Shettar Shreya S, Bagewadi Zabin K, Alasmary Mohammed, Mannasaheb Basheerahmed Abdulaziz, Shaikh Ibrahim Ahmed, Khan Aejaz Abdullatif
Department of Biotechnology, KLE Technological University, Vidyanagar, Hubballi, 580031, Karnataka, India.
Department of Medicine, College of Medicine, Najran University, 66462, Najran, Saudi Arabia.
Bioresour Bioprocess. 2025 Mar 21;12(1):21. doi: 10.1186/s40643-025-00860-1.
Enzyme deployment is proliferating extensively in industries owing to their environmentally friendly and easily degradable attributes. This article undertakes an exhaustive examination of wild subtilisin enzyme, covering purification, biochemical delineation, analytical techniques, and practical implementations. The purification methodology involved partial refinement, anionic exchange, and gel filtration chromatography, culminating in a purification factor of 3.406, corroborated by SDS-PAGE showcasing a molecular weight of ~ 42 kDa. Biochemical scrutiny unveiled the enzyme's response, with an optimal pH at 9 and temperature peak at 60 ℃. Various surfactants, metal ions, organic solvents and inhibitors exhibited notable efficacy. Substrate specificity and kinetics showcased the utmost specificity with N-Suc-F-A-A-F-pNA, registering K and V values of 0.731 ± 0.5 mM and 0.87 ± 9 × 10 U/mg, respectively. Different bioanalytical techniquesproffered insights into structural and biophysical facets. Practical applications encompassed goat skin depilation, feather disintegration, blood clot dissolution, exemplifying the enzyme's multifaceted utility. To embark upon the elucidation of structure-function relationships, a three-dimensional model was devised through homology modelling, leveraging existing subtilisin structures (PDB: 3WHI). Molecular docking score of - 8.8 kcal/mol and dynamic simulations augmented the comprehension of molecular interactions with N-Suc-F-A-A-F-pNA. This research significantly contributes to unravelling the biochemical intricacies of wild subtilisin and underscores potential industrial and biomedical prowess. Subtilisin can be explored for its thrombolytic potential in several cardiovascular diseases. It may aid in the management of thrombosis by dissolving blood clots in conditions like deep pulmonary embolism, myocardial infarction, ischemic strokes, and in atherosclerosis by breaking down fibrin in arterial plaques, thus preventing heart attacks and strokes.
由于酶具有环境友好且易于降解的特性，其在工业中的应用正在广泛增加。本文对野生枯草杆菌蛋白酶进行了详尽的研究，涵盖了纯化、生化特性描述、分析技术及实际应用。纯化方法包括部分精制、阴离子交换和凝胶过滤色谱，最终纯化因子为3.406，SDS-PAGE证实其分子量约为42 kDa。生化研究揭示了该酶的反应特性，最适pH为9，温度峰值为60℃。各种表面活性剂、金属离子、有机溶剂和抑制剂均显示出显著效果。底物特异性和动力学研究表明，该酶对N-Suc-F-A-A-F-pNA具有极高的特异性，其K值和V值分别为0.731±0.5 mM和0.87±9×10 U/mg。不同的生物分析技术为其结构和生物物理方面提供了见解。实际应用包括山羊皮脱毛、羽毛分解、血块溶解，体现了该酶的多方面用途。为了阐明结构-功能关系，通过同源建模利用现有的枯草杆菌蛋白酶结构（PDB：3WHI）设计了三维模型。-8.8 kcal/mol的分子对接分数和动态模拟增强了对与N-Suc-F-A-A-F-pNA分子相互作用的理解。本研究对揭示野生枯草杆菌蛋白酶的生化复杂性做出了重大贡献，并强调了其潜在的工业和生物医学优势。枯草杆菌蛋白酶可用于探索其在多种心血管疾病中的溶栓潜力。它可能有助于在深静脉肺栓塞、心肌梗死、缺血性中风等情况下溶解血块来管理血栓形成，并通过分解动脉斑块中的纤维蛋白来治疗动脉粥样硬化，从而预防心脏病发作和中风。
