本文選題：白腐真菌 + 漆酶��； 參考：《華中科技大學(xué)》2015年碩士論文

【摘要】：白腐真菌是一類能將木材腐爛成白色海綿狀團(tuán)塊的真菌,能夠在純系培養(yǎng)中有效地將木質(zhì)素徹底降解為CO2和H2O。漆酶是白腐真菌產(chǎn)生的一種具有巨大應(yīng)用價值和潛力的木質(zhì)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是本實驗室分離的一株能夠高效降解染料污染物的菌株,其降解能力與漆酶緊密相關(guān)。本論文以Ganoderma sp.En3為材料,對其分泌表達(dá)的三種漆酶同工酶進(jìn)行了分離純化,在此基礎(chǔ)上研究了不同漆酶同工酶的性質(zhì)及其差異性,探究了不同漆酶同工酶對不同結(jié)構(gòu)類型的合成染料及不同化學(xué)結(jié)構(gòu)的多環(huán)芳烴的降解作用。本論文主要研究結(jié)果如下:首先利用響應(yīng)面優(yōu)化法對Ganoderma sp.En3液體發(fā)酵漆酶產(chǎn)酶條件進(jìn)行了優(yōu)化。經(jīng)過響應(yīng)面優(yōu)化,Ganoderma sp.En3最優(yōu)產(chǎn)酶條件為葡萄糖46.468g/L,酵母浸膏6.490g/L和CuSO4 3.572mM。在這一條件下,經(jīng)實驗驗證得到的最高漆酶酶活為11.69±1.84U/mL,較優(yōu)化之前提高了47.23%。在以銅離子為誘導(dǎo)物的GYP培養(yǎng)基中,Ganoderma sp.En3能夠分泌產(chǎn)生4種漆酶同工酶,分別被命名為En3-Lac-1,En3-Lac-2,En3-Lac-3和En3-Lac-4。利用丙酮沉淀和多種層析手段,成功純化得到了三種漆酶同工酶En3-Lac-2,En3-Lac-3和En3-Lac-4,比活分別為329.03,382.73以及192.35U/mg Pr,總酶活回收率為22.94%,分子量分別為74,72和56 kDa。對三種漆酶同工酶的理化性質(zhì)進(jìn)行了比較,分離得到的三種漆酶具有相似的最適反應(yīng)pH和溫度,En3-Lac-2和En3-Lac-3的最適底物都是ABTS,而En3-Lac-4則對DMP具有最高的親和力。En3-Lac-2在三種漆酶同工酶中具有較強(qiáng)的熱穩(wěn)定性和pH穩(wěn)定性,En3-Lac-2對各種金屬離子和有機(jī)溶劑的耐受性也較其它兩種同工酶更強(qiáng)。利用純化的三種漆酶同工酶開展了對不同類型染料的降解研究,發(fā)現(xiàn)不同漆酶同工酶對于不同結(jié)構(gòu)類型染料的降解能力存在差異。En3-Lac-2對于三苯甲烷和靛藍(lán)類染料具有更強(qiáng)的降解能力,而En3-Lac-4對于偶氮和蒽醌類染料降解作用較好。en3-lac-3是三者之中降解能力相對最弱的漆酶同工酶,僅能降解甲基綠等少數(shù)幾種染料。染料降解的動力學(xué)研究表明,不同的漆酶同工酶具有不同的底物特異性。en3-lac-2對靛藍(lán)類染料靛藍(lán)胭脂紅(ic)的降解效率最高(kcat=4.03s-1),en3-lac-3和en3-lac-4則分別對甲基綠(mg)(kcat=2.58s-1)和雷瑪唑亮藍(lán)(rbbr)(kcat=1.26s-1)具有最高的降解效率。與en3-lac-2和en3-lac-3相比,en3-lac-4具有更寬的底物范圍,對于所選擇的十種染料,除酸性品紅(af)和活性藍(lán)5(rb5)外都有較好的降解能力。為了進(jìn)一步提高同工酶對染料的降解效果,比較了不同介體對en3-lac-4降解酸性品紅(af)的促進(jìn)作用,發(fā)現(xiàn)丁香醛具有最好的介導(dǎo)作用,可以使en3-lac-4降解af的降解率提高118倍。當(dāng)丁香醛作為介體添加到降解體系中,多數(shù)染料的降解率都達(dá)到或超過90%。添加丁香醛后,各個同工酶之間降解染料能力的差異性也被大大消除,降解時間曲線趨于一致。本研究分離得到的三種漆酶同工酶中,en3-lac-3自身對染料的降解能力較差,但是與其它兩種漆酶en3-lac-2和en3-lac-4存在明顯的協(xié)同降解作用,能夠促進(jìn)二者對偶氮染料的降解。這種協(xié)同效應(yīng)與同工酶之間的比例關(guān)系密切,同時en3-lac-3與en3-lac-4之間的協(xié)同作用效果更強(qiáng)。進(jìn)一步探究了三種漆酶同工酶對不同化學(xué)結(jié)構(gòu)的多環(huán)芳烴的降解作用,en3-lac-3和en3-lac-4對五種多環(huán)芳烴蒽、熒蒽、芴、菲和芘都具有較強(qiáng)的降解能力,24小時降解率都在85%以上。en3-lac-2對于幾種多環(huán)芳烴的降解能力都要弱于其它兩種漆酶同工酶,en3-lac-2對芘的降解能力最差,24小時降解率不足10%。三種漆酶同工酶對于多環(huán)芳烴的降解都符合一級反應(yīng)動力學(xué),比較一級反應(yīng)參數(shù)k0值發(fā)現(xiàn),en3-lac-2對幾種多環(huán)芳烴的降解容易程度依次為熒蒽、芴、菲、蒽、芘,en3-lac-3和en3-lac-4則大致為熒蒽、菲、芘、蒽、芴。綜上所述,本研究純化得到ganodermasp.en3三種漆酶同工酶,en3-lac-2具有較強(qiáng)的熱穩(wěn)定性和ph穩(wěn)定性,en3-lac-2對各種金屬離子和有機(jī)溶劑的耐受性也較其它兩種同工酶更強(qiáng)。不同漆酶同工酶對于不同結(jié)構(gòu)類型染料的降解能力存在差異。不同漆酶同工酶對偶氮染料的降解具有協(xié)同促進(jìn)作用。本研究結(jié)果有助于更深入地理解不同漆酶同工酶的功能及其相互關(guān)系,對于更好地將白腐真菌及其漆酶應(yīng)用于環(huán)境污染物降解等領(lǐng)域具有積極的促進(jìn)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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：X172;X703

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