【作者】 张燕新；

【导师】 沈标；

【作者基本信息】 南京农业大学 ， 微生物学， 2006， 硕士

【摘要】 嗜热菌是指能生活在50℃以上高温，最适生长温度在45～80℃以上的一类极端微生物。自从70年代Brock发现嗜热菌以来，日本、意大利、冰岛、美国等国家大力开发嗜热菌资源。我国各地分布着数量众多、生态环境各异的温泉及其他高温环境，具有丰富的嗜热菌资源。但从目前的相关报道看来，我国对于嗜热菌资源的研究开发还不是很多。本实验从云南腾冲的温泉淤泥和水样样品中分离到了18株嗜热菌，经过形态观察、生理生化特性和16S rDNA序列分析，除菌株LA5为放线菌外，其余都是Bacillus属或Geobacillus属的菌株。这些嗜热菌生长的最适温度为60～70℃。其中三株高温蛋白酶产生菌SB11、SB31和SC5，经初步鉴定都属于Geobacillus属，产高温蛋白酶的酶活力分别为35.6、26.1、26.6U／mL。虽然三菌株的最适生长温度为60℃，但其产生的蛋白酶的最适酶反应温度都在70℃以上，远高于一般动植物来源的蛋白酶。菌株LA5是一株高温放线菌。菌落乳黄色，呈放射状皱纹，形成黄色或白色的基质菌丝和孢子，孢子层较厚，没有气生菌丝，不产生可溶性色素。在LB、PDA、蛋白胨酪素培养基上生长良好，在甘油天冬酰胺、高氏一号、察氏、牛肉膏蛋白胨培养基上生长较差，在ISP4、淀粉酪素、甘油淀粉培养基上不生长。在光学、相差、电子显微镜下观察，发现基内菌丝上均有单个孢子，生长在不分支的孢子梗上。高温放线菌LA5可以发酵葡萄糖、果糖、木糖、纤维二糖，不能发酵阿拉伯糖、棉子糖、鼠李糖；V．P试验、M．R试验、苯丙氨酸脱氢酶试验、柠檬酸盐试验反应阳性，吲哚试验反应阴性；液化明胶，降解淀粉、酪素，不降解纤维素、果胶；能利用有机氮（酵母膏、牛肉膏、蛋白胨、尿素）和无机氮（硝酸铵、硫酸铵、氯化铵），利用乳糖、半乳糖最好，其他糖类（葡萄糖、蔗糖、果糖、木糖、麦芽糖）利用效果一般或较差；好氧；pH生长范围是6～10，最适pH是8.5；生长温度为50～75℃，最适生长温度为60～70℃；没有抗微生物活性物质的产生。高温放线菌LA5的化学组成为：细胞壁含有meso-DAP（Ⅲ型），但无特征性糖；主要的脂肪酸（≥10％）为iso-C_17∶0（27.66％）和C_16∶0（22.38％）；醌的组成成分为MK-7（66.58％）和MK-8（H₂）（33.42％）。16S rDNA序列分析表明，菌株LA5属于Planifilum属；DNA G+C mol％为56.8mol％；菌株LA5与Planifilum属仅有的两个种Planifilum fulgidum 500275和Planifilum fimeticola HO165进行DNA-DNA杂交，同源性分别为43.6％、52.7％。综合高温放线菌LA5的形态学特征、生理生化特征、化学组成分析和基因型特性，确认高温放线菌LA5是属于Planifilum的一个新种，命名为Planifilum yunnanesis sp．nov．。更多还原

【Abstract】 The extremophiles have unique genes, phisiological characteristics and metabolites compared with common microorganisms. Extremophiles are of interest to both basic and applied biology. Many extremophiles, in particular the hyperthermophiles, lie close to the "universal ancestor" of all extant life on Earth. This exciting realization has fueled much research on these organisms in order to understand the nature of primitive life forms, how the first cells "made a living" in Earth’s early days, and how early organisms set the stage for the evolution of modem life forms. Many thermophiles have been isolated around the world. In China, there are many different hot springs in which thermophiles usually live. But less thermophiles have been found.18 thermophilic strains were reported here isolated from hotsprings in Yunnan. Based on the their configurations, physiological and biochemistry features, 16S rDNA sequence,most of them belong to the genus Geobacillus and Bacillus except the strain LA5. The optimum growth temperature of 18 strains were 60-75℃. Three protease-producing strains(beloned to Geobacillus)SBll, SB31 and SC5 were studied in more detail. The protease activities were 35.6, 26.1, 26.6 U/mL respectively. Although they grew best at 60℃, their protease activities were highest at 70℃, higher than those from animals and plants.Strain LA5 was a thermoactinomyces.Its colony was cream-yellow with radial winkles. The isolate grew aerobically at temperature of 50～70℃and at pH 6～10. The aerobic mycelia wasn’t be observed. Single spores were produced along the substrate hypha. The strain LA5 utilized glucose, fructose, xylose, giutin, Casin and starch but not arabinose, raffinose, rhamnose, cellobiose, Hypoxanthine, Xanthine and L-Tyrosine. The strain LA5 could grow better on the following media: LB, PDA, Czapek and Starch-Casein,but weak on Glycerol-Asparagine and Gause’s Synthetic meadium.Chemotaxonomical analysis revealed the presence of meso-diaminopimelic acid, alaline and glutamic acid, indicating that the cell call chemotype belonged to the typeⅢ. Whole-cell sugars were found to be glucose, ribose, without any diagnostic sugars. The major fatty acids were iso-C17:0 (27.66%) and C16:0 (22.38%) and the predominant menaquinone were MK-7( 66.58 % )和MK-8( H2 )( 33.42 % )which were quitely different from those type strains of five genera in the family’Thermoactinomycetaceae’.DNA G+C% of strain LA5 was 56.8 mol%, close to those of Planifilum fulgidum 500275T (60.0 mol%) and Planifilum fimeticola H0165T(60.3 mol%) but much higher than those of others in the family ’Thermoactinomycetaceae’.DNA Hybridization outcome demonstrated the presence of 52.7%, 43.6% homogeneity between LA5 and Planifilum fimeticola H0165T and Planifilum fulgidum 500275T respectively.On the basis of phenotypic properties and phylogeny, strain LA5 should be placed in the genus Planifilum as a member of a novel species for which we propose the name Planifilum yunnanensis sp. nov.更多还原