南极低温降解菌Shewanella sp.NJ49羟化酶分离纯化及最适产酶条件研究

Separation and purification of hydroxylase from Antarctic psychrophilic bacterium Shewanella sp.NJ49 and optimum conditions for its enzyme production

羟化酶是细菌降解烷烃的关键酶,本文以南极低温降解菌Shewanella sp.NJ49为研究对象,通过硫酸铵沉淀、超滤浓缩及葡聚糖凝胶Sephadex G-75柱层析方法,分离纯化NJ49的羟化酶,获得了单一羟化酶组分,研究了温度、p H、表面活性剂和盐度等环境因素对NJ49羟化酶酶活活性影响。结果表明,羟化酶组分由α、β、γ三个亚基组成,分子质量分别为56,45和36 k D。NJ49最适产酶条件为:温度15℃、p H7.2、盐度55;不同类型表面活性剂对酶的活性影响效应不一,两性离子表面活性剂、阳离子表面活性剂、非离子表面活性剂对酶活没有作用,阴离子表面活性剂+非离子表面活性剂混和剂和阳离子表面活性剂+非离子表面活性剂混和剂能提高羟化酶的活性。

Hydroxylase is a key enzyme with respect to bacteria during hydrocarbon degradation. In this paper, we investigate the Antarctic psychrophilic bacteriumShewanella sp. NJ49. To obtain a single component of hydroxy-lase, we separated and purified hydroxylase from NJ49 in a series of steps, i.e., ammonium sulfate precipitation, ultrafiltration concentration, and column chromatography with Sephadex G-75 as the column media. In addition, we studied the relationships between the activity of NJ49 hydroxylase and environmental factors such as temperature, pH, surfactant, and salinity. We found that purified hydroxylase comprised subunitsα,β, andγ, with estimated molecular masses of 56, 45, and 36 kD, respectively. The optimum conditions for producing NJ49 hydroxylase were a temperature of 15℃, a pH of 7.2, and a salinity of 55‰. Different types of surfactants had different effects on enzyme activity. Although individual amphoteric, cationic, and nonionic surfactants did not inhibit enzyme activity, a mixture of anionic and nonionic surfactants, as well as a mixture of cationic and nonionic surfactants, improved the activity of the hydroxylase.