【摘要】：本文以從滇池底泥分離出來的能高效降解微囊藻毒素(microcystins,MCs)的鞘氨醇單胞菌(Sphingopyxis sp.)USTB-05為研究對象,通過研究USTB-05菌降解MCs的特性,利用碳納米管(Carbon Nanotubes,CNTs)加載細(xì)菌去除MCs,進(jìn)一步提高細(xì)菌降解MCs的效率;對USTB-05降解MCs基因進(jìn)行分子克隆、構(gòu)建重組菌、純化重組菌酶、檢測重組菌酶降解MCs產(chǎn)物,初步探索USTB-05菌降解MCs酶學(xué)途徑。具體研究內(nèi)容和結(jié)果如下:(1)USTB-05菌能在4 h內(nèi)將9.58 mg/L的微囊藻毒素-LR(MC-LR)完全降解;含蛋白濃度為350 mg/L的USTB-05菌無細(xì)胞提取液(Cell free extract,CE),能在8 h內(nèi)將11.6 mg/L的MC-LR完全降解;同時(shí)利用單壁碳納米管(Single-Walled Carbon Nanotubes,SWCNTs)和CE對MC-LR降解效率,比單獨(dú)使用USTB-05菌CE增強(qiáng)了10%;兩種CNTs加載相同濃度USTB-05菌酶降解MC-LR的平均速率比較:SWCNTs+USTB-05菌酶多壁碳納米管(Multi-Walled Carbon Nanotubes,MWCNTs)+USTB-05菌酶。(2)通過引物設(shè)計(jì)及目的基因的PCR擴(kuò)增等基因工程技術(shù),得到降解基因。通過構(gòu)建重組質(zhì)粒pT15/USTB-05-A、pET30a(+)/USTB-05-B和pET30a(+)/USTB-05-C,并將質(zhì)粒轉(zhuǎn)化至E.coli BL21(DE3),分別獲得重組菌A、重組菌B和重組菌C。(3)通過利用Ni-IDA親和柱對破碎后的重組菌A、重組菌B和重組菌C的CE分別進(jìn)行蛋白純化,純化后的蛋白采用Bradford法測定蛋白濃度,依次為1 mg/mL、0.2mg/mL、1.2 mg/mL。經(jīng)R250染色的SDS-PAGE膠評估,純化后的蛋白純度分別為95.4%、91.1%、91.6%。(4)同時(shí)利用CNTs和重組菌降解MCs的效率比單獨(dú)使用重組菌增強(qiáng)39%;含蛋白濃度350 mg/L重組菌CE能在2 h內(nèi)將11.6 mg/L的MC-LR完全降解;兩種CNTs加載相同濃度重組菌酶降解MC-LR的平均速率對比:MWCNTs+重組菌酶SWCNTs+重組菌酶。(5)通過對重組菌表達(dá)的3種高效酶降解MC-LR及產(chǎn)物的研究,使用高效液相色譜技術(shù),檢測MC-LR降解產(chǎn)物。結(jié)果表明,USTB-05菌的第一種酶能快速催化降解MC-LR,生成線性MC-LR。第二種酶能快速催化降解線性MC-LR,生成Adda-Glu-Mdha-Ala。而第三種酶不但能快速降解Adda-Glu-Mdha-Ala生成Adda,而且還能催化降解線性MC-LR生成產(chǎn)物Adda。
[Abstract]:In this paper, the sphingomonas (Sphingopyxis sp.) USTB-05, which can efficiently degrade microcystins (microcystins,MCs) from the sediment of Dianchi Lake, was used as the research object. The characteristics of MCs degradation by USTB-05 bacteria were studied, and the carbon nanotube (Carbon Nanotubes, was used. CNTs) loading bacteria to remove MCs, further improved the efficiency of MCs degradation by bacteria. The USTB-05 degrading MCs gene was cloned, the recombinant strain was constructed, the recombinant enzyme was purified, the MCs product was detected, and the enzymatic pathway of USTB-05 degrading MCs was preliminarily explored. The results were as follows: (1) USTB-05 could completely degrade LR (MC-LR) of 9.58 mg/L within 4 h; The cell-free extract (Cell free extract,CE) of USTB-05 containing protein concentration of 350 mg/L could completely degrade 11.6 mg/L MC-LR within 8 h. At the same time, the degradation efficiency of MC-LR by using single-walled carbon nanotubes (Single-Walled Carbon Nanotubes,SWCNTs) and CE was 10% higher than that of USTB-05 strain CE alone. Comparison of the average rate of degradation of MC-LR by two kinds of CNTs loaded with the same concentration of USTB-05 Bacillus enzyme: SWCNTs USTB-05 Bacillus Enzymatic Multiwalled carbon Nanotubes (Multi-Walled Carbon Nanotubes,) (2) the degradation gene was obtained by primer design and PCR amplification of the target gene. Recombinant plasmids pT15/USTB-05-A,pET30a () / USTB-05-B and pET30a () / USTB-05-C, were constructed and transformed into E.coli BL21 (DE3). Recombinant strain B and recombinant strain C. (3) the CE of broken recombinant strain A, recombinant strain B and recombinant strain C were purified by Ni-IDA affinity column, respectively. The purified protein was determined by Bradford method. 1 mg/mL,0.2mg/mL,1.2 mg/mL. in turn The purified protein was evaluated by R250 stained SDS-PAGE gel. The purity of purified protein was 91.6. (4) the degradation efficiency of MCs by CNTs and recombinant bacteria was 39% higher than that of recombinant bacteria alone. The recombinant strain CE with protein concentration of 350 mg/L could completely degrade 11.6 mg/L MC-LR within 2 h. Comparison of the average rates of degradation of MC-LR by two kinds of CNTs with the same concentration of Recombinant enzymes: MWCNTs Recombinase SWCNTs Recombinase. (5) High performance liquid Chromatography (HPLC) was used to study the degradation of MC-LR and its products by three kinds of high efficient enzymes expressed by recombinant bacteria. MC-LR degradation products were detected. The results showed that the first enzyme of USTB-05 could rapidly catalyze the degradation of MC-LR, to produce linear MC-LR.. The second enzyme can rapidly catalyze the degradation of linear MC-LR, to Adda-Glu-Mdha-Ala.. The third enzyme can not only rapidly degrade Adda-Glu-Mdha-Ala to Adda, but also catalyze the degradation of linear MC-LR product Adda..
【學(xué)位授予單位】：江西理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：X171.5

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