Structure-Based Design of Mucor pusillus Pepsin for the Improved Ratio of Clotting Activity/Proteolytic Activity in Cheese Manufacture

文献类型： 外文期刊

第一作者： Zhang, Jie

作者： Zhang, Jie;Sun, Yonghai;Luo, Quan;Zhang, Jie;Li, Zhuolin;Li, Tiezhu;Wang, Tuoyi

作者机构：

关键词： Aspartic proteinase;clotting activity;Mucor pusillus pepsin;proteolytic activity;site-directed mutagenesis;thermostability

期刊名称：PROTEIN AND PEPTIDE LETTERS （ 影响因子：1.89； 五年影响因子：1.528 ）

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收录情况： SCI

摘要： Previous theoretical studies have determined the intermolecular interactions between Mucor pusillus pepsin (MPP) and the key domain of kappa-casein, with the aim to understand the mechanism of milk clotting in the specific hydrolysis of kappa-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 kappa-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 kappa-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.

分类号： Q51

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