Biological Sciences Department, King Abdulaziz University, Saudi Arabia.
Int J Biol Macromol. 2013 Apr;55:169-75. doi: 10.1016/j.ijbiomac.2013.01.002. Epub 2013 Jan 9.
Novel keratinolytic enzyme (32kDa) secreted by a newly isolated Bacillus strain (Bacillus subtilis NRC3) cultivated in medium containing chicken feather meal was purified and partially characterized in a set of biochemical assays. The purification was carried out by applying a protocol of two successive chromatographic steps; cation exchange chromatography on CM-cellulose and gel filtration on sephadex G-75 columns. The purified enzyme showed a specific activity of 5233units/mg protein against 169units/mg protein for crude extract with 31 fold purification. The enzymatic activity of the purified keratinolytic enzyme stimulated by Na(+), K(+), Mg(2+), Ba(2+), Ca(2+), and inhibited by entire tested cations and metalloproteinase inhibitors, indicating that it belongs to metallo-keratinase enzymes. The optimum pH and temperature for the purified enzyme were (7.5, 8.0) and (50, 40°C) when using keratin azure and azocasein as substrates, respectively. The purified enzyme was highly stable at broad pH and temperature ranged (5-10) and (20-60°C), respectively. These results suggest that this keratinase may be a useful alternative and ecofriendly route for handling the abundant amount of waste feathers.
新型角蛋白酶（32kDa）由一株新分离的枯草芽孢杆菌（枯草芽孢杆菌 NRC3）在含有鸡毛粉的培养基中分泌，通过一系列生化测定对其进行了纯化和部分特性研究。纯化过程采用两步连续色谱步骤的方案进行；CM-纤维素上的阳离子交换色谱和 sephadex G-75 柱上的凝胶过滤。纯化后的酶对 169 单位/毫克蛋白的粗提物的比活为 5233 单位/毫克蛋白，具有 31 倍的纯化度。该角蛋白酶的酶活受 Na(+)、K(+)、Mg(2+)、Ba(2+)、Ca(2+)刺激，受所有测试的阳离子和金属蛋白酶抑制剂抑制，表明其属于金属角蛋白酶。当使用角蛋白azure 和偶氮酪蛋白作为底物时，纯化酶的最适 pH 和温度分别为（7.5、8.0）和（50、40°C）。该酶在较宽的 pH 和温度范围（5-10）和（20-60°C）下具有高度稳定性。这些结果表明，这种角蛋白酶可能是处理大量废羽毛的一种有用的替代和环保方法。
