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. Enterobacter cloacae (BC, pork), Bacillus paramycoides (P2, snook), and Pseudomonas aeruginosa (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 Pseudomonas aeruginosa CH1 as a particularly promising candidate for industrial applications requiring robust and sustained proteolytic activity.

Yellowfin tuna (Thunnus albacares) processing by-products are underutilized resources rich in high-quality proteins that can be valorized into bioactive ingredients. In this study, proteins from yellowfin tuna tail bones were hydrolyzed using either an endogenous protease extract recovered from tuna viscera or commercial trypsin. The endogenous extract was characterized and exhibited optimal proteolytic activity at pH 8 and 40 °C. Comparative bioactivity assessment showed that trypsin-derived hydrolysates exhibited significantly higher antioxidant capacity, with ABTS and DPPH values of 11.8 ± 0.04 and 13.3 ± 0.40 mg Trolox/g, respectively, compared with 10.1 ± 0.12 and 7.3 ± 0.21 mg Trolox/g for hydrolysates produced with endogenous enzymes. Trypsin hydrolysates also showed stronger dipeptidyl peptidase-IV (DPP-IV) inhibition (IC₅₀ = 0.83 ± 0.37 mg/mL) than endogenous-enzyme hydrolysates (IC₅₀ = 1.60 ± 0.47 mg/mL). Remarkably, hydrolysates generated with the endogenous enzymatic consortium exhibited exceptionally potent angiotensin-converting enzyme (ACE) inhibitory activity (IC₅₀ = 0.008 ± 0.004 mg/mL), outperforming trypsin hydrolysates (IC₅₀ = 0.01 ± 0.007 mg/mL). Neither hydrolysate showed evidence of acute toxicity in the Artemia salina model at the tested concentrations, supporting their favorable performance in this preliminary toxicity screening assay. Considering that nearly two-thirds of total fish biomass is discarded during processing, this integrated bioprocess demonstrates a sustainable strategy for the simultaneous valorization of skeletal and visceral waste streams. By employing endogenous enzymes as biocatalysts, this approach reduces dependence on commercial proteases, mitigates environmental burdens, and supports the development of a circular marine bioeconomy through the production of highly potent, peptide-rich functional ingredients with promising cardiovascular health applications. Further toxicological evaluation is required to confirm their safety for food and nutraceutical applications.