Caseinolytic Mechanism of Serine Protease MCP746 from Tamarillo (<i>Solanum betacea</i> Cav.) and Potential Application in Fresh Cheese

The serine protease MCP746 was previously found to have potential for cheese making in our previous research, but its caseinolytic mechanism is still unclear. This study aims to identify the cleavage sites of MCP746 on κ-casein (κ-CN), elucidate its caseinolytic mechanism, and evaluate the quality of the cheese. The Michaelis constant (<i>K</i>_m) and maximum hydrolysis rate (<i>V</i>_max) of MCP746 were 0.92 mg/mL and 833.33 U/min, respectively. MCP746 catalytic triad consists of Asp₁₄₅/His₂₀₂/Ser₅₃₁, enabling specific cleavage at Lys₁₃₃-Asn₁₃₄ of κ-CN. Additionally, compared to calf rennet, cheese produced with MCP746 demonstrated higher adhesiveness, springiness, and cohesiveness (<i>p</i> < 0.05). Cryogenic scanning electron microscopy (Cryo-SEM) revealed that cheese made with MCP746 exhibited smaller and denser pore structures. The content of bitter amino acids in cheese made with MCP746 (24.52%) was lower than that in the control (35.61%). These results illustrate that the plant protease MCP746 has promising applications in the production of fresh cheese.