【作者】 方华；

【导师】 黄俊；

【作者基本信息】 武汉理工大学 ， 材料学， 2009， 博士

【摘要】 2,4-二氯苯酚（2,4-dichlorophenol,DCP）是一种毒性较强且难降解的环境污染物。已被美国环保局和我国政府列为必须首先控制的有毒化合物,因此检测DCP含量对环境保护和人类健康具有重要的意义。本学位论文分别以磁性壳聚糖纳米复合粒子（magnetic chitosan nanoparticles,MCN）固定化漆酶、酞菁铜仿生酶为生物识别元件,构建了基于固定化漆酶、仿生酶催化的耗氧型光纤DCP传感器,实现了对环境污染物DCP的检测。本学位论文在以下几个方面进行了系统的研究。第一,对杂色云芝菌漆酶的培养和纯化条件进行了研究,制备了比活力为28.4 U/mg的漆酶;第二,采用反相悬浮聚合法制备了MCN,以其为载体固定漆酶,研究了固定化的最优条件,以2,2’-连氮-双-（3-乙基苯并噻吡咯啉-6-磺酸）（ABTS）为底物研究了游离漆酶和固定化漆酶的催化性能;第三,分别研究了漆酶催化DCP和漆酶-介体体系中漆酶催化DCP的机理,确定了漆酶和固定化漆酶催化DCP的最适条件以及漆酶-介体体系中漆酶和固定化漆酶催化DCP的最适条件;第四,制备3种金属酞菁,对金属酞菁催化氧化DCP的最适条件和机理进行了研究;第五,设计和构建了基于固定化漆酶、仿生酶催化的光纤DCP传感器,研究了传感器的性能。本学位论文的主要结论包括以下几个方面:（1）采用液体发酵法对培养杂色云芝菌漆酶的培养条件进行了优化,分离和纯化了杂色云芝菌漆酶,以ABTS为底物研究了其酶学性质。最优培养条件为:土豆汁20%,pH值为5.0,葡萄糖20.0 g/L,蛋白胨4.0g/L,CuSO₄ 1.0 mmol/L,KH₂PO₄ 3.0g/L,MgSO₄ 1.5 g/L,ZnSO₄ 0.5g/L,MnSO₄0.5 g/L,ABTS 1.0 mmol/L,Tween-80 0.10%,28℃,130 rpm/min摇瓶培养13d。采用超滤、硫酸铵分级、DEAE-Sepharose Fast Flow及Sephadex G-100柱层析对漆酶粗酶液进行分离纯化,分离纯化的漆酶酶粉的比活力为28.4 U/mg,分子量为64.4 kD,其同工酶分子量为35.8 kD。该漆酶催化ABTS的最佳pH值为2.0,最佳温度为55℃,25℃时漆酶催化ABTS的K_m值为7.78μmol/L,V_max为5.11×10^-5L·mol^-1·min^-1。该漆酶储存稳定性能良好,在-18℃放置1个月其酶活仍保持90.2%。（2）采用反相悬浮法制备了平均粒径约为104.5 nm的MCN。采用TEM、FI-IR对MCN的结构进行了表征。研究了MCN的磁性能及抗氧化性能。结果表明,MCN具有超顺磁性,矫顽力为10.27 Oe,抗氧化性良好,易于磁分离。以MCN为载体固定漆酶,研究了MCN固定漆酶的最适条件。固定化过程分为戊二醛活化和漆酶交联两步。戊二醛活化的最优条件为:50 mg MCN在pH7.0的磷酸缓冲液浸泡12 h,25℃时,在1%的戊二醛中活化2 h;漆酶交联的最优条件为:将活化好的MCN磁分离后,置于漆酶浓度为1.0 mg/mL的pH 6.0的磷酸缓冲液,4℃交联4 h。最优条件下制备的固定化漆酶固载率为28%,比活力为670 U/g。以ABTS为底物研究了固定化漆酶的酶学性质,结果表明,固定化漆酶催化ABTS的最适pH值为2.0,最适温度为50℃,25℃时K_m=23.38μmol/L,V_max=3.53×10^-5mol·L^-1·min^-1,固定化漆酶具有良好的热稳定性、操作稳定性和储存稳定性。（3）研究了漆酶催化氧化DCP的机理以及漆酶和固定化漆酶催化氧化DCP的最适条件,漆酶和固定化漆酶催化DCP的最佳pH均为5.0,最佳温度分别为45℃和55℃。动力学研究结果表明,漆酶对DCP的催化氧化符合一级反应动力学方程。在漆酶-ABTS介体体系中,ABTS起到了电子传递的作用,加入介体以后,DCP的氧化速度明显提高。游离漆酶催化DCP氧化的机理研究表明催化反应是由四个化学反应组成的,醌式中间体的生成速度与每个反应的速度常数有关,亦即与漆酶、介体、底物的浓度有关。在漆酶-介体体系中,游离漆酶和固定化漆酶催化DCP的最适pH和最适温度分别为4.5和50℃。（4）制备了3种金属酞菁（CuPc、MnPc、CoPc）,研究了金属酞菁对DCP的催化性能。金属酞菁催化DCP的最适pH为8.0的磷酸缓冲液,最适温度为55℃,最佳金属酞菁用量为5.0 mg·mL^-1。（5）分别以固定化漆酶、酞菁铜为生物识别元件,采用锁相放大技术构建了两种光纤DCP传感器,研究了传感器的性能并对DCP含量进行测定。结果表明,基于固定化漆酶催化的光纤DCP传感器的线性检测范围为1.0×10^-7-9.0×10^-5mol/L,检测下限为4.4×10^-8mol/L,响应时间为40 s;基于酞菁铜仿生酶催化的光纤DCP传感器线性检测范围在1.0×10^-6-9.0×10^-5 mol/L,检测下限为7.4×10^-7mol/L,响应时间为600 s。两种传感器均有较好的重复性和长期稳定性。将两种传感器应用于长江水和加入DCP的长江水的检测,并与高效液相色谱的测定结果进行了比较研究。结果表明,2种传感器的检测效果好,精度高。对于基于固定化漆酶催化的光纤DCP传感器,Mg²⁺、Ca²⁺的加入使得检测结果偏高,加入EDTA作螯合剂,能消除Mg²⁺、Ca²⁺的影响。在基于酞菁铜催化的光纤DCP传感器中加入Mg²⁺、Ca²⁺对检测结果没有影响。更多还原

【Abstract】 2,4-chlorophenol （DCP） is a kind of very toxic substance among the pollutants in the environment and they are widespread, difficult to degrade resistant and toxic refractory. It had been regarded by United States Environmental Protection Agency（US EPA） and our government. as the toxic organic substance to be specially controlled. Therefore, the detection of DCP concerntion is of crucial importance to environmental protection and human health.The main goal of this thesis is to investigate the fiber optic DCP biosensors to detect DCP concentration. Immobilized laccase and copper phthalocyanine were used respectively as the bio-recognition element to constructed fiber optical DCP biosensors based on oxygen consumption, and the detection of DCP by fiber optic DCP biosensors was realized.In this thesis, the following aspects have been studied systematically. Firstly, the culture and purification conditions of Coriolus versicolor had been investigated. Laccase with the specific activity of 28.4 U/mg was prepared. Secondly, magnetic chitosan nanoparticles（MCN） were prepared by reversed-phase suspension method and used to immobilize laccase. The optimum conditions of immobilization were studied and the catalytic properties of free laccase and immobilized laccase were studied by using ABTS as the substrate. Thirdly, the mechanisms of the DCP oxidation catalyzed in laccase system and laccase-ABTS [2,2-azinobis -（3-ethylbenzthiazoline-6-sulfonat）] mediator system were studied, the optimum conditions for DCP oxidation catalyzed by free laccase and immobilized laccase were investigated. Fourthly, three types of metal phthalocyanine were synthesized by benzene bitter wine-urea Law, the mechanisms and the optimum conditions of DCP oxidation catalyzed by metal phthalocyanine were investigated. Finally, fiber optic DCP biosensors based on enzyme catalysis were designed and constructed. The principle and the performance of the biosensors based on DCP catalysed by laccase or copper phthalocyanine were investigated, respectively. The main conclusions of this thesis include the following five aspects:（1） By means of liquid fermentation, the culture conditions of Coriolus versicolor were optimized. The laccase was isolated with the specific activity of 28.4 U/mg. The catalytic properties of laccase were studied using ABTS as the substrate.The optimal conditions were determined as: potato juice 20%, glucose 20.0 g/L, peptone 4.0 g/L, CuSO₄ 1.0 mmol/L, KH₂PO₄ 3.0 g/L, MgSO₄ 1.5 g/L, ZnSO₄ 0.5 g/L, MnSO₄ 0.5 g/L, ABTS 1.0 mmol/L, Tween-80 0.10 %, pH 5.0, 130 rpm/min, 28℃,13 days.Laccase was isolated and purified through ultra-filtration, followed by ammonium sulphate fractionation, DEAE-Sepharose Fast Flow and Sephedax G-100 column chromatography. The specific activity of laccase was 28.4 U/mg and the molecular mass of laccase was estimated to be 64.4 kDa by SDS-PAGE. The molecular weight of isoenzymes was 35.8 kD.The catalytic properties of laccase were studied using ABTS as the substrate. The optimum pH value was 7.0 and the optimum temperature was 55℃. The K_m and V_max values of laccase on ABTS at 25℃were 7.78umol/L and 5.11×10^-5 L·mol ^-1·min^-1, respectively. The purified laccase represents excellent storage stability and could maintain 90.2% of its initial activity after stored at -18℃for 28 days.（2） MCN were prepared by reversed-phase suspension method and were characterized by TEM, NMR, FT-IR. The magnetic properties and oxidation resistance were investigated. The results showed that MCN were almost superparamagnetic with Hc of 10.27 Oe. The oxidation resistance of MCN was good. With small Hc, MCN wrer easy to be separated from solution and reused.The laccase was immobilized on the surface of MCN by crosslinking method and the process included two steps: MCN activation by glutaraldehyde and cross-linking by laccase. The activation process by glutaraldehyde was under the following conditions: 50 mg MCN were dipped in the PBS（pH 7.0） buffer for 12 hours, then activated with 1% glutaraldehyde for 2h at 25℃. After activation and treatment by magnetic separation method, the crosslinking reaction with laccase was carried out at 4℃. The optimum pH, laccase concentration and reaction time of crosslinking reaction were 6.0, 1.0 mg/mL and 4h, respectively. The immobilization yield and the activity of the immobilized laccase were 28 %, 670 U/g, respectively.The catalytic properties of immobilized laccase were investigated by using ABTS as the substrate. The immobilized laccases exhibited the maximum enzyme activity at pH 2.0 and at 50℃. The K_m and V_max values of immobilized laccase at 25℃were 23.38μmol/L and 3.53×10^-5 mol·L^-1·min^-1, respectively. Also, the immobilized laccases had good thermal, store, operation stabilities.（3） The mechanism of the DCP oxidation catalyzed by laccase and the optimum catalytic conditions by free laccase and immobilized laccase were studied. For both free laccase and immobilized laccase, the optimum pH was 8.0. The optimal temperature of free laccase and immobilized laccase were 45℃, 55℃, respectively. The kinetic study indicates that the DCP oxidation catalyzed by laccase coincided with first-order kinetic equation.In the laccase-ABTS mediator system, ABTS is employed to shuttle electrons from the DCP to laccase. It was shown that by using ABTS as a mediator, laccase from Coriolus versicolor was able to increase the rate of DCP oxidation.The mechanism of DCP oxidation catalyzed by laccase indicated that the catalyze process contained four reactions. The producing rate of 2-chloro-1,4-benzoquinon had relations with each reaction’s rate constant, namely it was influenced by the concentration of laccase, mediator and substrate. For both free laccase and immobilized laccase in the laccase-ABTS mediator system, the optimum pH and temperature of DCP oxidiation catalyzed by laccase were 8.0, 50℃, respectively.（4） Three types of metal phthalocyanine were synthesized by benzene bitter wine-urea law. The mechanism and catalytic properties of DCP oxidation catalyzed by metal phthalocyanine were studied and the optimum catalytic conditions were as follows: pH 8.0, temperature 55℃, amount of metal phthalocyanin 5.0 mg/mL.（5） Two types of novel fiber optic DCP biosensors based on the DCP oxidation catalyzed by immobilized laccase or copper phthalocyanine were designed and fabricated. The performance of the biosensors was investigated. The linear detection range, detection limit and response time of the fiber optic DCP biosensor base on the catalysis of immobilized laccase were 1.0×10^-7-9.0×10^-5 mol/L, 4.4×10^-8 mol/L, 40 s respectively. The linear detection range, detection limit and response time of the biosensor base on the catalysis of copper phthalocyanine were 1.0×10^-6-9.0×10^-5 mol/L mol/L, 7.4×10^-7 mol/L, 600 s, respectively. Both of them showed good reproducibility and stability.Both of biosensors were used to detect the DCP concentration in the water from Yangtze river, and the results were compared those determined by HPLC, indicating that the detection of DCP concentration by these biosensors were reliable. The influences of interference ions Mg²⁺and Ca²⁺ on the detection of DCP concentration by these biosensors have been studied. For the fiber optic DCP biosensor base on immobilized laccase. The results were higher than the true values. The influences of Mg²⁺and Ca²⁺ were eliminated when EDTA was added. For the fiber optic DCP biosensor base on copper phthalocyanine, the result wouldn’t be influenced by interference ions Mg²⁺and Ca²⁺.更多还原