Application And Kinetic Analysis Of Laccase, Tyrosinase And Horseradish Peroxidase Catalyze Phenolic And Aniline Compounds

Objective: Phenolic or aniline compounds were important pollutants in the industrial wastewaters to seriously polluted water environment. Therefore, the rapid test for phenol and aniline compounds was an important task in environmental monitoring. This research developed a detecting method of phenolic and aniline compounds based on the kinetic parameters of the substrates of the three oxidases of laccase, tyrosinase and horseradish peroxidase. Catalytic reaction between the three oxidases and phenol and aniline compounds was characterized using spectrophotometric method.Methods: According to functional groups, thirteen phenols and six anilines were selected as representative test substrates for the three oxidases. Substrate selection was conducted and determined the absorption with spectral scanning of spectrophotometer. Catalytic reaction between the three oxidases and phenolic and aniline compounds was characterized using spectrophotometic method, which resulted 0-10 mg/L and 0-1 mg/L substrate reaction rate and calibration curve of substrate concentration and reaction rate. And then the non-volatile phenols in three kinds of coking wastewater were screened and the contents were detected. Tyrosinase was extracted and purified by ourselves from Agaricus bisporus.Results: ⑴In spectrophotometric method to analyze enzymatic reaction of phenols and anilines compounds, polyhydric phenol, aminophenol and multi-amine were the main substrates of laccase. Tyrosinase performed strong specificity for substrate and presented strong catalytic ability on catechol, pyrogallol acid and 2-aminophenol. Horseradish peroxidase have a broad substrate selectivity, and can catalyze monoamine, monophenol compounds which can not be oxidized by laccase. The calibration curves showed good first order kinetics linear relationship except benzidine determined by laccase with the correlation coefficient above 0.96. In addition to some of laccase linear range of detecting objects difficult to reach the national emission standards for phenols and anilines, the lower limit of detection linear range of tyrosinase and horseradish peroxidase can satisfy the national emission standards.⑵Using laccase method, the contents of catechol in three kinds of coking wastewater were respectively detected to be 190.5, 265.8 and 155.3 mg/L with recoveries ranged from 89.9% to 115.8%.⑶Using Agaricus bisporus as raw material, after a series step of homogenized, centrifuged, salt precipitation, dialysis, DEAE-Sepharose Fastflow chromatography, concentrated, Sephadex G-75 chromatography, and pure tyrosinase with high activity was obtained in the last. The optimum pH value for this enzyme to catalyze L-DOPA was 7.0 with the corresponding Km value was 0.182 mmol/L.Conclusion: A detecting method of phenolic and aniline compounds was developed based on catalytic reaction between the three oxidases and phenol and aniline compounds was characterized using spectrophotometric method. The method is simple and reliable which can conduct qualitative and semi-quantitative analysis for phenolic and aniline compounds in phenol wastewater. It also played a role in rapid screening for detecting the contents of phenolic and aniline compounds. The complementarity and cross-cutting was exsited for laccase, tyrosinase and horseradish peroxidase to select substrates. Especially, horseradish peroxidase can be used to detect the concentration of monophenol and monoamine, which provided basis for the research of enzyme biosensor with multi-enzyme and multi-channel.