Site-directed Mutation,Characterization And Application Of Extradiol Dioxygenase Tc3516

Extradiol dioxygenases is a class of enzymes widely present in nature that can cleave a variety of aromatic hydrocarbon substrates.Extradiol dioxygenases mainly rely on metal ions to complete the catalysis,which can effectively remove the toxic catechol pollutants accumulated in the environment,and has a positive significance for the restoration of the environment.At the same time,in industrial processing,petroleum smelting,bio-pharmaceuticals and other fields that are highly associated with aromatic hydrocarbons,extradiol dioxygenase can be used to remove the aromatic hydrocarbons which are be difficult to degraded in the production process.It's a critical part of industrial production and pollution control.In this study,the gene of Tc3516 was cloned from the genome of Thermomonospora curvata,then connected with plasmid p ET-28 a to construct the vector,which was successfully expressed in E.coli BL21?DE3?after transformation.The target protein was purified by Ni²⁺-NTA affinity chromatography,and its properties were characterized.After testing,the optimal reaction temperature of Tc3516 for 3-methylcatechol was 50?,the reaction buffer was 50 m M Gly-Na OH at p H = 9,and the specific activity was 3.70 U/mg.In order to specifically improve the catalytic activity of the enzyme for 3-methylcatechol,structural simulation and molecular docking of the protein receptor and substrate ligand were performed.Based on the docking results,near the active site of the enzyme,6 amino acid sties were selected for site-directed mutagenesis according to the principle of increasing the hydrophobicity of amino acids and reducing the steric hindrance of amino acid residues.After successful construction of mutants and characterization them,it was found that the mutant's catalytic ability was improved.Among them,the mutation result of D285 A was the most significant,its catalytic efficiency was increased by more than five times compared with the wild type,and each enzyme molecular can catalyze about 51 substrate molecules per minute.These also confirmed that sitedirected mutation based on molecular docking could improve the catalytic activity of the enzyme to a certain degree.In order to investigate the application of the most active mutant,SBA-15 was used as a carrier to bind D285 A to immobilize by physical adsorption,and a continuous flow reactor was manufactured to remove 3-methylcatechol in simulated sewage.By optimizing,the removal rate reached 96% at 45? with a sewage flow rate of 0.33 m L/s,and through the reactor three times it reached more than 80%.After ten treatments,the removal capacity of pollutants still remains at 60%.The continuous flow reactor based on the immobilized D285 A has a high catalytic efficiency and 3-methylcatechol removal rate,which can be further improved and expanded in scale.It has a positive application prospect in industrial large-scale sewage treatment.