本文選題：真菌漆酶　切入點(diǎn)：克百威　出處：《沈陽工業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：漆酶是一種來源廣泛、具有較好的底物專一性和穩(wěn)定性的環(huán)保型酶類，其可用于降解苯酚、多環(huán)芳烴化合物、氯化芳香化合物等多種污染物，，而通過有效的固定化技術(shù)可明顯提高漆酶的酶活穩(wěn)定性和重復(fù)利用率，增強(qiáng)漆酶的環(huán)境適應(yīng)性，使其在環(huán)保方面有著更廣泛的應(yīng)用空間。本文主要對(duì)真菌漆酶的固定化進(jìn)行了研究，考察了真菌產(chǎn)漆酶培養(yǎng)條件的優(yōu)化及固定化過程影響因素的確定，并將完成的固定化漆酶用于克百威與毒死蜱的降解過程中。該項(xiàng)研究為固定化漆酶在土壤修復(fù)中的實(shí)際應(yīng)用提供了一定的理論依據(jù)。 本研究選取土壤中殘留的克百威與毒死蜱兩種農(nóng)藥作為實(shí)驗(yàn)降解對(duì)象，首先在不同培養(yǎng)條件下對(duì)選取的三株真菌首先進(jìn)行產(chǎn)漆酶培養(yǎng)，通過漆酶酶活的測定確定出較優(yōu)的產(chǎn)酶條件；隨后采用包埋-交聯(lián)、吸附-包埋兩種復(fù)合方法分別對(duì)所得的游離態(tài)漆酶進(jìn)行固定化處理，并通過多因素試驗(yàn)確定出較適合的固定化條件；最后將固定化漆酶分別應(yīng)用于修復(fù)克百威與毒死蜱污染的土壤中，通過高效液相測定兩種農(nóng)藥的降解率，并考察在不同的環(huán)境條件下固定化漆酶對(duì)兩種農(nóng)藥的降解情況。 實(shí)驗(yàn)結(jié)果表明：較優(yōu)的真菌產(chǎn)酶培養(yǎng)條件為：5g浸濕秸稈+0.3g葡萄糖+0.025gMgSO4+0.0375gKH2PO4+0.03gCuSO4，同時(shí)確定比較合適的產(chǎn)酶培養(yǎng)時(shí)間為11～13天；包埋-交聯(lián)法制備固定化漆酶較合適的條件為：海藻酸鈉質(zhì)量百分濃度為3%，氯化鈣質(zhì)量百分濃度為1.5%，戊二醛質(zhì)量百分濃度為1%，交聯(lián)時(shí)間為6h，粗酶液體積為60ml，固定化時(shí)間為4h；包埋-吸附法固定化漆酶較合適的條件為：海藻酸鈉、活性炭質(zhì)量比例為1:1（g:g），氯化鈣質(zhì)量百分濃度為1.5%，粗酶液體積為80ml，固定化時(shí)間為6h；48h后包埋-交聯(lián)法制得的固定化C菌株漆酶和固定化Y菌株漆酶對(duì)土壤中殘留的克百威的降解率分別可達(dá)到75%左右和70%以上，對(duì)毒死蜱的降解率分別可達(dá)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菌株漆酶對(duì)土壤中殘留的克百威的降解率分別可達(dá)到86%左右和81%左右，對(duì)毒死蜱的降解率則分別可達(dá)到80%以上和77%左右；通過對(duì)固定化漆酶進(jìn)行環(huán)境影響因素的考察，得出在農(nóng)藥的初始濃度、環(huán)境pH值、環(huán)境溫度等變化幅度較大時(shí)，固定化漆酶都能保持一定的降解率，對(duì)環(huán)境變化具有較好的適應(yīng)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.
【學(xué)位授予單位】：沈陽工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：X592;Q814

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