability limits its application in actual industrial production.Therefore,improving the enzymatic properties of PETase through biotechnology effectively has significant value in PETase research,providing more efficient and stable enzyme for biodegradation of PET.Monomer of hydrophobic Tert-Butyl Methacrylate（TBMA）,hydrophilic Hydroxyethyl Methacrylate（HEMA）,positively charged monomer 2-（Dimethylamino）ethyl methacrylate（DMAEMA）and negatively charged monomer Methacrylic acid（MA）were selected for modification of PETase.The modification rate was low,monomer/enzyme molecule was about 2.9～5.4.Studies showed that the degradation activity of each polymer-modified PETase was higher than that of PETase（the ratio of the enzyme activity to PETase is 1.4-2.7）with bis（hydroxyethyl）terephthalate（BHET）as a substrate.Among them,DMAEMA-PETase had the highest activity,with 2.7 times higher than PETase,and its catalytic efficiency(k_cat/K_m)was 1.6 times that of PETase.The optimal temperature of polymer-modified PETases did not change significantly,but the thermal stability and p H tolerance of the modified enzyme were better than PETase,with TBMA-PETase possessed the highest thermal stability.Circular dichroism and fluorescence spectroscopy analysis showed that the secondary and tertiary structure of polymer-modified PETases were more compact and stable compared with PETase,which may be the main reason for the improved stability of polymer-modified PETases.In order to investigate effect of polymer-modified PETases in practical application,PET film was degraded continuously as a substrate for 6 days for degradation analysis.The four polymer-modified PETases showed significantly enhanced PET degradation performance at 30°C and 40°C.TBMA-PETase had the best degradation effect on PET film among four polymer-modified PETases,which was 2.6 times higher than PETase.On the one hand,the higher thermal stability of TBMA-PETase resulted in higher enzyme activity during the period of continuous degradation;on the other hand,the hydrophobicity of TBMA may enhanced the affinity of polymer-modified PETase with PET,improving the catalytic ability of TBMA-PETase to PET.In this research,the results showed that chemical modification is an effective approach to improve the catalytic performance of PETase.Choosing suitable modification methods and polymers for modification is expected to obtain PETase modified products with higher catalytic performance.