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

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

Volume: 22 Issue: 7

Author(s): Jie Zhang, Yonghai Sun, Zhuolin Li, Quan Luo, Tiezhu Li and Tuoyi Wang

Affiliation:

Keywords: Aspartic proteinase, clotting activity, Mucor pusillus pepsin, proteolytic activity, site-directed mutagenesis, thermostability.

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.

Cite this article as:

Zhang Jie, Sun Yonghai, Li Zhuolin, Luo Quan, Li Tiezhu and Wang Tuoyi, Structure-Based Design of Mucor pusillus Pepsin for the Improved Ratio of Clotting Activity/Proteolytic Activity in Cheese Manufacture, Protein & Peptide Letters 2015; 22 (7) . https://dx.doi.org/10.2174/0929866522666150526154648