Molecular and Functional Analysis of Proteases Isolated from Tropical Fish Visceral Waste via a Cost-Effective Dual-Buffer Technique

Fish processing wastes, particularly digestive organs, yield proteases such as trypsin, chymotrypsin, and elastase, which exhibit unique characteristics suitable for various industrial applications. This study focuses on enzyme recovery from various commercially important species prevalent in the fish market of West coast of India, including Rohu (Labeo rohita), Pangas (Pangasius sp.), Common Carp (Cyprinus carpio), Basa (Pangasius sp.), Pacu (Piaractus brachypomus), Mackerel (Rastrilliger kanagurta), and Croaker species. Proteases were extracted using NaCl and Tris-HCl buffers. Proteolytic activity was assessed using casein, collagen, and gelatin as substrates at optimal conditions, such as a reaction time of 10 min, pH 9, and temperature of 40 °C for collagen and gelatin and 60 °C for casein. SDS-PAGE analysis revealed molecular weights ranging from 20 to 60 kDa. Both NaCl and Tris-HCl buffer proteases were significantly inhibited by soya trypsin inhibitor (STI). NaCl buffer proteases exhibited strong inhibition by beta-mercaptoethanol, SDS, and hydrogen peroxide, with SDS-PAGE revealing band disappearance in the presence of beta-mercaptoethanol. The study highlights the potential of utilizing fish processing waste for enzyme recovery, promoting sustainable development, and minimizing environmental impact by transforming waste into valuable resources.