Department of Botany, Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh 221005, India.
Center for Systems Biology and Molecular Medicine, Yenepoya Research Center, Yenepoya (Deemed to be University), Mangalore 575018, India.
Int J Biol Macromol. 2024 Oct;278(Pt 4):135025. doi: 10.1016/j.ijbiomac.2024.135025. Epub 2024 Aug 24.
Keratin is one of the major components of solid waste, and the degradation products have extensive applications in various commercial industries. Due to the complexity of the structure of keratin, especially the disulfide bonds between keratin polypeptides, keratinolytic activity is efficient with a mixture of proteins with proteases, peptidases, and oxidoreductase activity. The present work aimed to create an engineered chimeric protein with a disulfide reductase domain and a protease domain connected with a flexible linker. The structure, stability, and substrate interaction were analyzed using the protein modeling tools and codon-optimized synthetic gene cloned, expressed, and purified using Ni-NTA chromatography. The keratinolytic activity of the protein was at its maximum at 70 °C. The suitable pH for the enzyme activity was pH 8. While Ni, Mg, and Na inhibited the keratinolytic activity, Cu, Ca, and Mn enhanced it significantly. Biochemical characterization of the protease domain indicated significant keratinolytic activity at 70 °C at pH 10.0 but was less efficient than the chimeric protein. Experiments using feathers as the substrate showed a clear degradation pattern in the SEM analysis. The samples collected from the degradation experiments indicated the release of proteins (2-fold) and amino acids (8.4-fold) in a time-dependent manner. Thus, the protease with an added disulfide reductase domain showed excellent keratin degradation activity and has the potential to be utilized in the commercial industries.
角蛋白是固体废物的主要成分之一，其降解产物在各种商业行业中有广泛的应用。由于角蛋白结构复杂，特别是角蛋白多肽之间的二硫键，角蛋白酶的活性与具有蛋白酶、肽酶和氧化还原酶活性的蛋白质混合物效率很高。本工作旨在创建一种具有二硫键还原酶结构域和蛋白酶结构域的工程嵌合蛋白，并用柔性接头连接。使用蛋白质建模工具和密码子优化的合成基因进行分析，该基因经过克隆、表达和 Ni-NTA 层析纯化。该蛋白的角蛋白酶活性在 70°C 时达到最大值。酶活性的适宜 pH 值为 8。虽然 Ni、Mg 和 Na 抑制角蛋白酶的活性，但 Cu、Ca 和 Mn 显著增强了其活性。蛋白酶结构域的生化特性分析表明，在 70°C 和 pH 值为 10.0 时具有显著的角蛋白酶活性，但效率低于嵌合蛋白。使用羽毛作为底物的实验在 SEM 分析中显示出明显的降解模式。降解实验中收集的样品表明，蛋白质（2 倍）和氨基酸（8.4 倍）以时间依赖的方式释放。因此，添加二硫键还原酶的蛋白酶具有优异的角蛋白降解活性，有望在商业行业中得到应用。
