嗜热菌属硫醇依赖性、氧化稳定丝氨酸碱性蛋白酶的纯化与特性分析
Purification and characterization of thiol dependent, oxidation-stable serine alkaline protease from thermophilic sp.
作者信息
Kamran Aysha, Ur Rehman Haneef, Ul Qader Shah Ali, Baloch Abdul Hameed, Kamal Mustafa
机构信息
Department of Biotechnology, University of Karachi, Karachi, Pakistan.
Department of Agriculture, Lasbela University of Agriculture, Water and Marine Sciences (LUAWMS), Uthal, Pakistan.
出版信息
J Genet Eng Biotechnol. 2015 Jun;13(1):59-64. doi: 10.1016/j.jgeb.2015.01.002. Epub 2015 Feb 26.
Alkaline serine protease was purified to homogeneity from culture supernatant of a thermophilic, alkaliphilic sp. by 80% ammonium sulphate precipitation followed by CM-cellulose and DEAE-cellulose ion exchange column chromatography. The enzyme was purified up to 16.5-fold with 6900 U/mg activity. The protease exhibited maximum activity towards casein at pH 8.0 and at 80 °C. The enzyme was stable at pH 8.0 and 80 °C temperature up to 2 h. The Ca and Mn enhanced the proteolytic activity up to 44% and 36% as compared to control, respectively. However, Zn, K, Ba , Co , Hg and Cu significantly reduced the enzyme activity. PMSF (phenyl methyl sulphonyl fluoride) completely inhibited the protease activity, whereas the activity of protease was stimulated up to two folds in the presence of 5 mM 2-mercaptoethanol. The enzyme was also stable in surfactant (Tween-80) and other commercial detergents (SDS, Triton X-100).
通过80%硫酸铵沉淀，随后进行CM-纤维素和DEAE-纤维素离子交换柱色谱法，从嗜热嗜碱菌的培养上清液中纯化出了碱性丝氨酸蛋白酶，使其达到了均一性。该酶的活性为6900 U/mg，纯化倍数达到了16.5倍。该蛋白酶在pH 8.0和80℃时对酪蛋白表现出最大活性。该酶在pH 8.0和80℃温度下长达2小时保持稳定。与对照相比，Ca和Mn分别将蛋白水解活性提高了44%和36%。然而，Zn、K、Ba、Co、Hg和Cu显著降低了酶活性。苯甲基磺酰氟（PMSF）完全抑制了蛋白酶活性，而在5 mM 2-巯基乙醇存在下，蛋白酶的活性被刺激提高了两倍。该酶在表面活性剂（吐温80）和其他商业洗涤剂（SDS、Triton X-100）中也很稳定。
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