Research On Semi-rational Design And Modification And Catalytic Mechanism Of 2,4-dichlorophenol Hydroxylase

2,4-dichlorophenoxyacetic acid(2,4-D)and 2,4-Dichlorophenol(2,4-DCP)are widely used in agricultural production and pharmaceutical synthesis.In the process of use,2,4-D and its intermediate product 2,4-DCP will accumulate in the soil and aboveground water and groundwater.Ingestion of 2,4-D at high doses will stimulate humans and lower mammals.The gastrointestinal tract also has damage to the central nervous system and liver.2,4-DCP accumulation will seriously affect the water environment,toxic to aquatic organisms,and irritating to human eyes and skin.Finding efficient and convenient pollution treatment methods is the focus of research today.The methods of microbial metabolic degradation have the advantages of mild operating conditions,economical savings,and no secondary pollution to the environment.It is one of the hot issues in current research.2,4-Dichlorophenol hydroxylase(tfdB enzyme)can catalyze the degradation of 2,4-DCP and is one of the key enzymes in the biodegradation process of 2,4-D.TfdB-JLU enzyme is a new type of tfdB enzyme,which has the characteristics of higher catalytic activity,wider substrate spectrum and cold adaptability.Compared with other tfdB enzymes,it is in 2,4-D and 2,4-DCP It has more advantages in the research of enzymatic degradation.However,the catalytic activity of the tfdB enzyme is generally very low,and the TfdB-JLU enzyme cannot meet the requirements of industrial production and application,so it is of great significance to design and modify the TfdB-JLU enzyme to improve its catalytic activity and stability.At the same time,TfdB-JLU enzyme,as a newly discovered tfdB enzyme,also requires in-depth study on the three-dimensional structure and catalytic mechanism of proteins.In this study,we first semi-rationally designed and modified the catalytic activity of TfdB-JLU for 2,4-DCP,based on the previous homology modeling and molecular simulation and the catalytic activity of TfdB-JLU for chlorophenols and biphenyls And specific research content.Optimized the enzyme activity detection method of TfdB-JLU,alanine scanning and free energy change to determine the target site.Computer simulation of the target site of saturation mutation TfdB-JLU and the use of binding free energy screening to predict the increase in the catalytic activity of 2,4-DCP have not been realized.The reason may be that the amino acid at the target site is in a region with less flexibility and is not suitable for mutation.Methionine 251(M251)is the amino acid at the entrance of the substrate binding site predicted in the previous study.This study verified its catalytic activity for 2,4-DCP after mutation to glycine(TfdB-JLU-M251G)The reason is that M251 is located in the region of greater flexibility in the TfdB-JLU structure and is located on the molecular channel.TfdB-JLU-M251 G makes the TfdBJLU structure more stable and expands the molecular channel.The structure determines the function,we try to analyze the crystal structure of TfdB-JLU.We initially screened nearly 2000 crystal culture conditions,and finally found 6 conditions that may be suitable for TfdB-JLU crystallization.However,based on the complexity of TfdB-JLU protein structure,we did not successfully analyze TfdBJLU crystallization,and subsequent optimization is required.Finally,we initially explored the catalytic product of TfdB-JLU.We found that the polarity of the catalytic product was greater than 2,4-DCP by thin layer chromatography analysis.The reduction of 2,4-DCP in TfdB-JLU catalytic reaction can be detected by liquid chromatography,indicating that TfdB-JLU can catalyze 2,4-DCP.When analyzing the composition of the product by LC-MS,only the peak of 3,5-dichlorocatechol can be seen in the output TIC diagram,which initially demonstrated the product composition of 2,4-DCP catalyzed by TfdB-JLU.In summary,the semi-rational design and modification of the catalytic activity of TfdB-JLU and the further exploration of the structural properties of key amino acids in this thesis,the crystal culture conditions and catalytic mechanism of TfdB-JLU are preliminary explored.These explorations have laid a solid foundation for the in-depth study of TfdB-JLU and are of great significance for the application of TfdB-JLU in the biodegradation of 2,4-D and 2,4-DCP pollution.