Isolation,Purification And Prokaryotic Expression Of Azoreductase From Thermophilic Anoxybacillus Sp.,and Investigation Of Its Active Sites

Azo dyes are widely used in textile,printing and dyeing,food and other industries,because of its special structure is difficult to control,its discharge of a large amount of wastewater to the environment caused serious pollution.The traditional physicochemical method has the advantages of high cost and easy to cause secondary pollution,while the biological method has the advantages of harmless to the environment and can achieve efficient treatment,which has become a research hotspot in recent years.The strain capable of degrading azo dye was selected from the soil near the printing and dyeing plant in the early stage of this study,and was identified by genome sequencing analysis as Anoxybacillus sp.,(named as PDR2).It is found that the strain has two kinds of azoreductase genes,and azoreductase plays an important role in the degradation of azo dyes.The optimal culture conditions for the high yield of azoreductase were determined by response surface optimization,and the intracellular azoreductase was obtained by protein separation and purification,prokaryotic expression and so on.Then,to understand more deeply the catalytic mechanism of azoreductase in the degradation of azo dyes,the binding patterns and action sites between azoreductase and FMN,NADH and different azo dyes were analyzed by homology modeling,AutoDock docking and active site prediction,and the possible modes of action were speculated.The results are illustrated as follows:(1)The whole genome sequencing analysis showed that Anoxybacillus sp.PDR2 contained two azoreductase genes-NADH-flavin oxidoreductase and NADH oxidoreductase,named as FMN and AZO.These two kinds of azoreductase were analyzed respectively and found that they were not related,but the protein structures both belong to the ?/? type structure.It was also found these two azoreductases participate in the riboflavin metabolic pathway by using NAD+or NADH as receptors,and also participate in the degradation of azo dyes by the strain Anoxybacillus sp.PDR2.(2)The response surface method was used to optimize the enzymatic production medium and culture conditions.Optimization results revealed that the optimum medium was Glucose 5.43 g/L,beef extract and peptone 5.82 g/L,NaH2PO4 3.5 g/L,KH2PO4 1.8 g/L,MgSO4 0.2 g/L,MnO4S 0.02 g/L,FeCl3 0.02 g/L.The optimum cultivation condition was temperature 53.52?,pH 7.2 and dye concentration 190.41 mg/L.It was found that the highest enzyme activity(634.20 U)of azoreductase was found to be 43.16%times higher than that of the original one(443.01 U)after optimization.Under these optimal conditions,a kind of active azoreductase was successfully purified by protein separation and purification technology,and it was proved to be the azoreductase FMN.(3)Furthermore,two kinds of azoreductase in the strain were successfully expressed by prokaryotic expression technique.To optimize the induction expression conditions of these two recombinant bacteria,the best induction conditions for AZO recombinant bacteria are:the amount of bacterium OD600 is 0.8 when the inducer is added,the inducer IPTG concentration is 0.2 mmol/L,the induction time is 24 h and the induction temperature is 16?,at which time the intracellular recombinant enzyme activity of intracellular recombinase reduced methyl red reaches 15.65 U/mg.The best induction conditions for FMN recombinant bacteria are:the amount of bacterium OD600 is 0.8 when the inducer is added,the inducer IPTG concentration is 0.4 mmol/L,the induction time and temperature is 10 h and 20?,at which time the intracellular recombinant enzyme activity of intracellular recombinase reduced methyl red reaches 18.66 U/mg.Under optimal induction expression conditions two azoreductases were isolated and purified by Ni+ affinity chromatography and detected a slight activity.(4)The molecular docking technique was used to study the binding patterns of two azo reductases with different structure azo dyes.the results showed that the two azo reductases had high affinity for the simple structure methyl red,and then showed better binding ability to congo red and direct black G.The key active sites of azo reductase were analyzed by analysis software.the results showed that Asn 104,Phe102,Ala121 and Phe105 in azoreductase FMN may play a vital role,and the Tyr74.Gly106 and His75 of azoreductase AZO plays an important role.Two active azoreductases were obtained from Anoxybacillus sp.PDR2 with azo dye degradation ability for the first time,and the structural characteristics and active sites of the two azoreductases were deeply investigated by bioinformatics analysis,molecular docking prediction and active site analysis.The present study can not only provide good genes for the construction of "new high efficiency super-degrading bacteria",but also provide new ideas for the mechanism analysis of thermophilic azoreductase.In addition,in the present study,two kinds of azoreductases were obtained from strains that can degrade azo dyes in thermophilic environment and their docking mechanisms were analyzed for the first time.This study can lay a foundation for the efficient treatment of azo dye wastewater and provide technical support for the industrial application of azo dye biodegradation.