Protease from banana stem postharvest (Musa x paradisiaca): Extraction, purification, and its potential milk-clotting...

Plant protease isolation has gained significant attention, particularly from agricultural waste, due to its promising potential for economic enhancement, sustainability, and greenness. While a previous study has focused on protease production from banana peels, the postharvest stem of bananas remains underexplored, which is more abundant as waste. This study aimed to extract and purify protease from the postharvest stems of banana (Musa x paradisiaca), investigating various factors influencing its activity. We optimized protease extraction using different pH buffer solutions and purified the enzyme through three-phase partitioning (TPP). Molecular weight analysis was conducted using SDS-PAGE, and the protease classification was determined using specific protease inhibitors. Optimal conditions for enzyme activity were determined concerning pH, temperature, metal ion effects, organic solvent effects, salinity, and storage conditions. The results indicated that the best extraction pH was 8.0, with purified proteases exhibiting molecular weights of approximately 18 kDa and 21 kDa, classified as serine proteases. Specific metal ions enhanced protease activity, while Ca²⁺ and Na⁺ negatively impacted it. Furthermore, the milk clotting activity (MCA) to protease activity (PA) ratio was notably higher compared to other reported plant coagulants, suggesting the potential of this novel protease from banana stems as a milk coagulant.