Structural Research And Molecular Modification Of A Novel PET Hydrolytic Enzyme

Polyethylene terephthalate(PET)is one of the most productive synthetic polymer,which is produced by polycondensation of terephthalic acid and ethylene glycol.Due to its excellent physical and chemical properties,PET has been widely used in industrial fields and daily life.Although PET brings convenience,the non-degradable PET wastes has caused serious environmental pollution problems,which aroused more and more attention to the degradability of PET waste.With the further study on the degradation of PET,microbial enzymatic degradation is considered as a "green" degradation method,which is expected to be applied to large-scale degradation of PET.So far,many microbial enzymes which could degrade PET have been found.Lipase and cutinase can hydrolyze low-crystallinity PET materials at higher temperature,but are unable to hydrolyze high-crystallinity PET materials effectively.Recently,a novel hydrolase PETase isolated from Ideonella sakaiensis,which can specifically and efficiently degrade PET at room temperature.However,the degradation efficiency of the enzyme needs to be further improved before applied to engineered large-scale degradation.In this work,we constructed petase gene and expressed PETase protein by E.coli system.Next,we attempt to get highly pure PETase protein by a variety of chromatography methods,and determined the high-quality structure of PETase by protein crystallography.By analyzing the three-dimensional structure of PETase and homologous hydrolase,we indicated a structural feature which is account for high specificity for PET.Lately,we study on the binding mode of PETase and 2PET by using molecular docking method,and proposed the catalytic hydrolysis mechanism of PETase.In addition,we further analyzed the amino acids near the binding pocket of PETase substrate based on the results of molecular docking,and designed some amino acid mutations to improve the degradation efficiency of the enzyme.These results would provide insights into further modification of PETase,which is aimed to develop more effective PET hydrolases.