【作者】 刘怡辰；

【导师】 沈标；

【作者基本信息】 南京农业大学 ， 微生物学， 2010， 博士

【摘要】 从我国东海近海污泥中分离筛选出一株高效降解烷烃的细菌,经形态、生理生化特性及其16S rDNA序列分析,将此菌株初步鉴定为食碱菌属(Alcanivorax sp.),命名为2B5。菌株2B5为革兰氏阴性,球形,能产生物表面活性剂,不能利用葡萄糖、蔗糖等碳源,以酵母粉为唯一氮源时生长较好。菌株2B5能够在96h内完全降解500 mg/L正十八烷,降解正十八烷的最适温度是30-37℃,最适pH值为6.0-7.0,最适NaCl浓度为2%-9%。菌株2B5能够降解原油中C13-C30的正构烷烃及姥鲛烷、植烷等支链烷烃,在人工海水中降解原油中烷烃的最适温度是30℃,最适接种量为1%,通气量越大越好,补加2 g/L NH4NO3和0.25 g/L酵母粉可以明显地提高原油中烷烃的降解率。由于其较高的烷烃降解率和较广的烷烃降解谱,菌株2B5可以应用于海洋石油污染的生物修复。从山东东营胜利油田油井下的油水混合物中分离筛选出一株嗜热烷烃降解菌株,经形态、生理生化特性及其16S rDNA序列分析,将其初步鉴定为土芽孢杆菌属(Geobacillus sp.),命名为MH-1。菌株MH-1为革兰氏阳性,长杆状,能产芽孢。菌株MH-1在培养96h后对正十六烷的降解率达92%以上,降解的最适温度是70℃,最适pH值为6.0,能够在6天内降解原油中的大部分中链烷烃及部分长链烷烃。首先从菌株2B5中克隆出alkB基因的保守片段,在此基础上通过SEFA-PCR方法成功克隆出alkB全长基因及其上、下游序列。氨基酸序列分析表明,该基因与具有完整ORF的Marinobacter sp. ELB17的putitave AlkB具有最高的同源性(65%),与经过功能验证的Alcanivorax borkumensis SK2的A1kB1的同源性为41.9%,存在四个含His的保守基序,分别为Hist-1、Hist-2、Hist-3和HYG-motif。进一步将该基因在P. fluorescens KOB2△1进行异源表达,恢复了P. fluorescens KOB2△1降解烷烃的功能,表明该基因就是烷烃羟化酶基因(GenBank登录号为No. FJ905614),能够氧化C14和C16烷。通过RT-PCR方法证明该alkB基因能够在指数生长期被正十八烷诱导表达。在alkB基因的上、下游序列中分别存在一个与AraC家族转录调控蛋白有关序列的同源性达58%的ORF和一个与Shewanella sp. W3-18-1菌株的siderophore-interacting protein的氨基酸序列的同源性达45%的ORF。从菌株2B5中成功克隆出细胞色素P450基因及其基因簇,该基因簇包含5个完整的ORF,它们分别与Limnobacter sp. MED 105菌株的transcriptional regulator和Alcanivorax dieselolei B-5T菌株的putative ferrodoxin、cytochrome P450 alkanehydroxylase、putative alcohol dehydrogenas、putative FAD-dependent oxidoreductase family protein的氨基酸序列的同源性达73%、98%、99%、99%和99%,将此5个ORF分别命名为transcriptional regulator, fdx、P450、alkJ和fdr。通过在E. coli BL21 (DE3)中构建多顺反子结构fdx、P450和fdr基因的共表达系统,检测P450基因的功能活性,但没有显示出活性。研究了Geobacillus sp. MH-1菌株中的alkB基因及其基因簇。采用普通PCR方法从Geobacillus sp. MH-1中获得alkB基因的同源物, alkB-geol、alkB-geo4和alkB-geo6,它们分别与Rhodococcus sp. Q15中alkB4、alkB3和alkB2基因高度同源。进一步采用SEFA-PCR的方法,获得alkB-geo6基因簇的完整序列(GenBank登录号为No. FJ977899),该基因簇包含putative transporter、alkB-geo6、rubA3、rubA4和putative regulatory protein。核苷酸和氨基酸序列分析表明,alkB-geo6基因簇的核苷酸序列与Rhodococcus sp. Q15和Rhodococcus sp. NRRL-16531的alkB2的基因簇的序列同源性高达98%。在菌株MH-1的AlkB-geo6中同样发现了四个含His的保守基序,分别为Hist-1、Hist-2、Hist-3和HYG-motif。菌株MH-1的AlkB-geo6和Rhodococcus sp. Q15及Rhodococcus sp. NRRL-16531的AlkB2氨基酸序列高度同源,只有4个氨基酸位点发生了变化。更多还原

【Abstract】 A bacterium strain,2B5, was isolated from the sea mud of the crude oil-polluted Donghai area in China, which was capable of utilizing alkane as the sole carbon source for its growth. This bacterium was identified preliminarily as Alcanivorax sp. based on its physiological characteristics and analysis of its 16S rDNA sequence. The strain 2B5 was a round-shaped, gram-negative bacterium which produced biosurfactant. The strain 2B5 degraded 500 mg/L octadecane in 96h. Surgars could not be metabolized by the strain 2B5. The optimal temperature and pH for strain 2B5 growth and octadecane degradation were 30～37℃and pH 6.0～7.0, respectively. NaCl is required for strain 2B5 growth and octadecane degradation with the optimal concentrations of 20 to 90 g/L. Strain 2B5 was able to degrade C13～C3o n-alkanes and branched alkanes (pristane and phytane) in crude oil as the sole carbon source. The optimal temperature and inoculum for strain 2B5 alkanes in crude oil degradation were 30℃and 1%, respectively. The aeration has little effect on degrading ability of the strain 2B5. NH4NO3 (2 g/L) and yeast extract (0.25 g/L) could improve degrading ability largely. Based on these results, it is obvious that the strain 2B5 could potentially be useful for the remediation of hydrocarbon pollution in the marine environment.An extremely thermophilic alkane-degrading bacterium, strain MH-1 was isolated from the deep subterranean petroleum reservoir in Shengli oil field in China. The strain MH-1 was rod-shaped gram-positive strain. Based on its physiological characteristics and analysis of its 16S rRNA gene sequence, the strain MH-1 was identified as Geobacillus sp. The strain MH-1 was able to grow at temperatures ranging from 50 to 72℃and effectively degraded hexadecane as the sole carbon source at 70℃. The strain MH-1 degraded alkanes with different length chain (C12～C31) in crude oil, but it preferentially degraded middle chain-alkanes.A novel alkane hydroxylase gene (GenBank Accession No. FJ905614), obtained by self-formed adaptor PCR (SEFA-PCR), showed 41.9% deduced amino acid sequence identity with AlkB1 of Alcanivorax borkumensis SK2. Functional heterologous expression of the alkB gene was achieved in Pseudomona fluorescens KOB2△1. Recombinant Pseudomonas containing the alkB gene of Alcanivorax sp.2B5 recovered the ability to grow on C14 and C16 n-alkanes. The three conserved histidine motifs and a fourth motif found in the alkane hydroxylases were identified in the AlkB of the strain 2B5. RT-PCR analysis showed that expression of the alkB gene was induced by octadecane. The immediately upstream sequence of the alkB gene was found to encode a peptide with 58% sequence similarity with a putative AraC family transcriptional regulator protein. In the downstream region of the alkB gene, an open reading fame was 45% similar to the deduced amino acid sequence of the siderophore-interacting protein in Shewanella sp. W3-18-1.Cytochrome P450 gene and its cluster were also cloned by the same method. The cluster included transcriptional regulator, fdx, P450, alkJ and fdr which had a 73%,98%, 99%,99% and 99% sequence similarity respectively with transcriptional regulator of Limnobacter sp. MED 105 and putative ferrodoxin cytochrome (Fdx), P450 alkane hydroxylase, putative alcohol dehydrogenas, putative FAD-dependent oxidoreductase family protein (Fdr) of Alcanivorax dieselolei B-5T. The fdx, P450 and fdr genes were ligated into expression vector pET29a, then transformed into E.coli BL21 (DE3) which could not degrade alkane s.Alkane hydroxylase system of Geobacillus sp. MH-1 was characterized by self-formed adaptor PCR (SEFA-PCR). It contained at least three alkane monooxygenase gene homologs such as alkB-geol, alkB-geo4 and alkB-geo6, which had a high sequence similarity with alkB4, alkB3 and alkB2 in Rhodococcus sp. Q15, respectively. AlkB-geo6 cluster (GenBank Accession No. FJ977899) included putative transporter, alkB-geo6, rubA3, rubA4 and putative regulatory protein, which had a high sequence similarity (98%) with the alkB2 cluster of Rhodococcus sp. NRRL B-16531 and Q15. The conserved Hist-1, Hist-2, Hist-3 and HYG-motif were also found in the AlkB-geo6 of strain MH-1. AlkB-geo6 in strain MH-1 had a very high amino acid sequence similarity with AlkB2 of Rhodococcus sp. Q15 and Rhodococcus sp. NRRL-16531. There was only four amino acids variation among them.更多还原