荧光假单胞杆菌胞外蛋白酶的纯化及热稳定性

从原料乳中分离获得一株荧光假单胞菌Rm12, 该菌株分泌的胞外蛋白酶具有很强的耐热性, 其发酵上清液经过硫酸铵沉淀、阴离子层析、疏水层析和分子排阻层析纯化得到SDS-PAGE均一蛋白酶, 经鉴定确认该酶是一种新的金属蛋白酶, 命名为Ht12, 对Ht12的基本特性和热稳定性进行了研究. 结果表明, 此蛋白酶分子量为45000, 含有较高的脯氨酸和二硫键, N末端氨基酸残基序列为MSKVKDKAIVSAAQAS, Mn2+对蛋白酶活力有促进作用. 该蛋白酶具有较高的热稳定性, 于160 ℃加热20 s, 保留活力3.8%.

Purication and Heat-stable Properties of a Novel Protease from Pseudomonads fluorescens Rm12

Heat-resistant proteases from psychrotrophic bacteria in raw milk may induce bitterness, gelation and hydrolyzation of sterilized milk. A novel extracellular heat-stable metalloprotease, named as Ht12, was found for the first time from Pseudomonads fluorescens Rm12,which was isolated from raw milk. Ht12 was purified to homogeneity from the culture supernatant via ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic chromatography, and size exclusion chromatography and its properties of enzymology and heat-stable properties was studied. This protease in its native state was identified as a monomer of 45000 Da, containing Pro and disulfide bonds and the N-terminal sequence was MSKVKDKAIVSAAQAS. Mn2+ has positive effect on activity. The protease has a higher heat resistance. After treatment of 160 ℃ for 20 s, the residual activity was 3.8%.