Research On The Non-specificity Of TfdB-JLU For Aromatic Compounds

Along with the advance of industrial development, people are constantly increasingdemand for aromatic compounds. But as contaminants, aromatic compounds havebecome a very serious threat both to the ecological environment and the biologicalhealth. So there is an urgent need for an efficient and environment friendly treatment todeal with these organic pollutant. The enzyme treatment for these organic pollutant haslots of advantage, and the application prospect is very broad.This method has become afocus in domestic and foreign research and development.TfdB-JLU is one such enzyme which can transform the aromatic compoundsefficiently. TfdB-JLU expresses in prokaryotic Escherichia-coli with simple operationand low cost. It can not only transform the most of aromatic compounds, and theconversion efficiency is also higher than the other enzymes.The first step to improve the efficiency of enzyme treatment method is to get moreactive enzymes. So first of all, optimizing the conditions of process for heterologousexpression of TfdB-JLU in Escherichia coli.The expression of TfdB-JLU in Escherichiacoli, is mainly in the form of inclusion bodies. In the condition of0.2mM IPTG,16℃we can get more soluble protein.Then broke the cell by Novagen BugBuster ProteinExtration Reagent instead of Ultrasonic broken.This method is conducive tomaintaining the stability of the enzyme, In order to get the more active protein. Thespecific activity of the enzyme is4.1times of the specific activity in the literature. Then2,4-DCP as a substrate, the conditions that concentration of TfdB-JLU12μg/ml,25℃,pH7.5, power of microwave400W, concentration of [EMIM][PF6]0.08mM andacetone can improve the activity of TfdB-JLU. Under the optimal conditions, the Kmvalue of TfdB-JLU is5μM, while the Vmax0.023μM/min on the2,4-DCP.Then we study the substrate specificity of TfdB-JLU and choose three kinds ofvery representative substrates whose pollution are very serious. They are chlorophenolcompounds, biphenyl compounds and indole compounds respectively, a total of39 different substrates. With the method of ultraviolet spectrophotometry, we compared theconversion efficiency and reaction rate of different substrates in different temperatureconditions, and verify the promoting effect of co-enzyme FAD on most of the selectedsubstrate.For going deep into the reaction from the perspective of structure, we dock themodel of TfdB-JLU with the selected aromatic substrate, by homology modeling andmolecular docking. On counts of no crystal structure of TfdB-JLU, we modeledaccording to the aminoacid sequence of the target protein and the selected template withhigh homology. At the same time, according to the analysis of the templates bindingsites, we confirmed the binding sites of co-enzyme NADPH and FAD. Then we dockedthe models with different substrates. The results show that the model with co-enzyme ofNADPH and FAD has lower binding free energy than that without co-enzyme, whichcan verify the promotion of co-enzyme. The results of different substrates are notideal.Further research is needed.