【作者】 于珊珊；

【导师】 刘东波；

【作者基本信息】 东北师范大学 ， 微生物， 2006， 硕士

【摘要】 从我国内蒙古查干诺尔盐湖泥土样品中分离得到一株产低温碱性蛋白酶的适冷菌,通过对其进行的包括形态,生理生化特性以及16srDNA序列的研究,初步确定为halomonas属的成员,并命名为Halomonas sp. SD-01。研究表明,该菌株为杆状,革兰氏阴性,周生鞭毛,有芽孢,菌体形状呈杆状,富集培养基上的菌落呈黄色。对该株菌的生长的适冷性和它产生的蛋白酶的耐低温的特性的研究结果表明:该菌株在15℃左右生长速率最高,在10℃左右酶的合成速率最高;该菌株所产蛋白酶催化酪蛋白水解的最适温度为30℃左右,最适pH为10.0左右,该蛋白酶低于50℃时稳定。故Halomonas sp. SD-01为一株适冷菌,所产蛋白酶为低温碱性蛋白酶。对这株菌产低温蛋白酶的发酵条件的研究结果表明,该菌株产生低温碱性蛋白酶的最适碳源为0.5%葡萄糖,最适氮源为酵母粉,最适盐度为10%,最适产酶时间为140h。在此基础上,采用离心、超滤、（NH₄）₂SO₄沉淀、DEAE-SephadexA25离子交换层析、Sephorose 6B柱层析、透析以及冻干等手段,对该菌株产生的低温碱性蛋白酶进行了分离与纯化。对该低温碱性蛋白酶的性质研究表明,该酶的最适反应温度为30℃,最适反应PH为10.0,在温度≤50℃的条件下保持稳定。SDS-PAGE分析结果表明该酶分子量为69183;该酶可被EDTA不完全抑制,对1%H₂O₂具有一定的耐受性,对5mmol/LSDS的耐受性较差;Mn²⁺, Cu²⁺对蛋白酶有较为明显的激活作用,Ag⁺, Al³⁺, Hg²⁺则能抑制酶活;该酶作用于Casein的Km为1.709mg/ml;等电点电泳结果表明,该酶的PI为7.5。更多还原

【Abstract】 One Gram-negative bacterial strain was isolated from saline soda lake in InnerMongolia. Based on polyphasic studies, including its morphology, physiological andbiochemical characteristics, chemotaxonomy and 16SrDNA sequence analysis, it wasa new species of genus Halomonas and nomeneultured it as Halomonas sp. SD-01.The Halomonas sp. SD-01 was Psychrotrophilic and excreted psychrophilicprotease. The optimum temperature for growth was about 15℃,and the highesttemperature for growth was 35℃.The highest specific synthetic rate of the proteaseoccurred at about 10℃.The optimum tempature of the protease activity for casincatalysis was about 35℃ and even at 0℃it still remain little protease activity forcasin catalysis,the optimum pH of the protease activity for casin catalysis was about10.0,which suggested the enzyme was a typical alkaline psychrophilic protease.The protease was purified from the cell-free supernatant by ammonium sulfateprecipitation, ion exchange chromatography and gel filtration chromatography.SDS-PAGE analyses revealed a single band of 69.2 Kda.The protease activity wasstimulated by Mn²⁺, Cu²⁺, and was inhibited by Ag⁺, Al³⁺, Hg²⁺. From the result thatEDTA showed obvious inhibiting effect on the enzyme, it can be suggested that metalions played an important role in the conservation of enzyme conformation for thecatalytic activity.更多还原