title: Structure-Based Design of Mucor pusillus Pepsin for the Improved Ratio of Clotting Activity/Proteolytic Activity in Cheese Manufacture.
abstract: 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 Ser(104) around the MPP-sensitive Phe(105)-Met(106) 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.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.forename: Yonghai
authors.initials: Y
authors.lastname: Sun
authors.forename: Zhuolin
authors.initials: Z
authors.lastname: Li
authors.forename: Quan
authors.initials: Q
authors.lastname: Luo
authors.affiliation: Institute of Agro-food Technology, Jilin Academy of Agricultural Sciences, 130033, Changchun, China. tiezhu_li@163.com.
authors.forename: Tiezhu
authors.initials: T
authors.lastname: Li
authors.forename: Tuoyi
authors.initials: T
authors.lastname: Wang
mesh_terms: D000818:Animals; D002122:Calcium Chloride / Q000494:pharmacology; D002364:Caseins / Q000378:metabolism; D002611:Cheese; D004305:Dose-Response Relationship, Drug; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D008892:Milk / Q000737:chemistry; D008958:Models, Molecular; D009089:Mucor / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D010434:Pepsin A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D059748:Proteolysis / Q000187:drug effects; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature
chemical_list: D002364:Caseins; D008670:Metals; D012965:Sodium Chloride; D010434:Pepsin A; D002122:Calcium Chloride