The Degradation Of Chlorpyrifos And Its Hydrolysis Product TCP By Laccase Mediator System

Chlorpyrifos is widely used for pest control in agriculture and to a lesser degree for indooruse or soil applications to control termites. The chlorpyrifos remained in the environment couldcause contamination in water, soil and air leading toxicity to human being and biota through foodchain. Chlorpyrifos was concerned because it is moderately toxic and used excessively posing ahigh contamination risk to soil and groundwater. Therefore, an ecofriendly and effectivetechnology was needed for treatment of these hazardous pesticides.Laccase is one of the enzymes produced by white-rot fungi. Laccase has been studied due toits ability to oxidize phenolic compounds. It was characterized by its remarkably wide substratespecificity and a variable range of oxidizable substrates. Laccase could catalyze the oxidation ofa wide variety of substrates, including monophenols, diphenols, polyphenols, aminophenols,methoxyphenols, aromatic amines and so on. The laccase mediator system had been successfullyapplied in different fields of biotechnology, such as paper pulp bleaching and delignification, thedegradation of polycyclic aromatic hydrocarbons, the decolorrization of textile dyes, thedetoxification of environmental pollutants and soil remediation.In this study, laccase (Coriolus versicolor) mediator system was used for the drgradation ofchlorpyrifos and its hydrolysis product3,5,6-trichioro-2-pyridinol (TCP). The enzymaticproperties of laccase, analysis methods of chlorpyrifos, the degradation of chlorpyrifos and TCPby laccase or laccase mediator systems, the degradation products of chlorpyrifos were studied.The main conclutions are as follows:(1) The enzymatic properties of laccase were studied. The optimum temperature and pH oflaccase were60℃and5.0respectively. Laccase showed stable when temperature arranged from25℃to35℃or pH at4.5to6.0.(2) The methods of UV spectrophotometry, gas chromatography (GC-ECD) and highperformance liquid chromatography (HPLC) for determination of chlorpyrifos were established.HPLC performed best. The analysis conditions for HPLC were described below: mobile phase ofmethanol:water(V:V)=90:10, flow rate of1mL/min, detector of VWD, detection wavelength of293nm, column temperature of25℃, sample volume of20μL. The recovery rate of chlorpyrifoswas between93.84%and98.25%, and RSD was0.30%to1.51%, when petroleum ether was theextraction solvent.(3) The degradation of chlorpyrifos by laccase and laccase mediator system were researched.The results showed that laccase had little effect on the degradation of chlorpyrifos without mediator. Vanillin and guaigcol promoted the degradation of chlorpyrifos by laccase, while2,6-dimethoxyphenol (2,6-DMP),2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate)(ABTS),violuric acid and1-hydroxybenzotriazole (HBT) had no obvious promotion. Vanillin performedmore effective than guaigcol. Vanillin is natural mediator which has environmental andecological advantage.The adding of manganese sulfate and Tween80promoted the degradationof chlorpyrifos by laccase/ABTS, but the degradation rate of chlorpyrifos was still insufficientideal.(4) Vanillin was chosen as the mediator for the degradation of chlorpyrifos by laccase.Firstly, factors affecting the degradation of chlorpyrifos such as reaction time, initial laccaseactivity, pH, temperature, mediator dosage and the initial chlorpyrifos concentration wereresearched. The results showed that when the initial chlorpyrifos concentration was25mg/L, itcould be degraded98%by laccase/vanillin system in24h with the initial laccase activity was0.05U/mL. The other conditions were as follows: the initial pH5.0, the temperature30℃, addedvanillin twice (molar rate of vanillin and chlorpyrifos was40and80respectively). Oxygen wasneeded for the degradation of chlorpyrifos by laccase/vanillin. The shaking speed and bufferconcentration had little impact on the degradation of chlorpyrifos by laccase/vanillin. Secondly,the dynamics of chlorpyrifos degradated by laccase/vanillin were studied. The half-life ofchlorpyrifos was6.6h with laccase/vanillin, while the half-life was34.6h without laccase andvanillin when the the initial concentration of chlorpyrifos was25mg/L. Finally, the products ofchlorpyrifos degradated by laccase/vanillin were studied. New peaks appeared in HPLC but nokind of degradation product of chlorpyrifos was detected by GC-MS.(5) The degradation of TCP by laccase and laccase mediator system were studied. First of all,HPLC analysis method for TCP was established. The analysis conditions were as follows: mobilephase of methanol:water:acetic acid(V:V:V)=80:18:2, flow rate of1mL/min, detector of VWD,detection wavelength of293nm, column temperature of25℃, sample volume of20μL. Therecovery rate of TCP was between98.25%and102.68%when methylene chlorde was theextraction solvent. Next, TCP was degradated by laccase and laccase-mediator system. Laccsehad a certain effect on the degradation of TCP without mediator. Mediators such as ABTS, HBT,violuric acid, vanillin and so on had no obvious promotion on the degradation of TCP by laccase.