Isolation And Characterization Of A Novel Esterase And Methane-oxidizing Bacterium From Freshwater

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 libraryThis 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 4-6×10³⁰ 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℃. Mn²⁺, Co²⁺, Hg²⁺, Zn²⁺, Fe³⁺ strongly inhibited the activity of EstY while Mg²⁺ 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 paddyThe 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 CO₂ 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 typeâ…¡(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.