固定化漆酶降解克百威与毒死蜱的研究

发布时间：2018-03-13 14:43

本文选题：真菌漆酶　切入点：克百威　出处：《沈阳工业大学》2015年硕士论文　论文类型：学位论文

【摘要】：漆酶是一种来源广泛、具有较好的底物专一性和稳定性的环保型酶类，其可用于降解苯酚、多环芳烃化合物、氯化芳香化合物等多种污染物，，而通过有效的固定化技术可明显提高漆酶的酶活稳定性和重复利用率，增强漆酶的环境适应性，使其在环保方面有着更广泛的应用空间。本文主要对真菌漆酶的固定化进行了研究，考察了真菌产漆酶培养条件的优化及固定化过程影响因素的确定，并将完成的固定化漆酶用于克百威与毒死蜱的降解过程中。该项研究为固定化漆酶在土壤修复中的实际应用提供了一定的理论依据。本研究选取土壤中残留的克百威与毒死蜱两种农药作为实验降解对象，首先在不同培养条件下对选取的三株真菌首先进行产漆酶培养，通过漆酶酶活的测定确定出较优的产酶条件；随后采用包埋-交联、吸附-包埋两种复合方法分别对所得的游离态漆酶进行固定化处理，并通过多因素试验确定出较适合的固定化条件；最后将固定化漆酶分别应用于修复克百威与毒死蜱污染的土壤中，通过高效液相测定两种农药的降解率，并考察在不同的环境条件下固定化漆酶对两种农药的降解情况。实验结果表明：较优的真菌产酶培养条件为：5g浸湿秸秆+0.3g葡萄糖+0.025gMgSO4+0.0375gKH2PO4+0.03gCuSO4，同时确定比较合适的产酶培养时间为11～13天；包埋-交联法制备固定化漆酶较合适的条件为：海藻酸钠质量百分浓度为3%，氯化钙质量百分浓度为1.5%，戊二醛质量百分浓度为1%，交联时间为6h，粗酶液体积为60ml，固定化时间为4h；包埋-吸附法固定化漆酶较合适的条件为：海藻酸钠、活性炭质量比例为1:1（g:g），氯化钙质量百分浓度为1.5%，粗酶液体积为80ml，固定化时间为6h；48h后包埋-交联法制得的固定化C菌株漆酶和固定化Y菌株漆酶对土壤中残留的克百威的降解率分别可达到75%左右和70%以上，对毒死蜱的降解率分别可达到73%左右和70%以上；包埋-吸附法制得的固定化C菌株漆酶和固定化Y菌株漆酶对土壤中残留的克百威的降解率分别可达到86%左右和81%左右，对毒死蜱的降解率则分别可达到80%以上和77%左右；通过对固定化漆酶进行环境影响因素的考察，得出在农药的初始浓度、环境pH值、环境温度等变化幅度较大时，固定化漆酶都能保持一定的降解率，对环境变化具有较好的适应能力。
[Abstract]:Laccase is a kind of environment-friendly enzymes with good substrate specificity and stability, which can be used to degrade phenol, polycyclic aromatic compounds, chlorinated aromatic compounds and other pollutants. The stability of laccase activity and the reutilization rate of laccase can be improved by immobilized laccase, and the environmental adaptability of laccase can be enhanced. In this paper, the immobilization of laccase of fungi was studied, and the optimization of culture conditions of laccase production of fungi and the determination of factors affecting the immobilization process were investigated. The immobilized laccase was applied to the degradation of carbofuran and chlorpyrifos, which provided a theoretical basis for the practical application of immobilized laccase in soil remediation. In this study, two kinds of pesticides remained in soil, carbofuran and chlorpyrifos, were selected as experimental degradation objects. Firstly, three strains of fungi were cultured for laccase production under different culture conditions. The optimum conditions for producing laccase were determined by the determination of laccase activity, and then immobilized laccase was immobilized by entrapping, crosslinking, adsorption-entrapment and immobilization of laccase. Finally, the immobilized laccase was applied to remediate the soil polluted by carbofuran and chlorpyrifos, and the degradation rate of the two pesticides was determined by high performance liquid phase. The degradation of two pesticides by immobilized laccase under different environmental conditions was investigated. The results showed that the optimum culture conditions were as follows: 0.3g glucose 0.025g MgSO _ 4 0.0375g KH _ 2PO _ 4 0.03g CuSO _ 4, and the optimum culture time was 11 ~ (13) days. The suitable conditions for preparing immobilized laccase by entrapment and crosslinking are as follows: sodium alginate 3%, calcium chloride 1.5%, glutaraldehyde 1%, crosslinking time 6 h, volume of crude enzyme 60 ml. The suitable conditions for immobilization of laccase by entrapment and adsorption are as follows: sodium alginate, sodium alginate, sodium alginate, sodium alginate, sodium alginate, Immobilized C strain laccase and immobilized Y strain laccase were obtained by mass ratio of 1: 1 g / g of activated carbon, 1.5% of calcium chloride, 80 ml of crude enzyme solution and 6 h of immobilized time after embedding and crosslinking. The degradation rates of Wei can reach about 75% and 70% respectively. The degradation rates of chlorpyrifos were about 73% and 70% respectively, and the degradation rates of immobilized C strain laccase and immobilized Y strain laccase were about 86% and 81%, respectively. The degradation rate of chlorpyrifos can reach more than 80% and 77% respectively. By investigating the environmental influence factors of immobilized laccase, it is concluded that when the initial concentration of pesticide, environmental pH value and environmental temperature change greatly, The immobilized laccase can maintain a certain degradation rate and has a good adaptability to environmental changes.
【学位授予单位】：沈阳工业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X592;Q814

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