Bioinformatics Aided Elucidation of Functional and Structural Attributes of <i>Bubalus Arnee Bubalis</i> (BAB) Prochymosin

ABSTRACT The aspartic protease prochymosin, found in the abomasum of Bubalus arnee bubalis (BAB), is pivotal in κ‐casein cleavage at the Phe105‐Met106 site, facilitating milk coagulation and cheese production. Initially synthesized as pre‐prochymosin, it undergoes post‐translational modifications to form active chymosin. Advanced bioinformatics approaches including BLAST, Gene Ontology (GO), structural modeling were used to characterize the structural and functional properties of prochymosin of BAB. The study identified prochymosin as a hydrophilic, low‐molecular‐weight protein with high sequence homology to bovine prochymosin homologs. Two conserved aspartic peptidase active sites confirm its classification within the aspartic protease family, essential for its enzymatic activity. GO analysis revealed its role in aspartic endopeptidase activity and proteolysis. Secondary structure analysis found a composition of 32.80% alpha helices, 44.18% random coils, and 23.02% beta strands. Post‐translational modification sites, including phosphorylation and glycosylation regions, along with intrinsic disorder zones, suggested regulatory mechanisms and functional flexibility. Protein–protein interaction (PPI) studies indicated significant roles within buffalo stomach proteolytic networks. This study underscores the evolutionary conservation and complexity of BAB prochymosin, providing a robust foundation for further structural and functional research, particularly in its application to dairy science. Future studies should focus on experimental validation of predicted structural features and post‐translational modifications, as well as comparative analyses with other ruminants to explore species‐specific adaptations.