摘要
本论文的研究工作涉及环境微生物学的两个不同领域,第一部分是长江淡水微生物宏基因组文库的构建以及新型脂肪酶的筛选;第二部分是稻田淡水中新型甲烷氧化菌株的分离。
1、长江淡水文库中新型脂肪酶的筛选
尽管微生物非肉眼可见,但其是地球生物群落的一个重要的组成部分。在自然环境下,微生物进行独特的却不可缺少的生物转化,产生对地球生命活动非常重要的生物物质,并展现出了地球生命的多样性。其是地球上已知种类最多、数量最大、分布最广的生物类群,据估计仅原核生物数量达4×10~(30)-6×10~(30)。
微生物可以产生两种不同的水解脂肪的酶,分别为酯酶(esterase)(EC 3.1.1.1)和“真正的”脂肪酶(lipase)(EC 3.1.1.3)。两者都可以将甘油三酯分解成脂肪酸和甘油以及进行其逆反应。微生物脂肪酶资源丰富,具有在有机溶剂中稳定性好、广泛的底物特异性、高度的位置选择性和异构体选择性、催化活性高而副反应少等特点,因此被广泛应用于食品加工、新型生物材料、生物传感器等领域。
但是由于目前环境中有99%的微生物都是不可(难)培养的,只有不到1%的微生物能够被人类认识并利用,所以近年来发展起来的宏基因组学就是利用分子生物学技术绕过培养分离方法来对微生物的多样性及其功能进行研究。目前已经通过此技术筛选到大量新的功能基因,例如编码脂肪酶、蛋白酶、腈水解酶、氧化还原酶和淀粉酶等活性物质的基因。长江作为我国主要的淡水资源,蕴含着极其丰富的微生物资源,必然包含着极其多样的活性物质。
本工作是结合了原位裂解微生物获取大片段DNA的方法对安庆长江水段(30°30'11.30 N,117°04'07.70 E)中的微生物资源进行挖掘,构建了宏基因组文库并对该环境中的脂肪酶基因资源进行筛选,得到了一种新型的脂肪酶。EstY含有423个氨基酸,其分子量为44kDa,等电点为7.28。EstY能水解对硝基苯乙酸(C2),对硝基苯丁酸(C4),对硝基苯辛酸(C8),对硝基苯癸酸(C10),对硝基苯月桂酸(C12),对硝基苯豆蔻酸(C14)和对硝基苯棕榈酸(C16)多种对硝基苯脂。其酶活的最适pH为9.0,最适温度为50℃。Mn~(2+)、Co~(2+)、Hg~(2+)、Zn~(2+)、Fe~(3+)强烈的抑制EstY的活性,但EstY却对Mg~(2+)表现出一定的依赖性。此外,EstY在10%浓度的乙醇,丙酮,异丙醇和二甲基亚砜的溶剂里不丧失活性。根据序列保守性和进化树分析,可以初步推断EstY和其相关的七个脂肪酶不属于任何一个已经的细菌脂肪酶家族。
2、稻田淡水中新型甲烷氧化菌株的分离
近年来,温室效应导致的环境问题近年来愈演愈烈,全球气候变暖的趋势日益明显,一些突出的环境问题如病虫害增加、海平面上升、气候冷暖反常、海洋风暴增多、土地干旱、沙漠化等无不与之密切相关。温室气体增多是造成温室效应的主要原因。通常所知,二氧化碳是主要的温室气体,对温室效应的贡献占到了60%以上。不容忽视的是,甲烷同样也是一种重要的温室气体,但以单位分子数而言,甲烷的温室效应要比二氧化碳大上25倍。从这一点上说,降低大气中甲烷的含量比降低二氧化碳对温室效应的防治更为有效。
甲烷氧化细菌是甲基氧化细菌的一个分支,其独特之处在于其能利用甲烷作为唯一的碳源和能源。几乎所有的甲烷氧化菌都是专性好氧甲烷氧化菌,甲烷氧化菌氧化甲烷最终生成二氧化碳,并在此过程中获得生长所需的能量。甲烷氧化菌的典型特征是含有甲烷单加氧酶,可催化甲烷氧化为甲醇,甲醇进一步氧化为甲醛,甲醛再同化为细胞生物量或通过甲酸氧化为二氧化碳重新回到大气的碳库中,即甲烷→甲醇→甲醛→甲酸盐→二氧化碳。甲烷氧化菌于1906年被首次分离,1970年Whittenbury等分离和鉴定了100多种能利用甲烷的细菌,奠定了现代甲烷氧化菌分类的基础。
本课题从合肥郊区(31°52'N,117°17'W)水稻田中分离了一株革兰氏阳性,短杆状,不运动,没有孢子形成的甲烷氧化菌。此菌为专性好氧菌,生长最适温度为30℃,最适pH为pH7.0-8.0,只能在以甲烷为碳源的培养基中生长。根据16SrRNA和pmoA序列比对,发现此菌属于typeⅡ(α-proteobacteria)甲烷氧化菌的其和一些甲基孢囊菌属(Methylocystis)的未鉴定的菌株有非常高的相似度,但不属于已发现的五个甲基孢囊菌属的任何一个种。因此,我们初步推断此菌为甲基孢囊菌属的一个新种(=CGMCC 1.5062=ATCC BAA-1775)。
My work mainly covers two different areas. One area is construction of metagenomic library from Yangtz River and screening for some novel function genes from the library; a sond area is the isolation of a new type of methane oxidizing bacterium from rice paddy.
1. Construction of metageomic library and screening for a novel esterase gene from the library
This work focus on construction of a bacterial artificial chromosome (BAC) library derived from Yangtze River in China(30°30'11.30 N, 117°04'07.70 E), screening for lipolytic activity, identifying an esterase-coding gene, expressed it in Escherichia coli and characterized the esterase.
Although invisible to the naked eye, prokaryotes are an essential component of the earth's biota. They catalyze unique and indispensable transformations in the biogeochemical cycles of the biosphere, produce important components of the earth's atmosphere, and represent a large portion of life's genetic diversity. The number of prokaryotes on earth is estimated to be 4-6×10~(30) cells.
Bacteria produce different classes of lipolytic enzyme, including carboxylesterases (EC 3.1.1.1), which hydrolyse small ester-containing molecules at least partly soluble in water; true lipases (EC 3.1.1.3), which display maximal activity towards water-insoluble long-chain triglycerides. Carboxylesterases are of importance in various biotechnological applications on account of their useful features, such as remarkable stability in organic solvents, broad substrate specificity, stereoselectivity, regioselectivity, and no requirement for cofactors.
Screening novel biocatalysts from isolated microorganisms using traditional cultivation techniques has limits in exploring the vast genetic diversity of environmental microorganisms because more than 99% of microbes present in various environments cannot be cultured. To access the genome resource of uncultured microorganisms, a recent approach is to screen directly novel biocatalysts from a "metagenomic library". Accordingly, the metagenome-based strategy has led to isolation of many novel biocatalysts, such as esterase, lipase, protease, oxidoreductase, nitrilase and amylase. Yangtz River has been shown to possess unique microbial diversity including members of various unculturable groups as the main fresh water resource in our country.
In this study, we characterize EstY from the metagenome library of Yangtz River. EstY had 423 amino acids with an estimated molecular mass of 44 kDa and pI of 7.28. It can hydrolyze various p-nitrophenyl esters (acetate, butyrate, caprate, caprylate, laurate, myristate and palmitate) and its best substrate was the p-nitrophenyl caprate (C8). The optimum pH for EstY activity was 9.0 and the optimum temperature was 50℃. Mn~(2+), Co~(2+), Hg~(2+), Zn~(2+), Fe~(3+) strongly inhibited the activity of EstY while Mg~(2+) was necessary for it. The activity remained in the presence of 10% alcohol, acetone, isopropanol and dimethyl sulfoxide (DMSO) separately. BLAST search on NCBI revealed that EstY had 7 closely related lipolytic enzymes. Sequence analysis showed that EstY, together with its 7 relatives, did not belong to any known lipolytic enzymefamily.
2. Isolation of a new type methane oxidizing bacterium from rice paddy
The earth's climate has changed dramatically over the last century and there is new and stronger evidence that most of the global warming observed the in the last 50 years is attributable to human activities, and the release of the greenhouse gas is one major reason. As we all know, carbon dioxide contributes most to global warming, but at the same time, methane is a kind of organic gas which can not be neglected. Methane is estimated to contribute about 26 times than that of carbon dioxide to climate change. Reduction of methane emissions would be more effective in reducing the global warming than reduction in CO_2 emissions.
Methane oxidizing bacteria (MOB) is a subset of a physiological group of bacteria known as methylotrophs. Methanotrophic bacteria are unique in their ability to utilize methane as a sole carbon and energy source. Almost all of them are obligate methane-oxidizing bacteria. MOB can oxidize methane and turn it to carbon dioxide at last, at the same time they obtain the energy essential for their growth. One typical characteristic of MOB is possessing mathane monooxygenase, which can catalyze the oxidation of methane to methanol.
In this study, a Gram-negative, non-motile, rod-shaped, methane-oxidizing bacterium, strain M261 was isolated from effluents of rice paddy in the suburb of Hefei in China (31°52' N, 117°17' W). Physiological characterization of the strain showed that the strain grew optimally at pH 7.0-8.0 and at moderate temperature (30℃). The strain only grew well with methane as carbon source. Both 16S rRNA and pmoA gene sequence analysis identified it as a novel methanotroph belonging to the typeⅡ(α- proteobacteria) and it was highly related to a number of non-characterized Methylocystis strains. On the basis of the phenotypic, phylogenetic and genotypic analyses, strain M261 primarily represents a novel species (=CGMCC 1.5062=ATCC BAA-1775) as the type strain.
引文
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