双加氧酶活力对细菌降解菲的指示作用

在液体培养基中选用两种石油降解细菌进行菲降解实验,研究了菲降解率和双加氧酶活力的变化。结果表明,菲降解率受其浓度的影响,当菲浓度为100mg·L-1时,其降解率为最高。而菲浓度高于100mg·L-1时,其降解率下降。实验发现在菲浓度为50～250mg·L-1条件下,细菌的双加氧酶活力与其菲的降解率存在较好的相关性,对细菌降解菲具有指示作用,可将双加氧酶活力作为菲降解率变化的评价指标。     

苯胺双加氧酶基因的克隆与序列分析

通过设计苯胺双加氧酶基因特异引物,以苯胺降解菌株ANA5基因组DNA为模板,PCR扩增出目的基因片断。然后利用粘粒pLAFR3作为载体,以E．coliEPI100作为受体,构建了菌株ANA5的基因组粘粒文库。以PCR扩增产物作为探针,通过菌落原位杂交筛选得到两个阳性克隆,经Southern杂交及亚克隆测序分析,初步确认克隆到苯胺双加氧酶基因。同时完成了苯胺双加氧酶基因atdA3A4A5序列的测定,并对其核苷酸及其推导的氨基酸序列进行分析,结果表明克隆到的苯胺双加氧酶基因与GenBank报道的基因有一定的差异,同时体现了该基因在进化上的保守性。     

深海多环芳烃降解菌新鞘氨醇杆菌H25的降解特性及降解基因

[目的]为了从深海环境中筛选新的多环芳烃降解菌,了解其降解基因及降解特性.[方法]以原油作为碳源从印度洋深海海水样品中富集筛选出降解能力较强的多环芳烃降解菌,并根据已报道的相关菌属的多环芳烃起始双加氧酶大亚基序列及侧翼序列设计兼并引物进行扩增.[结果]获得了1株能够高效降解原油、柴油及多种多环芳烃的菌株H25.经16S rDNA序列系统发育分析表明它属于新鞘氨醇杆菌属(Novosphingobium)(96%).并从该菌株中扩增获得2条相似度为91.0%双加氧酶基因片段.2条序列在NCBI上Blastn分析表明均与菌株N.aromaticivorans DSM12444T的降解质粒pNL1上的双加氧酶大亚基具有最高相似度,分别为99.6%和91.0%.根据pNL1上的双加氧酶序列设计引物获得了包含H25双加氧酶大亚基及上下游序列的2个基因片段H25 Ⅰ(2.9kb)和H25Ⅱ(4.5kb).另外,单碳降解实验表明H25对联苯、2-甲基萘、2,6-二甲基萘、菲、二苯并噻吩、二苯并呋喃等均有较好的降解能力.[结论]H25菌株是Novosphingobium属可能的新种.深海细菌在大洋环境多环芳烃污染的自然净化中起到一定作用,并在环境生物修复中有较大的应用前景.     

LB克隆系统的构建及在鞘氨醇单胞菌基因融合表达中的应用

为摆脱限制性酶切位点不足的限制,构建可灵活改变多基因融合方向的表达载体,基于ⅡS型和ⅡT型限制性内切酶LguⅠ和BbvC Ⅰ设计开发了 LB克隆系统.该克隆系统是以广宿主质粒pBBR1MCS-3为初始载体,利用 PCR的方法,在其多克隆位点区插入LB片段(GCTCTTCCTCAGC)构建得到的.LB片段含Lgu Ⅰ和B...     

苯胺双加氧酶基因的克隆与序列分析*

通过设计苯胺双加氧酶基因特异引物,以苯胺降解菌株ANA5基因组DNA为模板,PCR扩增出目的基因片断。然后利用粘粒pLAFR3作为载体,以E.coliEPI100作为受体,构建了菌株ANA5的基因组粘粒文库。以PCR扩增产物作为探针,通过菌落原位杂交筛选得到两个阳性克隆,经Southern杂交及亚克隆测序分析,初步确认克隆到苯胺双加氧酶基因。同时完成了苯胺双加氧酶基因atdA3A4A5序列的测定,并对其核苷酸及其推导的氨基酸序列进行分析,结果表明克隆到的苯胺双加氧酶基因与GenBank报道的     

鞘氨醇单胞菌的研究进展

鞘氨醇单胞菌(Sphingomonas)不仅细胞膜含有比脂多糖更疏水的鞘糖脂,而且具有高效的代谢调控机制和基因调控能力,使其在威兰胶合成、环境修复和促进植物生长等方面具有巨大的应用潜力。目前国内在鞘氨醇单胞菌代谢机制方面的研究尚无新突破。本文主要综述了鞘氨醇单胞菌的系统分类、基因组学、基因调控机制及其应用等方面的研究,从基因层面分析鞘氨醇单胞菌产威兰胶的合成机制,为后续鞘氨醇单胞菌高密度发酵、工业化生产等研究提供理论基础,以便进一步发掘其在生物技术上的应用潜力。     

微生物降解菲的机理研究进展

针对微生物降解菲的机理研究进展,论述了细菌、真菌在好氧、厌氧条件下代谢菲的产物以及推测的降解途径;在此基础上概括了催化反应的酶系以及编码酶系的基因簇。简要介绍了基因探针的应用,并结合本实验室的初步研究,指出了该领域有待深入探讨的问题。     

PAHs降解基因及降解酶研究进展

由于环境中的多环芳烃(PAHs)具有高遗传毒性和\"三致\"性(致癌、致畸和致突变),其生物降解基因和降解功能酶研究备受关注.多环芳烃双加氧酶是近年来研究较多的多环芳烃降解的关键酶系之一,主要由细菌产生,可通过氧化反应使多环芳烃开环生成小分子的中间产物并最终氧化成CO2和水.目前,有关这类酶的理化性质、结构特点、功能等的研究相继开展,本文对PAHs降解基因、降解酶的研究现状与发展趋势进行综述.     

香蕉rbcS基因启动子的克隆及序列分析

以巴西香蕉为材料,根据已经获得的香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基基因的全长cDNA序列设计1对专一引物,通过PCR扩增得到了香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基的基因组全长,序列长811 bp,含有2个内含子。根据其基因组序列设计引物,采用SEFA-PCR方法,以总DNA为模板克隆了香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基基因的启动子序列,长1 681 bp。用PLACE软件分析发现该序列具有启动子的基本元件TATA-box、CAAT-box,包含多个胁迫诱导元件,如光诱导元件、赤霉素、低温诱导元件、昼夜节律调控元件等。该序列的克隆与分析为进一步研究香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基基因的表达调控奠定了基础。     

拟南芥AtPSK3基因的克隆及序列分析

根据拟南芥基因组数据库提供的信息,首次以特异引物经PCR技术克隆到拟南芥硫肽激素α的一个前体基因———AtPSK3,并对其进行了测序。序列分析表明,所获得的AtPSK3基因全长为505bp,含有一个内含子和两个没有3′或5′非转译区的外显子,与数据库提供的序列比较,同源性为100%。     

微生物降解苯甲酸的研究进展

苯甲酸在工业中的广泛应用使其成为环境中的常见污染物,对微生物好氧降解苯甲酸的邻位途径、间位途径、龙胆酸途径和原儿茶酸途径及厌氧降解途径等进行总结,并对苯甲酸降解过程中发挥重要作用的苯甲酸双加氧酶的种类、不同组分及苯甲酸降解基因和调控基因的基因簇进行介绍,同时展望微生物降解污染物的发展方向。     

Identification,cloning, and characterization of a multicomponent biphenyl dioxygenase from <Emphasis Type="Italic">Sphingobium yanoikuyae</Emphasis> B1

Sphingobium yanoikuyae B1 utilizes both polycyclic aromatic hydrocarbons (biphenyl, naphthalene, and phenanthrene) and monocyclic aromatic hydrocarbons (toluene, m- and p-xylene) as its sole source of carbon and energy for growth. The majority of the genes for these intertwined monocyclic and polycyclic aromatic pathways are grouped together on a 39 kb fragment of chromosomal DNA. However, this gene cluster is missing several genes encoding essential enzymatic steps in the aromatic degradation pathway, most notably the genes encoding the oxygenase component of the initial polycyclic aromatic hydrocarbon (PAH) dioxygenase. Transposon mutagenesis of strain B1 yielded a mutant blocked in the initial oxidation of PAHs. The transposon insertion point was sequenced and a partial gene sequence encoding an oxygenase component of a putative PAH dioxygenase identified. A cosmid clone from a genomic library of S. yanoikuyae B1 was identified which contains the complete putative PAH oxygenase gene sequence. Separate clones expressing the genes encoding the electron transport components (ferredoxin and reductase) and the PAH dioxygenase were constructed. Incubation of cells expressing the dioxygenase enzyme system with biphenyl or naphthalene resulted in production of the corresponding cis-dihydrodiol confirming PAH dioxygenase activity. This demonstrates that a single multicomponent dioxygenase enzyme is involved in the initial oxidation of both biphenyl and naphthalene in S. yanoikuyae B1.     

Recombinant carbazole-degrading strains for enhanced petroleum processing

Biotechnological upgrading of fossil fuels is of increasing interest as remaining stocks of petroleum show increasing levels of contaminants such as heavy metals, sulfur and nitrogen-containing heteroaromatic compounds. Carbazole is of particular interest as a major petroleum component known to reduce refining yields through catalyst poisoning. In this study, the biotransformation of carbazole was successfully demonstrated in a liquid two-phase system, when solubilized in either 1-methylnaphthalene or in diesel fuel. The effects of solvent toxicity were investigated by expressing the carbazole-transformation genes from MB1332, a rifampicin-resistant derivative of Pseudomonas sp. LD2, in a solvent-resistant heterologous host, P. putida Idaho [1]. This solvent-resistant strain successfully degraded carbazole solubilized in 1-methylnaphthalene and in the presence of 10 vol% xylenes similar to the non-recombinant strain Pseudomonas sp. LD2. Identification of a suitable recombinant host, however, was essential for further investigations of partial pathway transformations. Recombinant P. putida Idaho expressing only the initial dioxygenase enzymes transformed carbazole to an intermediate well retained in the oil phase. Partial carbazole transformation converts carbazole to non-aromatic species; their effect is unknown on refinery catalyst poisoning, but would allow almost complete retention of carbon content and fuel value. Electronic Publication     

多环芳烃类化合物在土壤上的吸附

研究了几种多环芳烃化合物在土壤上的吸附行为．通过一个连续投药－取样试验装置,在没有任何其它有机试剂干扰的情况下,测定了荧蒽与菲在土壤上的吸附量．研究表明,这两种多环芳烃化合物在土壤上的吸附量与土壤有机质含量之间呈显著相关．对多环芳烃化合物的分子结构及理化特性,如辛醇－水分配系数、溶解度等参数与ＬｏｇＫｏｃ关系的研究发现多环芳烃化合物的ＬｏｇＫｏｃ与化合物的水溶性、辛酸－水分配系数以及分子结构中的苯环数线性相关．     

Metabolic characterisation of a novel vanillylmandelate-degrading bacterium

A newly isolated gram-negative bacterium, possibly Brevundimonas diminuta, utilised d,l-vanillylmandelate (d,l-VMA) as a sole carbon and energy source. The organism converted d,l-VMA to vanillylglyoxylate using a soluble NAD-dependent dehydrogenase specific for d-VMA and a dye-linked, membrane-associated l-VMA dehydrogenase. Vanillylglyoxylate was further metabolised by decarboxylation, dehydrogenation and demethylation to protocatechuate. A 4,5-dioxygenase cleaved protocatechuate to 2-hydroxy-4-carboxymuconic semialdehyde. Partially purified d-VMA dehydrogenase exhibited optimal activity at 30° C and pH 9.5 and had an apparent K _m for d-VMA of 470 μM. Although induced by several substituted mandelates, the enzyme had a narrow substrate specificity range with virtually no activity towards d-mandelate. Such properties render the enzyme of potential use in both diagnostic and biosynthetic applications. Received: 23 January 1996 / Accepted: 9 April 1996     

化石燃料生物脱有机氮研究展望

化石燃料中与有机硫相似的另一类孤对电子含氮有机化合物的存在对生产和环境造成许多危害。石油中的含氮有机化合物是影响炼油工艺、产品性能质量的主要因素。含氮有机化合物具有致癌、致突变性 ,燃烧后则以NOx的形式释放污染大气。化石燃料中所含的有机氮较有机硫更难以去除 ,常规的化学脱有机氮技术高压加氢法处理燃油能耗高 ,处理效果不理想等方面的缺陷使人们思考生物脱氮的可能性。考察了国内外近十多年来化石燃料生物脱有机氮工作的研究进展 ,包括模式有机氮化合物微生物的代谢途径 ,以及相应的代谢途径中的关键酶及其编码基因等方面的研究。