HR-PETase From Bacterium : Study On Biochemical Properties And Degradation Of Polyethylene Terephthalate (PET)

Polyethylene terephthalate（PET）is widely used in many fields such as food packaging,chemical industry,daily necessities,machinery industry and electronics industry due to its excellent performance.However,the difficult-to-degrade characteristics of PET have also caused huge pollution to the environment and threatened the survival of human beings.Therefore,enzymatic degradation of waste PET,an economical and environmentally friendly treatment method,has attracted the attention of researchers.However,the current enzymes that can be used to degrade PET have problems such as low activity,poor heat resistance,and unclear catalytic mechanisms,which limit their industrial application.This study obtained the new PET hydrolase HR-PETase from Bacterium HR29 as the research object,and investigated the preparation,enzymatic properties,the crystal structure and the application in PET degradation.This research provided not only an important basis for industrial applications,but also a structural foundation for in-depth understanding of the structure-function relationship of HR-PETase.The main results are as follows:（1）Preparation of HR-PETase and study of enzymatic properties of HR-PETaseThe prokaryotic expression vector of HR-PETase was constructed and expression in E.coli BL21（DE3）host.The HR-PETase recombinant protein was isolated and purified by metal chelate affinity chromatography and the yield was 6.27 mg/L.The SDS-PAGE analysis showed that the molecular weight of HR-PETase was about 29 k Da,and the purity is greater than 90%.The optimum reaction temperature of HR-PETase was 75°C and the optimum p H was 8.5towards p NPB,and the T_m was 95.6°C,indicating that HR-PETase was a high-temperature resistant hydrolase,which was stable in alkaline environment.On the other hand,HR-PETase had great resistance to most common metal ions and organic solvents,and surfactants can activate HR-PETase to a certain extent.Divalent metal ions such as Ca²⁺could significantly improve the thermal stability of HR-PETase.（2）Research on the degradation of PET by HR-PETaseThe hydrolysis effect of HR-PETase on PET film and PET powder were explored respectively.The optimum reaction temperature was 70℃and the optimum reaction p H was8.0 towards PET film and the PET powder.The reactions were carried out under these conditions for 24 h,the weight loss rate of the PET film reached 78.5%,and the degradation rate of the PET powder was 91.2%.The degradation products were determined to be TPA and MHET by HPLC.The specific activity of HR-PETase to degrade PET powder is 44.77mg TPAeq.h^-1.mg enzyme^-1.（3）Crystallization and structure analysis of HR-PETaseGel filtration chromatography was used to obtain higher purity HR-PETase wild-type protein,which was concentrated to 10 mg/m L,and the sitting drop method and hanging drop method were used to screen and optimize the protein crystals respectively.Finally,a crystal with a resolution of 1.24?was obtained under the conditions of 0.1 M Tris-HCl p H 8.4 and19.5%（v/v）ethanol.HR-PETase was a typicalα/βhydrolase,consisting of 8α-helices and 9β-sheets connected by loops,with a conservative catalytic triad Ser165-Asp210-His242 and a pair of C-terminal disulfide bonds:Cys275-Cys293.Using molecular docking to analyze the interaction between HR-PETase and the substrate analogue HEMT.The result showed that Trp190 stabilized the aromatic ring of the substrate throughπ-πstacking and Ser165 nucleophilic attacked on the carbonyl carbon of the substrate.The backbone NH groups of M132 and Y58 that constituted an oxyanion hole to stabilize the tetrahedral intermediate product.The catalytic core area of HR-PETase was almost exposed on the surface of the enzyme and was located in a long trench,which was conducive to contacting and catalyzing insoluble long-chain PET molecules.