Bioprospecting of Aerobic Bacteria with Proteolytic Potential Isolated from Animal and Water Sources in the Three Regions of Mainland Ecuador

The growing demand for efficient and cost-effective industrial proteases has intensified bioprospecting efforts in diverse ecosystems as a strategy to identify microorganisms with enhanced enzymatic performance. This study aimed to isolate, identify, and evaluate aerobic protease-producing bacteria from animal-protein matrices and water sources collected across the three continental regions of Ecuador, and to assess their suitability for industrial enzyme production A total of 34 bacterial strains were isolated and taxonomically assigned to the orders Enterobacterales, Pseudomonadales, and Bacillales. Proteolytic activity was evaluated using azocasein and casein assays after cultivation in an optimized medium containing 1% soybean paste as an inducer at 37 °C and 120 rpm for 72 h. <i>Enterobacter cloacae</i> (BC, pork), <i>Bacillus paramycoides</i> (P2, snook), and <i>Pseudomonas aeruginosa</i> (CH1, chontacuro) were identified as the most active protease producers from the Andean (Sierra), coastal (Costa), and Amazon regions, respectively. Production kinetics revealed marked strain-dependent differences. BC and P2 reached maximum proteolytic activity on day 4 followed by a decline, whereas CH1 peaked on day 2 and maintained stable activity over time, indicating superior enzymatic stability. Partial purification by gel-filtration chromatography (Sephadex G-100) yielded fractions with enhanced proteolytic activity, while SDS-PAGE analysis confirmed successful enrichment of protease-containing fractions. Overall, the results demonstrate that ecological origin strongly influences protease production and stability, and identify <i>Pseudomonas aeruginosa</i> CH1 as a particularly promising candidate for industrial applications requiring robust and sustained proteolytic activity.