Structure-based design of Mucor pusillus pepsin for the improved ratio of clotting activity/proteolytic activity in cheese manufacture

Previous theoretical studies have determined the intermolecular interactions between mucor pusillus pepsin (mpp) and the key domain of κ-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. © 2015 bentham science publishers.