Abstract
Previous theoretical studies have determined the intermolecular interactions between Mucor pusillus pepsin (MPP) and the key domain of -casein, with the aim to understand the mechanism of milk clotting in the specific hydrolysis of κ-casein by MPP for cheese making. Here, we combined the docking model with site-directed mutagenesis to further investigate the functional roles of amino acid residues in the active site of MPP. T218S replacement caused a low thermostability and moderate increase in the clotting activity. Mutations of three amino acid residues, T218A and T218S in S2 region and L287G in S4 region, led to a significant decrease in proteolytic activity. For T218S and L287G, an increase in the ratio of clotting activity to proteolytic activity (C/P) was observed, in particular 3.34-fold increase was found for T218S mutants. Structural analysis of the binding mode of MPP and chymosin splitting domain (CSD) of κ-casein indicated that T218S plays a critical role in forming a hydrogen bond with the hydroxyl group of Ser104 around the MPP-sensitive Phe105-Met106 peptide bond of κ- casein and L287G is partially responsible for CSD accommodation in a suitable hydrophobic environment. These data suggested that T218S mutant could serve as a promising milk coagulant that contributes to an optimal flavor development in mature cheese.
Keywords: Aspartic proteinase, clotting activity, Mucor pusillus pepsin, proteolytic activity, site-directed mutagenesis, thermostability.
Graphical Abstract
Related Journals
Current Protein & Peptide Science
Related Books
Frontiers in Protein and Peptide Sciences
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