Comprehensive biochemical, molecular and structural characterization of subtilisin with fibrinolytic potential in bioprocessing.

Abstract

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_m and V_max values of 0.731 ± 0.5 mM and 0.87 ± 9 × 10³ 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.