Infrared And Fluorescent Probe Labelling Of Halohydrin Dehalogenase And Preliminary Study On Its Structural Dynamics

Halohydrin dehalogenase not only degrade organic halides but also synthesize chiral epoxides and?-substituted alcohols.The catalytic mechanism and broad application of halohydrin dehalogenase have attracted much attention in recent years.The dynamic structural change of halohydrin dehalogenase is related to its biological function.Studying its structural dynamics helps to reveal the catalytic mechanism and improve modification efficiency.It is well known that spectroscopy has a wide range of applications in studying the dynamics of enzyme structures,and related research requires embedding appropriate spectral probes in enzyme proteins.Therefore,the labeling of the enzyme is particularly important.This study combines biological techniques and spectroscopy techniques to insert the infrared and fluorescent probes in the enzyme.Because the probe is very sensitive to the microenvironment,it can accurately reflect the dynamic structures of the enzyme.In this study,Naek is introduced into K140,K161 and P184 of halohydrin dehalogenase using stop codon method.The azide-modified enzyme is obtained by optimizing the expression system and affinity chromatography,and its catalytic properties and infrared spectrum are detected in the study.Experiment results show that:?1?184⁺mutant has the best activity and it holds the same thermal stability and optimum reaction temperature as the wild type.?2?in the FT-IR detection,the peaks of the three mutants locate at 2131cm^-1,2137cm^-1,2183cm^-11 respectively,because the polarities of the azide group in the halohydrin dehalogenase are different.?3?high concentration of GdHcl unify the structures of the enzyme,and the infrared peak is more concentrated.However,the infrared absorbance of the enzyme remain unchanged when reacted with chloride ions,so fluorescent probe labeling and Tryptophan mutant are carried out in the subsequent experiments.Based on the research above,the azide group is inserted into halohydrin dehalogenase by SPAAC reaction,and enzyme activity and fluorescence spectroscopy are detected.The study also constructs Tryptophan mutant by site-directed mutation and detect the enzyme activity of all mutants.Experiment results show that:?1?the fluorescence efficiency of the enzyme can reach 49.03%,and the mutant has the maximum fluorescence emission peak at 670 nm.The maximum fluorescence intensity locates at 1469 and the click reaction process does not lose the enzyme vitality at all.?2?in halide ion binding experiment,Cl^-and Br^-can affect the relative fluorescence intensity of the halohydrin dehalogenase,which proves that 184 site is involved in the enzyme conformation changes when performing catalytic functions.In summary,this study constructed an infrared and fluorescent probe for halohydrin dehalogenase,which lays a foundation for studying the structural kinetics of halohydrin dehalogenase.