Bacillus subtilis S1-4, isolated from chicken feather could efficiently degrade feathers by secreting several extracellular proteases. In order to get insight into the individual protease involved in keratin hydrolysis, a keratinase designed as BsKER71 was cloned and expressed in Bacillus subtilis WB600. In silico analysis revealed that BsKER71 protein contained a mature protein of 36.1 kDa. Further, purified BsKER71 could hydrolyze a variety of natural proteins, such as fibrous protein, collagen protein, casein, keratin and bovine serum albumin. In addition, this keratinase exhibited high enzyme activity in a wide range of pH and optimal pH of 10.0 and 9.0 in the hydrolysis of casein and keratin, respectively. Similarly, the optimal temperature was 55 °C and 50 °C for the hydrolysis of above two substrates, respectively. The hydrolytic activity was significantly inhibited by phenylmethanesulfonyl fluoride (PMSF), indicating the presence of serine residue in the active site. Moreover, ethylenediaminetetraacetic acid (EDTA) and phenanthroline moderately inhibited the hydrolytic activity. The catalytic activity was stimulated by Mg2+ and Ca2+, but greatly inhibited by Cu2+. Furthermore, several chemicals exhibited different effects on the hydrolysis of casein and keratin by BsKER71. These results provided a better understanding of BsKER71 from feather degrading bacterium B. subtilis S1-4.

中文翻译：

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羽毛降解细菌枯草芽孢杆菌S1-4新型角蛋白酶BsKER71的重组表达和生化特性。

从鸡羽毛中分离出的枯草芽孢杆菌S1-4可通过分泌几种细胞外蛋白酶有效降解羽毛。为了深入了解参与角蛋白水解的单个蛋白酶，克隆了设计为BsKER71的角蛋白酶并在枯草芽孢杆菌WB600中表达。在计算机分析中发现，BsKER71蛋白含有36.1kDa的成熟蛋白。此外，纯化的BsKER71可以水解多种天然蛋白，例如纤维蛋白，胶原蛋白，酪蛋白，角蛋白和牛血清白蛋白。另外，该角蛋白酶在酪蛋白和角蛋白的水解中分别在宽的pH范围和10.0和9.0的最佳pH中显示出高的酶活性。同样，水解上述两种底物的最佳温度分别为55°C和50°C。苯甲磺酰氟（PMSF）显着抑制了水解活性，表明在活性位点存在丝氨酸残基。此外，乙二胺四乙酸（EDTA）和菲咯啉可适度抑制水解活性。Mg2 +和Ca2 +刺激了催化活性，但Cu2 +大大抑制了催化活性。此外，几种化学物质对BsKER71水解酪蛋白和角蛋白的作用不同。这些结果提供了来自羽毛降解细菌枯草芽孢杆菌S1-4的BsKER71的更好理解。Mg2 +和Ca2 +刺激了催化活性，但Cu2 +大大抑制了催化活性。此外，几种化学物质对BsKER71水解酪蛋白和角蛋白的作用不同。这些结果提供了来自羽毛降解细菌枯草芽孢杆菌S1-4的BsKER71的更好理解。Mg2 +和Ca2 +刺激了催化活性，但Cu2 +大大抑制了催化活性。此外，几种化学物质对BsKER71水解酪蛋白和角蛋白的作用不同。这些结果提供了来自羽毛降解细菌枯草芽孢杆菌S1-4的BsKER71的更好理解。