鞘氨醇单胞菌USTB-05降解微囊藻毒素-LR特性及酶学途径研究
发布时间：2018-11-10 07:28
【摘要】：本文以从滇池底泥分离出来的能高效降解微囊藻毒素(microcystins,MCs)的鞘氨醇单胞菌(Sphingopyxis sp.)USTB-05为研究对象,通过研究USTB-05菌降解MCs的特性,利用碳纳米管(Carbon Nanotubes,CNTs)加载细菌去除MCs,进一步提高细菌降解MCs的效率;对USTB-05降解MCs基因进行分子克隆、构建重组菌、纯化重组菌酶、检测重组菌酶降解MCs产物,初步探索USTB-05菌降解MCs酶学途径。具体研究内容和结果如下:(1)USTB-05菌能在4 h内将9.58 mg/L的微囊藻毒素-LR(MC-LR)完全降解;含蛋白浓度为350 mg/L的USTB-05菌无细胞提取液(Cell free extract,CE),能在8 h内将11.6 mg/L的MC-LR完全降解;同时利用单壁碳纳米管(Single-Walled Carbon Nanotubes,SWCNTs)和CE对MC-LR降解效率,比单独使用USTB-05菌CE增强了10%;两种CNTs加载相同浓度USTB-05菌酶降解MC-LR的平均速率比较:SWCNTs+USTB-05菌酶多壁碳纳米管(Multi-Walled Carbon Nanotubes,MWCNTs)+USTB-05菌酶。(2)通过引物设计及目的基因的PCR扩增等基因工程技术,得到降解基因。通过构建重组质粒pT15/USTB-05-A、pET30a(+)/USTB-05-B和pET30a(+)/USTB-05-C,并将质粒转化至E.coli BL21(DE3),分别获得重组菌A、重组菌B和重组菌C。(3)通过利用Ni-IDA亲和柱对破碎后的重组菌A、重组菌B和重组菌C的CE分别进行蛋白纯化,纯化后的蛋白采用Bradford法测定蛋白浓度,依次为1 mg/mL、0.2mg/mL、1.2 mg/mL。经R250染色的SDS-PAGE胶评估,纯化后的蛋白纯度分别为95.4%、91.1%、91.6%。(4)同时利用CNTs和重组菌降解MCs的效率比单独使用重组菌增强39%;含蛋白浓度350 mg/L重组菌CE能在2 h内将11.6 mg/L的MC-LR完全降解;两种CNTs加载相同浓度重组菌酶降解MC-LR的平均速率对比:MWCNTs+重组菌酶SWCNTs+重组菌酶。(5)通过对重组菌表达的3种高效酶降解MC-LR及产物的研究,使用高效液相色谱技术,检测MC-LR降解产物。结果表明,USTB-05菌的第一种酶能快速催化降解MC-LR,生成线性MC-LR。第二种酶能快速催化降解线性MC-LR,生成Adda-Glu-Mdha-Ala。而第三种酶不但能快速降解Adda-Glu-Mdha-Ala生成Adda,而且还能催化降解线性MC-LR生成产物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..
【学位授予单位】：江西理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X171.5
本文编号：2321797
[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..
【学位授予单位】：江西理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X171.5
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