Semi-rational Modification Of PET Degrading Enzyme And Study On Its Molecular Mechanism

Attention has been increasingly paid to the environmental pollution caused by the difficult degradation of plastics.Polyethylene terephthalate(PET)is one of the most popular plastic and the most difficult plastic to degrade.With the continuous improvement of directed evolution technology,biocatalysis tends to become one of the key technologies to solve the plastic problem.In this study,LCC-ICCM,which has the highest activity of PET degradation reported at present,was taken as the research subject.Firstly,the expression and ability of PET degradation of LCC-ICCM were preliminarily studied.The results showed that the better induction temperature of LCC-ICCM was 30℃.The better reaction conditions for degradation of PET films were 70℃,p H 8.0,and the reaction conversion rate reached 86% after 96 h.In order to understand better the industrial application of LCC-ICCM,the metal activity of LCC-ICCM were analyzed.The results show that metal ions have a certain influence on the enzyme activity and its binding with the substrate.To improve the activity and stability of the enzyme,the semi-rational design of LCC-ICCM was carried out by molecular docking,binding energy decomposition and site analysis.Six sites M166,R107,R108,D98,H164 and T211 were selected for saturation mutation and overlapping mutations.From structural analysis,M166 forms oxygen anion holes with Trp190 and Tyr95,which is mutated to destroy the protein structure and lead to decreased enzyme activity.H164 is located next to the catalytic triplet Ser165,and the size change of amino acid residues greatly affects the spatial position of Ser165,so the mutation is not conducive to the enzyme reaction.Mutations in R107 and R108 disrupt the α-helix structure of the proteins,resulting in reducing enzyme activity.The results stated that most of the mutations at site 98 and some of the mutations at site 211 showed high activity.Among the mutations,the degradation efficiency of PET in the better mutant LCC-ICCM-211 F is 1.5 times higher than that of the wild type,and the optimal reaction temperature to p-nitrophenol acetate is 5℃ higher than that of the wild type.According to the structure analysis,it is speculated that 98 site is located opposite the 243 site which has the function of stability with 243 site to boost the PET combined with enzyme.The mutation of 211 could provide larger space for the substrate binding,which is the key site to affect the enzyme activity.The temperature stability test of mutants show that 98 site is the key site affecting the temperature stability of the enzyme.The optimal p H and p H stability of LCC-ICCM-211 F shows that the optimal p H of the mutant is 8 and has a very high p H stability.In this study,by the semi-rational design of PET degrading enzyme LCC-ICCM,the key sites of affecting enzyme activity and stability were screened out,and the influence of active sites on PET degradation was preliminarily explored,which laid a foundation for the subsequent study of cutinase.