红灯食烷菌(Alcanivorax hongdengensis)黄素结合单加氧酶(AlmA)的基因克隆及其烷烃诱导表达

【目的】研究海洋烷烃降解菌新种模式菌株Alcanivorax hongdengensis A-11-3降解长链烷烃的分子机制。【方法】PCR克隆编码黄素结合单加氧酶的基因序列,利用生物信息学软件对序列进行分析,运用RT-PCR和实时荧光定量PCR技术分析基因在不同烷烃诱导下的表达水平。【结果】从菌株A-11-3中克隆获得了两个黄素结合单加氧酶基因片段(almA1和almA2)。它们编码的氨基酸序列与菌株Acinetobacter sp.DSM17874的AlmA同源性分别为58.6%和53.2%。实时荧光定量PCR分析表明,almA1基因只在长链烷烃(C28-C32)的诱导下上调表达,而almA2基因中能在更宽范围的长链烷烃(C24-C34)和支链烷烃诱导下上调表达。两者均在C9-C22的烷烃诱导下没有上调表达。【结论】黄素结合单加氧酶可能是A-11-3降解长链烷烃和支链烷烃的关键酶。     

一株耐碱高效长链烷烃降解菌C24MT1的筛选及其降解特性

【背景】石油被称为“液体黄金”,人类的工业生产活动在利用其创造巨大社会价值的同时,也对自然环境造成了严重的污染。微生物修复技术是现阶段治理石油类污染有效的手段之一,具有经济、高效、无二次污染等优点。【目的】从受石油污染的土壤中分离高效降解长链烷烃正二十四烷的菌株,探究其降解特性及在微生物修复中的应用前景。【方法】通过形态学及16S rRNA基因测序进行菌株鉴定,采用气相色谱法检测菌株对正二十四烷的降解效果,并结合气相色谱-质谱(gas chromatography-mass spectrometer, GC-MS)分析降解中间产物以推测其潜在代谢途径。【结果】筛选到一株可高效降解正二十四烷的菌株C24MT1,经鉴定为不动杆菌属(Acinetobacter)。该菌株最适降解条件为30 °C、pH 9.0、盐度2 g/L,该条件下生长7 d对9 g/L正二十四烷的降解率高达86.63%;与此同时,菌株在强碱性环境(pH 11.0)中生长良好(OD₆₀₀为0.39)并保持较高烷烃降解率(75.38%),对极端环境具备较强的耐受能力;对降解中间产物进行分析,推断菌株代谢长链烷烃正二十四烷的途径可能包括末端氧化及次末端氧化。【结论】不动杆菌C24MT1具有良好的环境适应能力及烷烃降解能力,在后续微生物菌剂开发和石油类污染土壤的环境修复领域具有巨大的应用前景。本研究可为盐碱地区高浓度石油类污染土壤的修复提供优良菌种,并进一步丰富石油烃类生物降解的菌种资源库。     

柴油降解基因工程菌的构建及降解特性

【目的】构建柴油降解基因工程菌,提高柴油降解速率,研究p450基因在柴油降解过程中的作用。【方法】将Alcanivorax borkumensis SK2的p450基因合成后,连接至烷烃响应表达载体p Com8中,构建该基因的表达载体p450-SK2/p Com8,并将其转入大肠杆菌DH5α中,通过SDS-PAGE检测该基因在大肠杆菌DH5α中的表达,并将重组质粒p450-SK2/p Com8转入柴油降解菌Acinetobacter sp.Y9中,构建基因工程菌p450-SK2/Y9,研究工程菌p450-SK2/Y9对柴油的降解特性及p450基因在构建的工程菌p450-SK2/Y9中的表达。【结果】PCR、酶切及测序结果表明重组质粒p450-SK2/p Com8构建正确。当柴油诱导浓度大于1%时,目的基因在大肠杆菌DH5α中的蛋白表达量较大,且随着诱导时间的延长而呈增加趋势。通过PCR检测构建的基因工程菌p450-SK2/Y9中的p450基因表明,工程菌构建正确,利用单菌株降解柴油时,宿主菌Y9与工程菌p450-SK2/Y9的柴油降解效率未见明显差异,但工程菌p450-SK2/Y9在构建的菌群中对柴油降解的促进效果明显。SDS-PAGE结果表明,p450基因在构建的工程菌p450-SK2/Y9中能得到准确表达,在混合菌中的表达量高于单菌株。【结论】柴油降解基因工程菌在混合菌群中对柴油降解具有促进作用,而在单菌株情况下未见促进作用,且p450基因的蛋白表达在混合菌中也高于单菌株,这对于提高柴油的降解速率及研究p450基因在柴油降解过程中的作用机理具有一定意义。     

高效石油烃降解菌不动杆菌(Acinetobacter sp.BZ-15)的筛选、鉴定及其降解性能研究

本研究旨在从土壤中筛选高效石油烃降解菌株,并对其系统分类和降解特性进行研究,为石油污染的原位修复奠定基础.该研究从滨州油井溢油污染土壤样品中分离得到一株高效石油烃降解菌株BZ-15,对菌株BZ-15进行形态观察、16S r RNA基因序列分析及系统发育树分析;对该菌株的生长特性进行研究;通过GC-MS分析其对原油组分中不同碳原子饱和烃的降解特性;同时研究吐温-20对其生长及降解特性的影响;对该菌株中的烷烃羟化酶基因alk M进行了克隆.结果表明,菌株BZ-15为不动杆菌属(Acinetobacter sp.)细菌,在LB培养基中其代时为3.25 h,添加吐温-20代时为2.67 h,吐温-20可促进菌株BZ-15生长;该菌株可降解C13~C28碳链长度饱和烃,饱和烃降解率为61.0%,添加吐温-20饱和烃降解效率为52.2%,吐温-20可抑制菌株BZ-15降解饱和烃;菌株BZ-15存在烷烃羟化酶基因alk M,通过末端氧化途径对饱和烃进行降解.     

一株菊酯类农药降解菌的分离鉴定及其降解酶基因的克隆

摘要：【目的】筛选分离高效降解菊酯类农药的光合细菌,研究其降解特性,并对该菌株中降解酶基因进行克隆与初步分析。【方法】根据分离菌株的细胞形态结构、活细胞光吸收特征、生理生化特征及其16S rDNA序列系统发育分析鉴定降解菌,气相色谱法测定该菌株降解菊酯类农药的能力,PCR方法克隆降解酶基因。【结果】菌株PSB07-21属红假单胞菌属（Rhodopseudomonas sp.）,其降解最佳条件为3000 lx、35℃、pH 7,在此条件下培养15 d对600 mg/L甲氰菊酯、氯氰菊酯、联苯菊酯降解率分别为     

一株降解纤维素放线菌的产纤维素酶基因克隆与表达

【背景】纤维素在自然界中储量丰富,但天然纤维素的难降解性成为广泛应用纤维素资源的壁垒,近年来利用微生物来降解纤维素成为热点研究。【目的】筛选分离得到一株具有降解纤维素功能的放线菌菌株Lb1,通过全基因组测序确定其产纤维素酶关键基因5676,对基因5676进行克隆转化,使其在大肠杆菌中进行表达。【方法】通过基因工程技术将产纤维素基因连接到表达质粒上并导入表达菌株,对其降解纤维素生成葡萄糖的能力进行探究。【结果】将Lb1菌株的16S rRNA基因进行比对,确定菌株Lb1属于链霉菌属,命名为Streptomyces sp. Lb1。成功构建出纤维素酶表达载体,并且导入表达菌株大肠杆菌BL21(DE3),重组菌株的产纤维素酶能力大于空载菌株。【结论】通过基因工程技术成功克隆出产纤维素酶基因,从而表达纤维素酶,为今后利用微生物降解纤维素的大规模应用提供参考。     

近海柴油降解菌群的构建及其对柴油的降解特性

【目的】实施近海柴油污染的生物治理。【方法】以柴油为唯一碳源,从深圳港口海域富集筛选柴油降解菌;采用复配、正交试验等方式构建混合菌群;通过单因素试验研究环境因素对菌群降解柴油的影响;使用气相色谱-氢火焰检测器(GC-FID)分析降解前后柴油各组分的变化;通过生理生化试验和16S rRNA基因序列分析对菌株进行鉴定。【结果】获得了16株柴油降解菌,7 d内对柴油的降解率最高达40.8%;选择菌株C1-8、C2-10、C3-13构建了混合菌群CQ1,投加量分别为0.5%、2.0%和1.0%,CQ1对柴油去除率比单菌提高了10%以上;CQ1的最适环境条件为：温度30 °C、pH 7.6、摇床转速220 r/min、柴油浓度20 g/L,优化后9 d内对柴油去除率达60%以上;GC-FID结果显示,菌群CQ1可降解大部分C11?C27的正构烷烃,对C21?C27的烷烃降解可达100%。经鉴定,菌株C1-8、C2-10和C3-13分别为微杆菌(Microbacterium sp.)、剑菌(Ensifer sp.)和变异棒杆菌(Corynebacterium variabile)。【结论】CQ1在近海柴油污染的生物修复中具有良好的应用前景。     

石油中长链烷烃微生物降解及分子机制研究进展

中长链烷烃是石油烃中的重要组成部分,由于其疏水性强、黏度大、化学活性低、难降解,是地下原油黏度大、石油采收率低、泄漏后长期污染生态环境的重要原因,因此成为提高石油采收率和石油污染环境治理中的重要降解目标。微生物降解中长链烷烃作为一种新型高效的绿色技术日益受到重视。本文总结了微生物降解中长链烷烃的间期适应与转运过程,与转运过程相关的膜蛋白,微生物好氧与厌氧降解的代谢途径,以及好氧降解过程中的基因调控机制,并对微生物降解中长链烷烃的研究方向提出了展望,以期为后续的相关研究工作提供参考。     

玉米赤霉烯酮降解菌的鉴定及其降解特性

【背景】玉米赤霉烯酮(Zearalenone,ZEN)是一种具有类雌激素作用的霉菌毒素,常会污染谷物和饲料,严重威胁动物和人类的健康。生物脱毒作为理想的去除ZEN的方法,广受关注,然而相关菌株较少,仍有待进一步筛选。【目的】明确一株玉米赤霉烯酮降解菌的生物学分类地位,并优化其赤霉烯酮降解菌降解条件。【方法】通过菌株的16S rRNA基因序列比对,构建系统发育进化树,并开展了相关培养条件的单因素优化和玉米赤霉烯酮降解动力曲线的绘制。【结果】实验菌株WLB-29经鉴定为斯塔普氏菌属(Stappia),其16S rRNA基因序列在GenBank上登录号为MT196321,该序列与模式菌株Stappia indica B106T相似性最高为97.47%,初步确定为斯塔普氏菌属潜在新种。单因素优化表明,菌株降解玉米赤霉烯酮的最佳条件为LB培养基、37℃培养、pH 8.0、2%接种量和玉米赤霉烯酮初始浓度为10mg/L,在此条件下培养144h后,玉米赤霉烯酮的降解率最高可达92.56%。【结论】菌株WLB-29具有较好的ZEN降解作用,为进一步解析菌株降解ZEN作用机理提供了研究基础,也为进一步开发利用菌株开展ZEN的生物脱毒提供了新的菌株资源。     

一株高效烷烃降解菌Acinetobacter sp. LAM1007的分离鉴定及降解特性

【目的】挖掘高效烷烃降解菌,为后续石油烃污染修复工程提供优良菌种资源。【方法】以正十六烷为唯一碳源,将大庆石油污染土样中分离筛选到的高效烷烃降解菌经形态观察、生理生化试验、细胞化学组分及16SrRNA基因序列分析等方法进行初步鉴定与系统分类;同时通过单因素试验研究环境因素(温度、pH、接种量和转速)以及不同初始浓度的正十六烷(0.1%、0.3%、0.5%、1.0%、1.5%、2.0%,体积比)对菌株降解效率的影响。【结果】筛选到一株高效烷烃降解菌LAM1007,经初步鉴定该菌株为不动杆菌属(Acinetobacter)。该菌株在添加正十六烷的无机盐培养基中的最适降解条件为:30°C,pH 7.0,接种量1%(体积比),转速180 r/min,在该条件下浓度为0.3%(体积比)的正十六烷60 h内降解率高达90%。【结论】菌株LAM1007是一株在石油烃污染修复方面极具应用潜力的高效烷烃降解菌。     

Multiple alkane hydroxylase systems in a marine alkane degrader, Alcanivorax dieselolei B-5

Alcanivorax dieselolei strain B-5 is a marine bacterium that can utilize a broad range of n-alkanes (C(5) -C(36) ) as sole carbon source. However, the mechanisms responsible for this trait remain to be established. Here we report on the characterization of four alkane hydroxylases from A. dieselolei, including two homologues of AlkB (AlkB1 and AlkB2), a CYP153 homologue (P450), as well as an AlmA-like (AlmA) alkane hydroxylase. Heterologous expression of alkB1, alkB2, p450 and almA in Pseudomonas putida GPo12 (pGEc47ΔB) or P. fluorescens KOB2Δ1 verified their functions in alkane oxidation. Quantitative real-time RT-PCR analysis showed that these genes could be induced by alkanes ranging from C(8) to C(36) . Notably, the expression of the p450 and almA genes was only upregulated in the presence of medium-chain (C(8) -C(16) ) or long-chain (C(22) -C(36) ) n-alkanes, respectively; while alkB1 and alkB2 responded to both medium- and long-chain n-alkanes (C(12) -C(26) ). Moreover, branched alkanes (pristane and phytane) significantly elevated alkB1 and almA expression levels. Our findings demonstrate that the multiple alkane hydroxylase systems ensure the utilization of substrates of a broad chain length range.     

Identification of novel genes involved in long-chain n-alkane degradation by Acinetobacter sp. strain DSM 17874

Acinetobacter sp. strain DSM 17874 is capable of utilizing n-alkanes with chain lengths ranging from that of decane (C10H22) to that of tetracontane (C40H82) as a sole carbon source. Two genes encoding AlkB-type alkane hydroxylase homologues, designated alkMa and alkMb, have been shown to be involved in the degradation of n-alkanes with chain lengths of from 10 to 20 C atoms in this strain. Here, we describe a novel high-throughput screening method and the screening of a transposon mutant library to identify genes involved in the degradation of n-alkanes with C chain lengths longer than 20, which are solid at 30 degrees C, the optimal growth temperature for Acinetobacter sp. strain DSM 17874. A library consisting of approximately 6,800 Acinetobacter sp. strain DSM 17874 transposon mutants was constructed and screened for mutants unable to grow on dotriacontane (C32H66) while simultaneously showing wild-type growth characteristics on shorter-chain n-alkanes. For 23 such mutants isolated, the genes inactivated by transposon insertion were identified. Targeted inactivation and complementation studies of one of these genes, designated almA and encoding a putative flavin-binding monooxygenase, confirmed its involvement in the strain's metabolism of long-chain n-alkanes. To our knowledge, almA represents the first cloned gene shown to be involved in the bacterial degradation of long-chain n-alkanes of 32 C's and longer. Genes encoding AlmA homologues were also identified in other long-chain n-alkane-degrading Acinetobacter strains.     

Functional analysis of long-chain n-alkane degradation by Dietzia spp

The genetic background of long-chain n-alkane degradation was investigated in detail in strain E1, a member of the genetically unexplored Dietzia genus. A suicide vector carrying a 518-bp alkB fragment was site-specifically integrated into the E1 chromosome, and the full alkB, as well as its chromosomal environment was sequenced after plasmid rescue experiments. Four out of the nine putative genes were strongly induced by long-chain n-alkanes in wild-type E1. ORF4 encoded a natural fusion protein consisting of an integral membrane alkane hydroxylase and a rubredoxin domain. The significance of the alkB-rub gene in n-alkane degradation was investigated in phenotypic tests, and the disruption mutant strain exhibited severely impaired growth on n-C(20) alkane carbon source. The mutation was successfully complemented with the expression of intact AlkB-Rub protein, the full-length form of which was detected by simultaneous immunoblotting. The presented data furnish the first experimental evidence of the in vivo existence of an AlkB-Rub natural fusion protein, which plays a major role in long-chain n-alkane degradation.     

Assessing the role of alkane hydroxylase genotypes in environmental samples by competitive PCR

AIMS: A molecular tool for extensive detection of prokaryotic alkane hydroxylase genes (alkB) was developed. AlkB genotypes involved in the degradation of short-chain alkanes were quantified in environmental samples in order to assess their occurrence and ecological importance. METHODS AND RESULTS: Four primer pairs specific for distinct clusters of alkane hydroxylase genes were designed, allowing amplification of alkB-related genes from all tested alkane-degrading strains and from six of seven microcosms. For the primer pair detecting alkB genes related to the Pseudomonas putida GPo1 alkB gene and the one targeting alkB genes of Gram-positive strains, both involved in short-chain alkane degradation (相似文献   

Diversity of flavin-binding monooxygenase genes (almA) in marine bacteria capable of degradation long-chain alkanes

Many bacteria have been reported as degraders of long-chain (LC) n-alkanes, but the mechanism is poorly understood. Flavin-binding monooxygenase (AlmA) was recently found to be involved in LC-alkane degradation in bacteria of the Acinetobacter and Alcanivorax genera. However, the diversity of this gene and the role it plays in other bacteria remains unclear. In this study, we surveyed the diversity of almA in marine bacteria and in bacteria found in oil-enrichment communities. To identify the presence of this gene, a pair of degenerate PCR primers were was designed based on conserved motifs of the almA gene sequences in public databases. Using this approach, we identified diverse almA genes in the hydrocarbon-degrading bacteria and in bacterial communities from the surface seawater of the Xiamen coastal area, the South China Sea, the Indian Ocean, and the Atlantic Ocean. As a result, almA was positively detected in 35 isolates belonging to four genera, and a total of 39 different almA sequences were obtained. Five isolates were confirmed to harbor two to three almA genes. From the Xiamen coastal area and the Atlantic Ocean oil-enrichment communities, a total of 60 different almA sequences were obtained. These sequences mainly formed two clusters in the phylogenetic tree, named Class I and Class II, and these shared 45-56% identity at the amino acid level. Class I contained 11 sequences from bacteria represented by the Salinisphaera and Parvibaculum genera. Class II was larger and more diverse, and it was composed of 88 sequences from Proteobacteria, Gram-negative bacteria, and the enriched bacterial communities. These communities were represented by the Alcanivorax and Marinobacter genera, which are the two most popular genera hosting the almA gene. AlmA was also detected across a wide geographical range, as determined by the origin of the bacterial host. Our results demonstrate the diversity of almA and confirm its high rate of occurrence in hydrocarbon-degrading bacteria, indicating that this gene plays an important role in the degradation of LC alkanes in marine environments.