白腐真菌Ganoderma sp.En3漆酶同工酶的性质及其对染料和多环芳烃降解研究

发布时间：2018-07-10 06:05

本文选题：白腐真菌 + 漆酶　；参考：《华中科技大学》2015年硕士论文

【摘要】：白腐真菌是一类能将木材腐烂成白色海绵状团块的真菌,能够在纯系培养中有效地将木质素彻底降解为CO2和H2O。漆酶是白腐真菌产生的一种具有巨大应用价值和潜力的木质素降解酶,主要以同工酶的形式在胞外分泌。白腐真菌Ganoderma sp.En3是本实验室分离的一株能够高效降解染料污染物的菌株,其降解能力与漆酶紧密相关。本论文以Ganoderma sp.En3为材料,对其分泌表达的三种漆酶同工酶进行了分离纯化,在此基础上研究了不同漆酶同工酶的性质及其差异性,探究了不同漆酶同工酶对不同结构类型的合成染料及不同化学结构的多环芳烃的降解作用。本论文主要研究结果如下:首先利用响应面优化法对Ganoderma sp.En3液体发酵漆酶产酶条件进行了优化。经过响应面优化,Ganoderma sp.En3最优产酶条件为葡萄糖46.468g/L,酵母浸膏6.490g/L和CuSO4 3.572mM。在这一条件下,经实验验证得到的最高漆酶酶活为11.69±1.84U/mL,较优化之前提高了47.23%。在以铜离子为诱导物的GYP培养基中,Ganoderma sp.En3能够分泌产生4种漆酶同工酶,分别被命名为En3-Lac-1,En3-Lac-2,En3-Lac-3和En3-Lac-4。利用丙酮沉淀和多种层析手段,成功纯化得到了三种漆酶同工酶En3-Lac-2,En3-Lac-3和En3-Lac-4,比活分别为329.03,382.73以及192.35U/mg Pr,总酶活回收率为22.94%,分子量分别为74,72和56 kDa。对三种漆酶同工酶的理化性质进行了比较,分离得到的三种漆酶具有相似的最适反应pH和温度,En3-Lac-2和En3-Lac-3的最适底物都是ABTS,而En3-Lac-4则对DMP具有最高的亲和力。En3-Lac-2在三种漆酶同工酶中具有较强的热稳定性和pH稳定性,En3-Lac-2对各种金属离子和有机溶剂的耐受性也较其它两种同工酶更强。利用纯化的三种漆酶同工酶开展了对不同类型染料的降解研究,发现不同漆酶同工酶对于不同结构类型染料的降解能力存在差异。En3-Lac-2对于三苯甲烷和靛蓝类染料具有更强的降解能力,而En3-Lac-4对于偶氮和蒽醌类染料降解作用较好。en3-lac-3是三者之中降解能力相对最弱的漆酶同工酶,仅能降解甲基绿等少数几种染料。染料降解的动力学研究表明,不同的漆酶同工酶具有不同的底物特异性。en3-lac-2对靛蓝类染料靛蓝胭脂红(ic)的降解效率最高(kcat=4.03s-1),en3-lac-3和en3-lac-4则分别对甲基绿(mg)(kcat=2.58s-1)和雷玛唑亮蓝(rbbr)(kcat=1.26s-1)具有最高的降解效率。与en3-lac-2和en3-lac-3相比,en3-lac-4具有更宽的底物范围,对于所选择的十种染料,除酸性品红(af)和活性蓝5(rb5)外都有较好的降解能力。为了进一步提高同工酶对染料的降解效果,比较了不同介体对en3-lac-4降解酸性品红(af)的促进作用,发现丁香醛具有最好的介导作用,可以使en3-lac-4降解af的降解率提高118倍。当丁香醛作为介体添加到降解体系中,多数染料的降解率都达到或超过90%。添加丁香醛后,各个同工酶之间降解染料能力的差异性也被大大消除,降解时间曲线趋于一致。本研究分离得到的三种漆酶同工酶中,en3-lac-3自身对染料的降解能力较差,但是与其它两种漆酶en3-lac-2和en3-lac-4存在明显的协同降解作用,能够促进二者对偶氮染料的降解。这种协同效应与同工酶之间的比例关系密切,同时en3-lac-3与en3-lac-4之间的协同作用效果更强。进一步探究了三种漆酶同工酶对不同化学结构的多环芳烃的降解作用,en3-lac-3和en3-lac-4对五种多环芳烃蒽、荧蒽、芴、菲和芘都具有较强的降解能力,24小时降解率都在85%以上。en3-lac-2对于几种多环芳烃的降解能力都要弱于其它两种漆酶同工酶,en3-lac-2对芘的降解能力最差,24小时降解率不足10%。三种漆酶同工酶对于多环芳烃的降解都符合一级反应动力学,比较一级反应参数k0值发现,en3-lac-2对几种多环芳烃的降解容易程度依次为荧蒽、芴、菲、蒽、芘,en3-lac-3和en3-lac-4则大致为荧蒽、菲、芘、蒽、芴。综上所述,本研究纯化得到ganodermasp.en3三种漆酶同工酶,en3-lac-2具有较强的热稳定性和ph稳定性,en3-lac-2对各种金属离子和有机溶剂的耐受性也较其它两种同工酶更强。不同漆酶同工酶对于不同结构类型染料的降解能力存在差异。不同漆酶同工酶对偶氮染料的降解具有协同促进作用。本研究结果有助于更深入地理解不同漆酶同工酶的功能及其相互关系,对于更好地将白腐真菌及其漆酶应用于环境污染物降解等领域具有积极的促进作用。
[Abstract]:White rot fungus is a kind of fungi that can decompose wood into white cavernous mass. It can effectively degrade lignin into CO2 and H2O. laccase in pure line culture. It is a kind of lignin degrading enzyme with great application value and potential produced by white rot fungi. It is mainly in the form of Isozyme in exocytosis. White rot fungus Ganoderma Sp.En3 is a strain which can efficiently degrade dye contaminants in our laboratory. Its degradation ability is closely related to laccase. In this paper, three kinds of laccase isozymes were purified and purified by Ganoderma sp.En3. On this basis, the properties and differences of different laccase isozymes were studied. The degradation of polycyclic aromatic hydrocarbons with different structural types of synthetic dyes and different chemical structures by different laccase isozymes. The main research results in this paper are as follows: first, the enzyme production conditions of Ganoderma sp.En3 laccase were optimized by response surface optimization. The optimal enzyme production conditions of Ganoderma sp.En3 were optimized by response surface optimization. Under the condition of glucose 46.468g/L, yeast extract 6.490g/L and CuSO4 3.572mM., the highest laccase activity was 11.69 + 1.84U/mL, and Ganoderma sp.En3 could produce 4 laccase isozymes in the GYP medium with copper ion as the inducer before optimization, and were named En3-Lac-1, En3-, respectively. Lac-2, En3-Lac-3 and En3-Lac-4. have successfully purified three laccase isozymes En3-Lac-2, En3-Lac-3 and En3-Lac-4, respectively, 329.03382.73 and 192.35U/mg Pr, and the total enzyme activity recovery rate is 22.94%, and the molecular weight is 74,72 and 56 kDa. on the physical and chemical properties of the three laccase isozymes. Three kinds of laccase have similar optimum reaction pH and temperature, and the most suitable substrates for En3-Lac-2 and En3-Lac-3 are ABTS, while En3-Lac-4 has the highest affinity for DMP,.En3-Lac-2 has strong thermal stability and pH stability in three laccase isozymes, and En3-Lac-2 is tolerated to various metal ions and organic solvents. The degradation of different types of dyes was carried out by using three kinds of laccase isozyme, the degradation ability of different laccase isozymes for different types of dyes was found to be different.En3-Lac-2 for the degradation of triphenyl methane and indigo dye, and En3-Lac-4 for pairs of dyes. The degradation of nitrogen and anthraquinone dyes is better.En3-lac-3 is the weakest laccase isozyme in the three, only a few dyes such as methyl green can be degraded. The kinetic study of dye degradation shows that different laccase isozymes have different substrate specific.En3-lac-2 degradation of indigo indigo carmine (IC). The highest efficiency (kcat=4.03s-1), en3-lac-3 and en3-lac-4 have the highest degradation efficiency for methyl green (kcat=2.58s-1) and rema blue (rbbr) (kcat=1.26s-1). Compared with en3-lac-2 and en3-lac-3, en3-lac-4 has a wider substrate range, for the selected ten dyes, except for acid fuchsin (AF) and active blue 5 (RB5). Better degradation ability. In order to further improve the degradation effect of isozymes to dye, the promotion of different mediators on en3-lac-4 degradation of acid fuchsin (AF) was compared. It was found that syringaldehyde had the best mediating effect, which could increase the degradation rate of en3-lac-4 degradation AF by 118 times. When Ding Xiangquan was added to the degradation system as a medium, most of them were added to the degradation system. After the degradation rate of the dyes reached or exceeded the 90%., the difference of the ability to degrade dyes between various isozymes was greatly eliminated and the degradation time curves tended to be consistent. Among the three laccase isozymes isolated in this study, the degradation ability of en3-lac-3 itself was poor, but with the other two laccase en3-lac-2 and en3-l AC-4 has a significant synergistic effect, which can promote the degradation of azo dyes by the two. This synergistic effect is closely related to the proportion of isozymes, and the synergistic effect between en3-lac-3 and en3-lac-4 is stronger. Further explore the degradation of polycyclic aromatic hydrocarbons with three laccase isozymes for different structure of polycyclic aromatic hydrocarbons. En3-lac- 3 and en3-lac-4 have strong degradation ability for five polycyclic aromatic hydrocarbons anthracene, fluorene, fluorene, phenanthrene and pyrene. The degradation rate of 24 hours is above 85%. The degradation ability of several polycyclic aromatic hydrocarbons is weaker than that of the other two laccase isozymes. The degradation ability of en3-lac-2 to pyrene is the worst, and the degradation rate of 10%. three kinds of laccase isozyme is less than 24 hours. The degradation of polycyclic aromatic hydrocarbons conforms to the first order reaction kinetics. Comparing the K0 values of first order reaction parameters, the degradation of several polycyclic aromatic hydrocarbons by en3-lac-2 is in turn fluoranthene, fluorene, phenanthrene, anthracene, pyrene, en3-lac-3 and en3-lac-4 are roughly fluoranthene, phenanthrene, pyrene, anthracene, fluorene. This study purified three kinds of laccase of ganodermasp.en3. The enzyme, en3-lac-2 has strong thermal stability and pH stability, en3-lac-2 is more tolerant to various metal ions and organic solvents than the other two isozymes. Different laccase isozymes have different degradation ability for different structure type dyes. Different laccase isozyme has synergistic effect on the degradation of azo dyes. The results are helpful to understand the functions and relationships of different laccase isozymes more deeply, and have a positive effect on the application of white rot fungi and their laccase to the degradation of environmental pollutants.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X172;X703

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