【作者】 王全富；

【导师】 李光友； 缪锦来；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2006， 博士

【摘要】 南极具有独特的地理及气候特征,其主要特点是变化极大的光照辐射、季节性的光照时间;常年极低的水温(通常在–1.8℃~2.0℃),在冬季最低温度可以达到–50℃;高盐度环境(海水中的盐度一般为34‰~35‰),海冰中的盐囊和盐通道的盐度可达到150‰,因此形成了一个酷寒、强辐射和高盐度的自然环境。为了适应极端的环境,生存于其中的微生物必然具备了相应独特的生理生化和分子机制。因此南极微生物不仅是研究在低温条件下生物适应性的极好材料,而且也是获得在低温条件下具有较大催化能力的适冷酶类的最佳来源。对南极微生物及其产生适冷酶的研究有着重要的生物学意义;同时,由于适冷酶能在低温下有效发挥催化作用的特点,在生物工程领域有着广阔的应用前景。本研究以南极海冰嗜冷菌Colwellia sp.NJ341为研究对象,对该菌株分泌的胞外适冷丝氨酸蛋白酶进行系统的研究,同时对该菌株的低温适应性也予以初步探讨,以期为南极微生物的深入研究和开发应用提供科学依据。(1)采用水解酪蛋白平板法对本试验室保存的260株南极低温微生物产适冷蛋白酶情况进行测定,发现107株具有蛋白酶活性,其中5株菌(NJ276、NJ341、NJ16-70、NJ345、NJ5-9)所产的蛋白酶活性高于45 U/ml。除NJ5-9菌株外,对其余4株菌所产蛋白酶的部分酶学特性进行研究,发现蛋白酶的酶学性质及酶的种类和数量在种间存在差异。菌株NJ276、NJ16-70分泌的蛋白酶接近中温蛋白酶,NJ341、NJ345分泌的蛋白酶属于适冷蛋白酶。随后,对5株菌进行了16S rRNA分子鉴定,菌株NJ276、NJ5-9、NJ16-70、NJ345均属于假交替单胞菌属(Pseudoalteromonas sp.);菌株NJ341属于科尔韦尔氏属(Colwellia sp.)。(2)以国内外研究较少且只分泌一种适冷蛋白酶的嗜冷菌Colwellia sp.NJ341作为研究材料,进行酶的优化培养、分离纯化及酶学性质的研究。结果表明,菌株NJ341发酵培养基的初始pH和通气量的最适值,分别为pH7~9和50~120更多还原

【Abstract】 Antarctica, due to its specific geographical position and climate, which characterized by strong seasonal changes in light and UV intensity, low-temperature (–50℃in winter), high dissolved oxygen and high-salinity (150‰salinity in sea ice saline channels), is considered an extreme environment on the earth. To be colonized in this extreme environment, microorganisms form gradually a series of physiological, chemical and molecular mechanisms. Therefore, Antarctic microorganisms provide a useful model system for studying cold adaptation, and also are the optimal sources of cold-adapted enzymes. It is of not only greatly biological value to study on these microorganisms and their cold-adapted enzymes, but also important practical value due to their potential for biotechnological applications. Consequently, selecting Colwellia sp.NJ341 as a research material, we describe the optimization of its cold-adapted protease production using RSM, purification and characterization of its cold-adapted protease, and unveil strain’s low-temperature adaptation mechanisms. The results are summarized as follows.(i) Protease activity of the cultures from 260 strains of Antarctic bacteria was qualitatively screened using the method of flat proteolysis casein Niujin. The results showed that there were 107 strains formed clear zones, meaning that they secreted the extracellular protease and protease activity of five strains NJ276, NJ341, NJ5-9, NJ16-70 and NJ345 was higher than 45 U/ml. The growth and producing protease characteristics of four Antarctic psychrophilic bacteria were studied, and the result showed that there were different among the strains. Proteases from NJ276 and NJ16-70 were mesophilic proteases, while NJ341 and NJ345 proteases were cold-adapted proteases. The 16S rRNA gene sequences homology and phylogenetic analysis showed that NJ276、NJ5-9、NJ16-70、NJ345 belonged to the described genus Pseudoalteromonas sp., and NJ341 belonged to the genus Colwellia sp.(ii) Antarctic psychrophilic bacteria Colwellia sp.NJ341 was chosen for the further更多还原