Biochemical, thermodynamic, and structural characterization of a collagenolytic protease fromMucor subtilissimusUCP 1262.

Collagenolytic proteases have attracted increasing attention due to their ability to hydrolyze collagen and their relevance in biotechnological processes. In this study, a collagenolytic protease produced by Mucor subtilissimus UCP 1262 was characterized through an integrated evaluation of its biochemical properties, thermal-inactivation thermodynamics, and conformational behavior. The enzyme was produced by solid-state fermentation using wheat bran and purified by DEAE-Sephadex A50 ion-exchange chromatography. Optimal collagenolytic activity was observed at pH 7 and 40 °C, and approximately 50% of the initial activity was retained after 24 h at 30-40 °C, indicating moderate thermal stability. Fluorescence spectroscopy revealed marked pH- and temperature-dependent changes in fluorescence intensity and emission maxima, with incomplete structural recovery after cooling. Thermodynamic analysis further supported the occurrence of temperature-dependent destabilization. Overall, the results provide new insight into the functional and conformational stability of the collagenolytic protease from M. subtilissimus UCP 1262 and support its further investigation for biotechnological applications under mild processing conditions.