Biotechnological Application of New Cold-Active Protease From Aeromonas salmonicida subsp. salmonicida EDT1

This study involved the isolation of ten psychrophilic bacterial strains from cold water in Söğütlü village, Erzurum. Following isolation, the strains were characterized using molecular and conventional methods. On the basis of the results of Petri dish assays, Aeromonas salmonicida subsp. salmonicida EDT1 (GenBank accession no: PP068881) exhibited the highest protease activity. The cold-active protease obtained from A. salmonicida subsp. salmonicida EDT1 was partially purified using a one-step, three-phase partitioning (TPP) method under the following conditions: pH 9.0; a ratio of crude extract to t-butanol of 1.0:1.5; and 80% saturated ammonium sulfate. This resulted in a yield of 244% and a purification fold of 42. The molecular weight of the enzyme was found to be approximately 39.44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The optimal pH and temperature for the protease were 9.0 and 5°C, respectively. Although enzymatic activity increased after 60 min at 5°C, it gradually declined thereafter. Protease activity increased in the presence of Mg²⁺ (1 mM), Na⁺ (5 mM), and Mn²⁺ (10 mM) by 253%, 213%, and 169%, respectively. Phenylmethylsulfonyl fluoride (PMSF) significantly inhibited the enzyme, reducing its activity to 15%. After 1 h of incubation, activity increased in the presence of 50% acetone and 50% isopropanol to 393% and 256%, respectively. SDS increased protease activity by 336%. The enzyme exhibited a K_m of 0.751 mg/mL and a V_max of 43.29 µmol/mL/min for casein. The enzyme retained substantial activity after exposure to various commercial detergents. Purified EDT1 protease effectively removed wet and dried blood, as well as grass stains. The enzyme-detergent combination was most effective after 1 h of incubation.