Isolation and Characterization of a Novel Acid Protease from Striped Marlin (Kajikia audax) Stomach with Potential as a Rennet Substitute in Dairy Processing

This study investigated the isolation, characterization, and optimization of acid protease from the stomach of the Striped Marlin (Kajikia audax). The gastrointestinal tract—including the stomach, pyloric caeca, pancreas, and intestine—was analyzed for size, weight, volume, and enzymatic activity. The stomach was selected for further study based on its high enzymatic potential. Subsequent analyses included proximate composition, acidified gastric extract (AGE), protease activity, total protein, and the effects of pH, temperature, metal ions, SDS-PAGE, and milk-clotting ability. Proximate analysis of the stomach showed crude protein (16.39%), crude lipid (4.89%), moisture (73.96%), ash (1.05%), and carbohydrates (3.71%). The extraction yield was 33.33%, and protease activity was significantly higher (1,013.823 ± 2.541 U/mL, P < 0.05) than in other organs. A 30% ammonium sulfate fraction yielded optimal specific activity (3.7116 ± 0.023 U/mg), with a 2.3-fold purification and 45% yield. Biochemical characterization revealed peak activity at pH 2.0 and 50 ± 1°C, with the enzyme retaining over 80% activity between 45–55°C. At 5 mM concentration, MgCl₂ enhanced activity (443% ± 12%, P< 0.01), while NaCl and CaCl₂ were inhibitory (37% and 48% relative activity, respectively). Milk-clotting activity, evaluated using 12% (w/v) reconstituted skim milk, was optimal at pH 3.0–4.0 and 50°C, with a clotting/proteolytic ratio of 1.8. The dialyzed enzyme maintained >70% activity after 1 hour at 45°C and showed consistent milk-clotting, suggesting its potential as a rennet substitute. This marine-derived protease offers promising applications in cheese production.