Characterization of a Novel Milk-Clotting Aspartic Protease from Penicillium Sp. and Structural Explanation for Its High Milk-Clotting Index.

A novel milk-clotting enzyme isolated from Penicillium sp. ACCC 39790 (PsMCE) was prepared by heterologous expression. The recombinant PsMCE had an apparent molecular mass of 45 kDa and exhibited maximum casein hydrolysis activity at pH 4.0 and 50 °C. The PsMCE activity was enhanced by calcium ions and strongly inhibited by pepstatin A. Through hydrolysis pattern and cleavage site analyses, the milk-clotting activity of PsMCE was related to its specific hydrolysis between Phe105 and Met106 in the κ-casein proteins. The structural basis of PsMCE was characterized using homology modeling, molecular docking, and interactional analysis. The P1' region of PsMCE is critical for its selective binding to the hydrolytic site in κ-casein, and the hydrophobic forces play a decisive role in the specific cleavage of Phe105 and Met106. These interactional analyses between PsMCE and the ligand peptide clarified the fundamentals of its high milk-clotting index (MCI). PsMCE could be applied in cheese making due to its thermolability and high MCI value as a potential milk-clotting enzyme.