漆酶介体体系对毒死蜱及其水解产物TCP的降解研究
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摘要
毒死蜱是目前全世界生产量和销售量最大的杀虫剂品种之一，也是世界卫生组织唯一许可的有机磷品种，被广泛用于农业和城市卫生害虫的防治。有机磷农药长期大规模地生产和使用，不仅对环境造成严重污染，而且在食物及饲料中的残留会通过食物链对人类及养殖动物产生毒害作用。因此，人们需要寻找环境友好、安全实用的净化措施消除有机磷农药的污染，妥善地解决有机磷农药生产使用和环境保护的问题。
白腐菌降解酶系中的漆酶，具有广泛的底物作用范围，它可催化氧化多种酚类和芳香胺类化合物，也可降解其它难降解物质。目前，漆酶介体体系已成功应用于不同领域，包括纸浆漂白、木素生物降解、多环芳烃降解、染料废水脱色、环境污染物脱毒、土壤修复等。
本研究利用杂色云芝漆酶（Coriolus versicolor）介体体系对有机磷杀虫剂毒死蜱进行降解，研究了漆酶酶学性质、毒死蜱检测方法、漆酶介体体系对毒死蜱及其水解产物3,5,6-三氯-2-吡啶酚（TCP）的降解，并对毒死蜱降解产物进行了初步检测分析，主要研究结果如下：
（1）研究了漆酶的酶学性质，漆酶的最适温度为60℃，最适pH值在5.0左右，当温度在25～35℃、pH在4.5～6.0范围内时，漆酶的稳定性较好。
（2）建立了毒死蜱测定的紫外分光光度法、气相色谱法（GC-ECD）和高效液相色谱法（HPLC），其中高效液相色谱法最佳，具体分析条件：流动相为甲醇:水(V:V)=90:10，流速为1mL/min，检测器为VWD，检测波长为293nm，柱温为25℃，进样体积为20μL。萃取溶剂为石油醚，毒死蜱的加标回收率为93.84%～98.25%，变异系数为0.30%～1.51%。
（3）研究了漆酶及漆酶介体体系对毒死蜱的降解，研究结果表明，当不加介体时，漆酶对毒死蜱的降解作用较小；香草醛对漆酶降解毒死蜱的促进作用最大，且香草醛为天然介体，具有环境和生态优势；愈创木酚对漆酶降解毒死蜱也有一定的促进作用；2,6-二甲氧基酚（2,6-DMP）及最常用的三种人工合成介体2,2′-联氮-二(3-乙基-苯并噻唑-6-磺酸)（ABTS）、1-羟基苯并三唑（HBT）和紫脲酸对漆酶降解毒死蜱均没有促进作用。MnSO4和Tween80对漆酶/ABTS降解毒死蜱有一定的促进作用，但毒死蜱的降解率仍旧不高。
（4）选择香草醛作为漆酶降解毒死蜱的介体，首先，采用单因素法，研究了反应时间、漆酶初始酶活、反应温度、pH、介体投加量、毒死蜱初始浓度等因素对漆酶/香草醛降解毒死蜱的影响，研究结果表明，当毒死蜱初始浓度为25mg/L，反应体系中漆酶的初始酶活为0.05U/mL，分两次投加香草醛，香草醛与毒死蜱的摩尔比分别为40和80，在30℃、pH5.0的条件下，24h内毒死蜱的降解率可达98%。向反应体系中通O2，可提高漆酶/香草醛对毒死蜱的降解率；缓冲溶液浓度和摇床转速对漆酶/香草醛降解毒死蜱的影响很小。其次，对漆酶/香草醛降解毒死蜱的动力学进行了研究，当毒死蜱初始浓度为25mg/L时，在漆酶/香草醛的作用下，毒死蜱的降解半衰期为6.6h；当不加漆酶不加香草醛时，毒死蜱的半衰期为34.6h。最后，对毒死蜱的降解产物进行了初步检测分析，通过HPLC分析，HPLC谱图上有新峰出现；通过GC-MS分析，没有检测到毒死蜱的降解产物。
（5）研究漆酶及漆酶介体体系对毒死蜱水解产物TCP的降解。首先，建立了TCP测定的HPLC法，具体分析条件为：流动相为甲醇:水:冰醋酸=80:18:2(V:V:V)，流速为1mL/min，检测器为VWD，检测波长为293nm，柱温为25℃，进样体积为20μL。当萃取溶剂为二氯甲烷时，TCP的加标回收率在98.25%～120.68%。然后，利用漆酶及漆酶介体体系对TCP进行降解，漆酶本身对TCP有一定的降解能力，ABTS、HBT、紫脲酸、香草醛等介体对漆酶降解TCP均没有明显的促进作用。
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.
白腐菌降解酶系中的漆酶，具有广泛的底物作用范围，它可催化氧化多种酚类和芳香胺类化合物，也可降解其它难降解物质。目前，漆酶介体体系已成功应用于不同领域，包括纸浆漂白、木素生物降解、多环芳烃降解、染料废水脱色、环境污染物脱毒、土壤修复等。
本研究利用杂色云芝漆酶（Coriolus versicolor）介体体系对有机磷杀虫剂毒死蜱进行降解，研究了漆酶酶学性质、毒死蜱检测方法、漆酶介体体系对毒死蜱及其水解产物3,5,6-三氯-2-吡啶酚（TCP）的降解，并对毒死蜱降解产物进行了初步检测分析，主要研究结果如下：
（1）研究了漆酶的酶学性质，漆酶的最适温度为60℃，最适pH值在5.0左右，当温度在25～35℃、pH在4.5～6.0范围内时，漆酶的稳定性较好。
（2）建立了毒死蜱测定的紫外分光光度法、气相色谱法（GC-ECD）和高效液相色谱法（HPLC），其中高效液相色谱法最佳，具体分析条件：流动相为甲醇:水(V:V)=90:10，流速为1mL/min，检测器为VWD，检测波长为293nm，柱温为25℃，进样体积为20μL。萃取溶剂为石油醚，毒死蜱的加标回收率为93.84%～98.25%，变异系数为0.30%～1.51%。
（3）研究了漆酶及漆酶介体体系对毒死蜱的降解，研究结果表明，当不加介体时，漆酶对毒死蜱的降解作用较小；香草醛对漆酶降解毒死蜱的促进作用最大，且香草醛为天然介体，具有环境和生态优势；愈创木酚对漆酶降解毒死蜱也有一定的促进作用；2,6-二甲氧基酚（2,6-DMP）及最常用的三种人工合成介体2,2′-联氮-二(3-乙基-苯并噻唑-6-磺酸)（ABTS）、1-羟基苯并三唑（HBT）和紫脲酸对漆酶降解毒死蜱均没有促进作用。MnSO4和Tween80对漆酶/ABTS降解毒死蜱有一定的促进作用，但毒死蜱的降解率仍旧不高。
（4）选择香草醛作为漆酶降解毒死蜱的介体，首先，采用单因素法，研究了反应时间、漆酶初始酶活、反应温度、pH、介体投加量、毒死蜱初始浓度等因素对漆酶/香草醛降解毒死蜱的影响，研究结果表明，当毒死蜱初始浓度为25mg/L，反应体系中漆酶的初始酶活为0.05U/mL，分两次投加香草醛，香草醛与毒死蜱的摩尔比分别为40和80，在30℃、pH5.0的条件下，24h内毒死蜱的降解率可达98%。向反应体系中通O2，可提高漆酶/香草醛对毒死蜱的降解率；缓冲溶液浓度和摇床转速对漆酶/香草醛降解毒死蜱的影响很小。其次，对漆酶/香草醛降解毒死蜱的动力学进行了研究，当毒死蜱初始浓度为25mg/L时，在漆酶/香草醛的作用下，毒死蜱的降解半衰期为6.6h；当不加漆酶不加香草醛时，毒死蜱的半衰期为34.6h。最后，对毒死蜱的降解产物进行了初步检测分析，通过HPLC分析，HPLC谱图上有新峰出现；通过GC-MS分析，没有检测到毒死蜱的降解产物。
（5）研究漆酶及漆酶介体体系对毒死蜱水解产物TCP的降解。首先，建立了TCP测定的HPLC法，具体分析条件为：流动相为甲醇:水:冰醋酸=80:18:2(V:V:V)，流速为1mL/min，检测器为VWD，检测波长为293nm，柱温为25℃，进样体积为20μL。当萃取溶剂为二氯甲烷时，TCP的加标回收率在98.25%～120.68%。然后，利用漆酶及漆酶介体体系对TCP进行降解，漆酶本身对TCP有一定的降解能力，ABTS、HBT、紫脲酸、香草醛等介体对漆酶降解TCP均没有明显的促进作用。
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.
引文
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