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 S & ouml;& gbreve;& uuml;tl & uuml; 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 degrees C, respectively. Although enzymatic activity increased after 60 min at 5 degrees 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 Km of 0.751 mg/mL and a V-max of 43.29 mu mol/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.