Thumar Jignasha, Singh S P
Department of Biosciences, Saurashtra University, Rajkot-360005, Gujarat, India.
J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Jul 1;854(1-2):198-203. doi: 10.1016/j.jchromb.2007.04.023. Epub 2007 Apr 25.
An alkaline protease from a salt-tolerant alkaliphilic Streptomyces clavuligerus was purified to homogeneity by 141-fold with a yield of 12% using two-step method of salt precipitation and ion exchange chromatography on DEAE cellulose. The apparent molecular mass was 49+/-2 kDa and the enzyme appeared as monomer based on SDS and Native-PAGE. The temperature optimum was 70 degrees C with significant stability at 60-80 degrees C for more than 60 min. The enzyme was active over the pH range of 8.5-11, with an optimum at 10-11. The serine nature of the protease was confirmed by PMSF inhibition. The enzyme was highly resistant against chemical denaturation and displayed varied effects towards metal ions. The results are significant as extremozymes are difficult to purify and therefore, a two-step purification of alkaline protease from relatively less explored group of actinomycetes is quite appealing.
采用盐析和DEAE纤维素离子交换色谱两步法，从耐盐嗜碱棒状链霉菌中纯化出一种碱性蛋白酶，纯化倍数达141倍，产率为12%，该酶达到了均一性。根据SDS和非变性聚丙烯酰胺凝胶电泳结果，其表观分子量为49±2 kDa，且该酶以单体形式存在。该酶的最适温度为70℃，在60 - 80℃具有显著稳定性，可保持60分钟以上。该酶在pH值8.5 - 11范围内具有活性，最适pH值为10 - 11。苯甲基磺酰氟抑制实验证实了该蛋白酶的丝氨酸性质。该酶对化学变性具有高度抗性，并且对金属离子表现出不同的效应。由于极端酶难以纯化，因此从相对较少研究的放线菌中两步纯化碱性蛋白酶的结果具有重要意义。
