一株紅球菌（Rhodococcus sp. strain p52）降解二苯并呋喃途徑基因的表達(dá)及功能驗(yàn)證
發(fā)布時(shí)間：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統(tǒng)稱,是對(duì)人體健康和環(huán)境有很大威脅的持久性有機(jī)污染物,這些物質(zhì)在焚燒、紙漿漂白和化學(xué)生產(chǎn)等過(guò)程中作為副產(chǎn)物產(chǎn)生,并且在環(huán)境中穩(wěn)定存在。與物理化學(xué)技術(shù)相比,生物降解法成本低,不會(huì)產(chǎn)生二次污染,通過(guò)生物修復(fù)去除二VA英始終是國(guó)內(nèi)外的研究熱點(diǎn),并被認(rèn)為是未來(lái)的發(fā)展方向。本文以課題組前期分離獲得的一株含兩個(gè)二VA英降解質(zhì)粒的紅球菌(Rhodococcus sp.strain p52)作為研究對(duì)象,對(duì)其降解二苯并呋喃(DF)途徑中外二醇雙加氧酶與水解酶基因的表達(dá)及功能進(jìn)行研究。通過(guò)全基因組序列分析發(fā)現(xiàn),該菌株含有兩套能夠以有角度雙加氧方式代謝DF的雙加氧酶系統(tǒng),兩套雙加氧酶系統(tǒng)分別位于兩個(gè)不同的質(zhì)粒上,其編碼基因簇分別包含起始雙加氧酶基因dfdA和dbfA,外二醇雙加氧酶基因dfdB和flnD,水解酶基因dfdC和fnE。本文通過(guò)PCR擴(kuò)增技術(shù)分別得到DF降解途徑中外二醇雙加氧酶基因dfdB和fnD、水解酶基因d 和 flnE,并以 pET-32a(+)為載體,以Escherichia coli DH5α,Escherichia coliBL21(DE3)和Rosetta gamiTM2(DE3)plysS 為宿主菌株,進(jìn)行基因的克隆及異源表達(dá)。通過(guò)聚丙烯酰胺凝膠電泳(SDS-PAGE)檢測(cè)表達(dá)產(chǎn)物,結(jié)果顯示,與同樣經(jīng)過(guò)IPTG誘導(dǎo)的含有空載體的宿主菌相比,在連接有目的基因的重組菌株的蛋白質(zhì)電泳圖譜中,均有明顯的特異性蛋白條帶,其分子量大小與理論預(yù)測(cè)值相符,且基因dfdB、dfdC和fnE的表達(dá)蛋白主要存在于上清液中,以可溶性蛋白的形式存在,而外二醇雙加氧酶基因fnD的表達(dá)產(chǎn)物以包涵體的形式存在于沉淀中�；谝陨涎芯�,本文進(jìn)一步驗(yàn)證了外二醇雙加氧酶DfdB和水解酶DfdC的生物學(xué)功能,同時(shí)研究了溫度、pH、金屬離子對(duì)外二醇雙加氧酶DfdB活性的影響。為獲得具有生物活性的目的蛋白,對(duì)于外二醇雙加氧酶基因flnD的表達(dá)產(chǎn)物進(jìn)行了包涵體的復(fù)性研究。以上研究結(jié)果表明,外二醇雙加氧酶基因dfdB編碼開環(huán)裂解酶,該酶能夠催化2,3-二羥基聯(lián)苯的開環(huán)裂解,產(chǎn)生HPDA,而基因dfdC編碼水解酶,該酶能夠催化HPDA的水解。在溫度為30℃,pH值為8時(shí),外二醇雙加氧酶DfdB具有最高生物活性,不同金屬離子對(duì)酶活性產(chǎn)生不同程度的抑制作用。外二醇雙加氧酶基因fnD以包涵體的形式大量表達(dá),經(jīng)包涵體變性復(fù)性后可成為可溶性蛋白,但其生物學(xué)功能尚有待進(jìn)一步驗(yàn)證。綜上所述,本研究從分子遺傳學(xué)角度探究了二VA英的降解機(jī)理,為開發(fā)利用及改造微生物有效去除二VA英等污染物提供了理論依據(jù),為環(huán)境中二VA英等污染物的生物修復(fù)打下基礎(chǔ)。
[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 偽 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，
本文編號(hào)：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 偽 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，
本文編號(hào)：2149272
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