The Construction And Molecular Dynamics Simulation Of Carbohydrate-binding Module-PET Hydrolase Fusion Protein

A large number of plastic products are discarded or put into landfill after use,and plastic waste flows into the environment with human activities finally,which has caused serious pollution.PET is one of the most widely used plastics,and it is difficult to achieve the degradation of PET.Meanwhile,current methods can not achieve the green and efficient recycling of PET.In recent years,biocatalytic PET degradation mediated by PET hydrolase has received widespread attention as it is a promising method with no pollution and mild conditions.However the catalytic efficiency of PET hydrolases is not sufficient for their practical application.Thus it is urgent to develop new PET hydrolases to solve the problem of PET pollution.One of the key reasons for the low catalytic efficiency of PET hydrolases is the poor PET-binding ability,and several carbohydrate-binding modules(CBM)have been proved to effectively bind with PET membrane.The fusion protein of CBMs and PET hydrolases can enhance the PET-binding ability of the enzyme and improve the catalytic efficiency.Nevertheless,it is very difficult to screen suitable CBMs from a large family,as the mechanism of CBMs binding to PET membrane is still unclear.In this study,we chose a high-efficiency PET hydrolases namly leaf and branch compost cutinase(LCC)as the model,and its mutant ICCG(F243I/D238C/S283C/Y127G)was constructed by site-directed mtagenesis.Then four CBMs were screened out and corresponding fusion proteins with ICCG were successfully constructed.Finally,the possible binding sites of CBMs with PET and the relevant mechanism were analyzed by molecular dynamics simulation.The optimal p H and temperature of LCC were measured to be 9.0 and50 ℃,respectively,and it exhibited favorable thermal stability;The optimal p H of ICCG was also 9.0,and the optimal temperature was 60 ℃,which was slightly higher than that of LCC,indicating that ICCG had better thermal stability.The scanning electron microscope analysis for the PET surface morphology showed that ICCG had higher degradation efficiency on PET film than LCC.Subsequently,four CBMs were screened and employed to construct fusion proteins of CBMs and ICCG.The PET tetramer was used as the ligand to finish molecular docking with four CBMs,and appropriate positions were selected as the initial conformation for molecular dynamics simulation.By calculating binding free energy and energy decomposition,the key residues and the main interaction between the key residues and PET tetramer have been obtained.Based on the analysis,van der Waals forces were primarily responsible for driving the binding of PET to CBMs.Aromatic residues and proline with pyrrole ring played an important role in the binding of these two components,and arginine residue with positive could also promote the binding of enzyme with PET.In conclusion,based on the strategy of combining computer-aided design and in vitro screening,this study constructed CBM-PET hydrolase fusion protein that significantly improved the degradation ability of PET.It provided a good idea for the rational design of high efficiency PET hydrolases and the degradation and recycling of PET.However,due to the impact of COVID-19 in Changchun,the PET degradation activity of fusion protein was failed to complete.In the follow-up,the PET degradation activity of fusion protein will be systematically evaluated,and the PET hydrolases with better catalytic ability will be constructed based on computer-aided design and rational design.