一株红球菌（Rhodococcus sp. strain p52）降解二苯并呋喃途径基因的表达及功能验证
发布时间：2018-07-28 06:56
【摘要】：二VA英是多氯代二苯并二VA英和多氯代二苯并呋喃的统称,是对人体健康和环境有很大威胁的持久性有机污染物,这些物质在焚烧、纸浆漂白和化学生产等过程中作为副产物产生,并且在环境中稳定存在。与物理化学技术相比,生物降解法成本低,不会产生二次污染,通过生物修复去除二VA英始终是国内外的研究热点,并被认为是未来的发展方向。本文以课题组前期分离获得的一株含两个二VA英降解质粒的红球菌(Rhodococcus sp.strain p52)作为研究对象,对其降解二苯并呋喃(DF)途径中外二醇双加氧酶与水解酶基因的表达及功能进行研究。通过全基因组序列分析发现,该菌株含有两套能够以有角度双加氧方式代谢DF的双加氧酶系统,两套双加氧酶系统分别位于两个不同的质粒上,其编码基因簇分别包含起始双加氧酶基因dfdA和dbfA,外二醇双加氧酶基因dfdB和flnD,水解酶基因dfdC和fnE。本文通过PCR扩增技术分别得到DF降解途径中外二醇双加氧酶基因dfdB和fnD、水解酶基因d 和 flnE,并以 pET-32a(+)为载体,以Escherichia coli DH5α,Escherichia coliBL21(DE3)和Rosetta gamiTM2(DE3)plysS 为宿主菌株,进行基因的克隆及异源表达。通过聚丙烯酰胺凝胶电泳(SDS-PAGE)检测表达产物,结果显示,与同样经过IPTG诱导的含有空载体的宿主菌相比,在连接有目的基因的重组菌株的蛋白质电泳图谱中,均有明显的特异性蛋白条带,其分子量大小与理论预测值相符,且基因dfdB、dfdC和fnE的表达蛋白主要存在于上清液中,以可溶性蛋白的形式存在,而外二醇双加氧酶基因fnD的表达产物以包涵体的形式存在于沉淀中。基于以上研究,本文进一步验证了外二醇双加氧酶DfdB和水解酶DfdC的生物学功能,同时研究了温度、pH、金属离子对外二醇双加氧酶DfdB活性的影响。为获得具有生物活性的目的蛋白,对于外二醇双加氧酶基因flnD的表达产物进行了包涵体的复性研究。以上研究结果表明,外二醇双加氧酶基因dfdB编码开环裂解酶,该酶能够催化2,3-二羟基联苯的开环裂解,产生HPDA,而基因dfdC编码水解酶,该酶能够催化HPDA的水解。在温度为30℃,pH值为8时,外二醇双加氧酶DfdB具有最高生物活性,不同金属离子对酶活性产生不同程度的抑制作用。外二醇双加氧酶基因fnD以包涵体的形式大量表达,经包涵体变性复性后可成为可溶性蛋白,但其生物学功能尚有待进一步验证。综上所述,本研究从分子遗传学角度探究了二VA英的降解机理,为开发利用及改造微生物有效去除二VA英等污染物提供了理论依据,为环境中二VA英等污染物的生物修复打下基础。
[Abstract]:DiVA is a combination of PCDDs and PCDFs. They are persistent organic pollutants that pose a significant threat to human health and the environment and are incinerated. Pulp bleaching and chemical production are produced as by-products and exist stably in the environment. Compared with physical and chemical technology, biodegradation method has low cost and can not produce secondary pollution. Removing diVA by bioremediation has always been a hot spot at home and abroad, and is considered to be the future development direction. In this paper, we studied the expression and function of dibenzofuran (DF) pathway of Rhodococcus rubrum (Rhodococcus sp.strain p52) containing two diVA degradation plasmids (Rhodococcus sp.strain p52). The whole genome sequence analysis showed that the strain contained two sets of double oxygenase systems which could metabolize DF in an angular double oxygenation manner, and the two double oxygenase systems were located on two different plasmids, respectively. The coding gene cluster consists of dfdA and dbfA, dfdB and flnDand dfdC and FNE, respectively. The coding gene cluster is composed of the original dioxygenase gene dfdA and dbfA, the exogenous diol dioxygenase gene dfdB and flnD. the hydrolase gene dfdC and fnE respectively. In this paper, the dfdB and FND genes, the hydrolase genes d and flnE of DF degradation pathway were obtained by PCR amplification, respectively. Using pET-32a () as vector, Escherichia coli DH5 伪 Escherichia coliBL21 (DE3) and Rosetta gamiTM2 (DE3) plysS were used as host strains for gene cloning and heterologous expression. The expression product was detected by polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that compared with the host bacteria with empty vector induced by IPTG, the protein electrophoresis of the recombinant strain with the target gene was found in the electrophoretic map of the recombinant strain. There were obvious specific protein bands, and their molecular weight was in agreement with the predicted value. The expression of dfdBndC and fnE in supernatant was mainly found in supernatant, and the expression of dfdBndC and fnE was in the form of soluble protein. The fnD expression product of diol dioxygenase gene existed in the precipitate as inclusion body. Based on the above studies, the biological functions of exogenous diol dioxygenase (DfdB) and hydrolase DfdC (DfdC) were further verified, and the effects of temperature (pH) and metal ions' external diol dioxygenase (DfdB) activity were studied. In order to obtain the target protein with biological activity, the inclusion bodies were refolded into the flnD expression products of exogenous diol dioxygenase gene. The results showed that dfdB encodes a ring-opening lyase, which can catalyze the ring-opening cleavage of 2C3- dihydroxybiphenyls to produce HPDAs, while the gene dfdC encodes a hydrolase, which can catalyze the hydrolysis of HPDA. When the temperature was 30 鈩，
本文编号：2149272
[Abstract]:DiVA is a combination of PCDDs and PCDFs. They are persistent organic pollutants that pose a significant threat to human health and the environment and are incinerated. Pulp bleaching and chemical production are produced as by-products and exist stably in the environment. Compared with physical and chemical technology, biodegradation method has low cost and can not produce secondary pollution. Removing diVA by bioremediation has always been a hot spot at home and abroad, and is considered to be the future development direction. In this paper, we studied the expression and function of dibenzofuran (DF) pathway of Rhodococcus rubrum (Rhodococcus sp.strain p52) containing two diVA degradation plasmids (Rhodococcus sp.strain p52). The whole genome sequence analysis showed that the strain contained two sets of double oxygenase systems which could metabolize DF in an angular double oxygenation manner, and the two double oxygenase systems were located on two different plasmids, respectively. The coding gene cluster consists of dfdA and dbfA, dfdB and flnDand dfdC and FNE, respectively. The coding gene cluster is composed of the original dioxygenase gene dfdA and dbfA, the exogenous diol dioxygenase gene dfdB and flnD. the hydrolase gene dfdC and fnE respectively. In this paper, the dfdB and FND genes, the hydrolase genes d and flnE of DF degradation pathway were obtained by PCR amplification, respectively. Using pET-32a () as vector, Escherichia coli DH5 伪 Escherichia coliBL21 (DE3) and Rosetta gamiTM2 (DE3) plysS were used as host strains for gene cloning and heterologous expression. The expression product was detected by polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that compared with the host bacteria with empty vector induced by IPTG, the protein electrophoresis of the recombinant strain with the target gene was found in the electrophoretic map of the recombinant strain. There were obvious specific protein bands, and their molecular weight was in agreement with the predicted value. The expression of dfdBndC and fnE in supernatant was mainly found in supernatant, and the expression of dfdBndC and fnE was in the form of soluble protein. The fnD expression product of diol dioxygenase gene existed in the precipitate as inclusion body. Based on the above studies, the biological functions of exogenous diol dioxygenase (DfdB) and hydrolase DfdC (DfdC) were further verified, and the effects of temperature (pH) and metal ions' external diol dioxygenase (DfdB) activity were studied. In order to obtain the target protein with biological activity, the inclusion bodies were refolded into the flnD expression products of exogenous diol dioxygenase gene. The results showed that dfdB encodes a ring-opening lyase, which can catalyze the ring-opening cleavage of 2C3- dihydroxybiphenyls to produce HPDAs, while the gene dfdC encodes a hydrolase, which can catalyze the hydrolysis of HPDA. When the temperature was 30 鈩，
本文编号：2149272
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