Functional evaluation of a Staphylococcus vitulinus protease: Biochemical properties, structural adaptation, and application in myofibrillar proteins.

An extracellular protease was obtained from Staphylococcus vitulinus and exhibited optimal activity at pH 8.0 and 50 °C. Mn2+ (10 mmol/L) significantly enhanced its activity, while Zn2+, Cu2+, Fe2+, and Fe3+ exhibited inhibitory effects (P < 0.05). The Km and Vmax values of the protease were 9.67 mg/mL and 27.5 U/mL·min, respectively, and the Ea was 16.5 kJ/mol. The protease from S. vitulinus exhibited ΔH* of 13.94 kJ/mol, ΔG* of 39.18 kJ/mol, and ΔS* of -70.9 J/(mol·K). Fluorescence and ultraviolet spectroscopic analyses demonstrated that variations in pH and temperature altered protease activity by affecting its tertiary structure, which is primarily associated with tyrosine and tryptophan residues. At the secondary structure level, the decline in protease activity was associated with the decrease in α-helices and β-sheets as well as the increase in random coils. Proteolysis catalyzed by S. vitulinus protease resulted in a significant increase in the degree of hydrolysis, solubility, and peptide concentration of myofibrillar proteins (MPs) (P < 0.05), with the highest hydrolysis rate observed within 0-30 min. Both the variety and content of free amino acids increased with the extension of hydrolysis time (P < 0.05). Protease treatment promoted the formation of volatile compounds derived from MPs, including alcohols, esters, aldehydes, acids, and ketones. Six volatile compounds with variable importance in projection values greater than 1 were selected. This study aimed to develop a protease with excellent proteolytic capacity and flavor-enhancing efficacy, and to elucidate its underlying mechanism from the perspective of amino acid metabolic pathways.