【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)加載細菌去除MCs,進一步提高細菌降解MCs的效率;對USTB-05降解MCs基因進行分子克隆、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,比單獨使用USTB-05菌CE增強了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)計及目的基因的PCR擴增等基因工程技術(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)同時利用CNTs和重組菌降解MCs的效率比單獨使用重組菌增強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..
【學位授予單位】：江西理工大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：X171.5

【參考文獻】

相關(guān)期刊論文 前10條

1 李冬冬;杜爾登;李志宏;張申耀;彭明國;李華杰;;基于EEMs和PARAFAC分析典型碳材料吸附水中DOM特征研究[J];環(huán)境工程;2016年11期

2 張西軒;李曄;王亞航;杜康龍;張真;阮海華;;蠟樣芽孢桿菌ColR75E膠原酶的表達、純化及酶學性質(zhì)研究[J];中國生物工程雜志;2015年10期

3 鐘鳳;吳暉;劉思利;唐語謙;;可降解玉米赤霉烯酮的重組過氧化物酶A4-Prx的純化與活性研究[J];現(xiàn)代食品科技;2015年08期

4 張雪;華霄;許琪;楊瑞金;范宇婷;;低溫β-半乳糖苷酶分離純化及酶學性質(zhì)研究[J];食品與機械;2014年03期

5 游狄杰;陳曉國;向薈圯;歐陽潦;楊冰;;微囊藻毒素降解菌Paucibacter sp.CH菌的分離鑒定及其降解特性[J];環(huán)境科學;2014年01期

6 盧嬋;鄭璞;孫志浩;;L-谷氨酸氧化酶的克隆表達、純化及酶學性質(zhì)研究[J];中國生物工程雜志;2013年06期

7 杜靜;錢玉婷;徐躍定;張建英;常志州;;太湖和滇池表層水體水質(zhì)與水華藍藻生物質(zhì)生化性狀的關(guān)系[J];長江流域資源與環(huán)境;2013年02期

8 謝維;吳涓;李玉成;何席偉;杜洋;王光云;;一株微囊藻毒素降解菌的篩選及鑒定[J];生物學雜志;2012年06期

9 盧健;谷金鈺;;超聲波除藻技術(shù)設(shè)備研究及推廣前景[J];中國水利;2012年20期

10 ;Cloning and expression of the first gene for biodegrading microcystin LR by Sphingopyxis sp.USTB-05[J];Journal of Environmental Sciences;2012年10期

相關(guān)碩士學位論文 前4條

1 吳迪;藻毒素降解酶家族的克隆表達及酶學性質(zhì)的研究[D];華中師范大學;2014年

2 歐陽潦;CJ5菌降解MCLR的酶學途徑研究[D];武漢理工大學;2014年

3 胡越;X20菌的鑒定及其對MCLR的酶學降解途徑研究[D];武漢理工大學;2012年

4 尹玉芬;一株太湖土著細菌對微囊藻毒素生物降解的初步研究[D];南京農(nóng)業(yè)大學;2010年

，

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