Study On Decolorization Characteristics Of Cationic Blue Dye By A Bacillus And Its Enzymatic Properties Of Azoreductase

Azo dyes are complex aromatic compounds containing azo bonds,which are widely used in a variety of industries such as printing and dyeing,biological dyeing,leather,medicine,paper making,food and cosmetics,accounting for 70%of the total dye production per year.Azo dyes are easily lost during production and use,and their retention in the environment can cause toxicity and mutagenicity to humans and animals.Due to the special chemical structure of azo dyes,it is relatively stable in the environment.It is difficult to remove azo dyes by general physical or chemical methods,and the cost is high.The treatment of azo dye wastewater by microbial degradation is economical,effective and environmentally friendly.Therefore,it is of great significance to study the properties of azo dye decolorizing bacteria and their enzymatic characteristics for the development of biological treatment methods.In this study,the decolorization characteristics of azo dye decolorizing bacteria ZHT-7,the activity of azoreductase and the coding genes were studied in order to provide scientific basis for the application of azo dye decolorizing bacteria ZHT-7.In this study,azo dye decolorization strain ZHT-7 was selected as the research object.The decolorization characteristics and influencing factors of azo dye Cationic Blue by strain ZHT-7 were studied by decolorization curve.The azoreductase of strain ZHT-7 was extracted by ultrasonication,and the extraction effect under different conditions was compared.Response surface methodology was used to optimize the extraction scheme.By studying the influence of NADH,p H and temperature on the determination of azoreductase activity,the optimum quantitative detection system of azoreductase activity was established with the aim of controlling good detection precision.The effects of temperature and metal ions on the activity of azoreductase were studied by using this detection system.The azoreductase gene of strain ZHT-7 was sequenced and analyzed by PCR and molecular cloning.The results showed that the decolorization characteristics of azo dye Cationic Blue by strain ZHT-7 were stable,and the Cationic Blue could not be used as the sole carbon source.Extracellular metabolites(EM)and decolorizing metabolites(DM)could inhibit the decolorization of Cationic Blue by strains,and only adding EM and DM could also decolorize Cationic Blue.The decolorization of strains was promoted by 10?300 mg/L of Mg²⁺,0.1?1 mg/L of Cu²⁺and 10?200 mg/L of Ca²⁺in the whole decolorization process.The inhibition of decolorization by 0.1?2 mg/L of Mn²⁺increased with increasing concentration.The operating parameters of ultrasonic fragmentation had an important impact on the amount of protein released by cells,and the total time of ultrasound had the greatest impact.Quadratic equation models of protein concentration and parameters were obtained by multiple regression analysis.Under the optimal extract conditions(ultrasonic power as 87 w,total ultrasonic time as 17.5 min and single ultrasonic time as1.5 s),the concentration of protein was 31.47 mg/L.Relative standard deviation of determination was only 3.76%in optimal detection system of azoreductase activity,where the concentration of NADH,p H value and temperature was 2.0 mmol/L,7 and37°C,respectively.The azoreductase activity of highly efficient decolorizing strain ZHT-7 was distributed both inside and outside the cell.The azoreductase activity of strain ZHT-7maintained high activity in the temperature range of 20?45?.The azoreductase activity of strain ZHT-7 was inhibited by 0.1?2 mg/L of Mn²⁺and Cu²⁺,and the inhibition increased with the increase of Mn²⁺and Cu²⁺concentration.The azoreductase gene of strain ZHT-7 was detected and it was found that the azoreductase gene of Bacillus thuringiensis strain ZHT-7 was located on its chromosome.Sequencing analysis of its azoreductase gene revealed that the sequences with higher identity were derived from Bacillus compared with the sequences already existing in Gen Bank.