Screening, Purification, and Characterization of Protease Inhibitors From Different Legume Seeds With Cytotoxic and Antimicrobial Effects

ABSTRACT This study reports the purification and characterization of protease inhibitors (PIs) from three Egyptian legumes: soybean ( Glycine max ), cowpea ( Vigna unguiculata ), and chickpea ( Cicer arietinum ). Their antimicrobial and cytotoxic activities were also assessed. PIs were extracted using different buffers: 0.02 M HCl yielded the highest activity for soybean and cowpea, while 0.3 M NaCl was optimal for chickpea. Ammonium sulfate precipitation at 0%–90% saturation was best for soybean and chickpea, whereas 0%–60% was most effective for cowpea. Purification via DEAE‐Sepharose showed that 0.25 M NaCl fractions had the highest total and specific activity across all legumes. Sephadex G‐50 further enhanced specific PIs activity. Electrophoresis revealed bands at 25 kDa (soybean), 45 kDa (cowpea), and 35 kDa (chickpea). PIs showed variable cytotoxicity depending on legume, lyophilization, cell line, and exposure time. Lyophilized soybean PIs significantly reduced human hepatocellular carcinoma (HepG2) cell viability after 48 h, while nonlyophilized chickpea PIs also affected HepG2. Cowpea PIs notably induced human colorectal adenocarcinoma (HT‐29) cell death at higher concentrations; instead, human lung adenocarcinoma (A549) cells were generally less sensitive, except at high cowpea PI doses. All lyophilized PIs inhibited Bacillus subtilis but were inactive against Staphylococcus epidermidis and Pseudomonas stutzeri .