Establishment Of High-throughput Rapid Screening Method For B-etherase LigF And Its Catalytic Mechanism Analysis

The lignin is mainly composed of syringyl(S),guaiacyl(G)and phydroxyphenyl(H)units,and the three basic structural units are cross-coupled by various chemical means,wherein ?-O-4 The number of ether bonds accounts for 50%to 70%of all connections.Therefore,the cleavage of the(3-0-4 ether bond is essential for biodegrading lignin.Sphingobium sp.strain SYK-6 is one of the most studied bacteria,and the ?-etherase(LigF)metabolic pathway with glutathione(GSH)as a coenzyme plays a vital role and has attracted widespread attention.It is studied that the ?-ether bond cleavage efficiency directly affects the biodegradation of lignin.The Kate E.Helmich team has successfully reported the crystal structure of LigF,which is of great significance for further research on LigF.In this study,a high-throughput,rapid screening method was established for the in vitro enzymatic reaction of LigF.Mutant strains with different enzyme activities were screened by constructing a LigF mutant library and subjected to high performance liquid chromatography(HPLC)calibration.Molecular dynamics simulations were used to analyze mutants with different enzyme activities,and the catalytic mechanism was understood.1.In this study,the model compound 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol(GVL)was used as the initial substrate in Ca-Dehydrogenase(LigD)catalyzes the formation of LigF substrate 1-(3,4-dimethoxyphenyl)-3-hydroxy-2-(2-methoxyphenoxy)acetone(GVG),using thin The layer chromatography silica gel plate was subjected to scraper recovery and detected by HPLC to have a purity of about 98.2%and a concentration of 20 mM.Due to the consumption of GSH by the LigF catalytic process,the enzyme activity is characterized by the GSH residue in the detection system.Based on this characteristic,a high-throughput and rapid screening method for LigF is established.2.The LigF mutant library was constructed by error-prone PCR.By comparing the restriction enzyme construction and circular mutation construction efficiency,it was found that the transformation efficiency and mutation success rate of the circular mutation construction method were higher than the enzyme digestion construction method,and the mutant library was completed by the circular mutation construction method.Construct.The mutant library was screened by high-throughput,rapid screening method,and the selected mutants were corrected by HPLC to obtain mutants with significantly different enzyme activities.Among them,the mutant AA4 showed an increase in enzyme activity,and the other mutants showed a decrease in enzyme activity.The catalytic efficiency of the mutant AA4 was about 3.6 times that of LigF,and the other mutants were decreased to different extents.3.Molecular docking and molecular dynamics simulation analysis of differential mutants F1,F6,AA4,AE4 and original LigF.The binding force of LigF to the small molecule of the substrate is mainly hydrogen bonding,and the binding ability of the mutant AA4 to the small molecule of the substrate is mainly hydrophobic interaction and hydrogen bonding,and the substrate molecules are also hydrophobic,which makes the two easier to combine.To facilitate the occurrence of the reaction.The apparent kinetic constant of the mutant was determined.