Structural Analysis of a Novel Aspartic-Type Endopeptidase from Moringa oleifera Seeds and Its Milk-Clotting Properties

Abstract

A novel aspartic-type endopeptidase was previously obtained from Moringa oleifera seeds; however, its specific milkclotting properties have remained unclear. Here, we used various biophysical and molecular simulation approaches for characterizing the structure and function of the aspartic-type endopeptidase. The endopeptidase was preferentially active toward κ-casein (CN) and hydrolyzed it more than calf rennet; however, its ability to hydrolyze α-CN and β-CN was weaker than that of calf rennet. The endopeptidase cleaved κ-CN at Gln135−Asp136 and generated a 15 588.18 Da peptide with 135 amino acids. We further simulated the docking complex of the endopeptidase and κ-CN and found out that they possibly combined with each other via hydrogen bonds. The flocculation reaction between the endopeptidase and κ-CN indicated that milk coagulation occurred within 60 min. Overall, our observations suggest that the aspartic-type endopeptidase can be a potential rennet alternative for cheese making.

Identification of Di/Tripeptide(s) With Osteoblasts Proliferation Stimulation Abilities of Yak Bone Collagen by in silico Screening and Molecular Docking

“…Also, the values of the α 2 chain were 2.467 and -2.733, respectively ( Figure 1B ). The larger positive value indicated the greater hydrophobicity, while the smaller negative value indicated the greater hydrophilicity ( 37 ). This result suggested that unwinding yak bone collagen-I could be soluble in water and hydrolyzed by proteases, which was consistent with previous studies ( 21 , 38 , 39 ).…”

Characterization of a Novel Milk-Clotting Aspartic Protease from Penicillium sp. and Structural Explanation for its High Milk-Clotting Index

“…High PA leads to low MCI, which directly affects cheese yield, flavor, and texture. 6,11,16 Given that calf chymosin is considered the mosteffective MCE, it is often compared with other alternative coagulants. 4,7 In this study, although the MCI of PsMCE was lower than that of calf chymosin, it was higher than the commercial MCEs from R. miehei, 4 M. pusillus, 19 and Cynara scolymus.…”

“…The PsMCE structure was mainly composed of β-sheets, which are consistent with the structure of aspartic proteases from other species. 16,21 The Ramachandran plot analysis of the selected PsMCE structure showed that over 92.7% of the regions residues were within the most favored, 6.1% of the residues were in the additional allowed region, 1.2% of the residues were in the generously allowed regions, and no residues were in the disallowed region (Figure 5B). These results indicate that the structural model of PsMCE is extremely of good quality and can be used for the following analysis.…”

“…pusillus, and C. parasitica have been used commercially in cheese production. ,, However, these fungal MCEs usually have higher thermostability, which extends the milk protein hydrolysis process during cheese maturation and forms bitter peptides. , Several researchers have discovered that Penicillium species are also important sources of MCEs . These enzymes usually have low thermostability, showing promising application in cheese production.…”

“…To the best of our knowledge, the proteolytic patterns of these milk-clotting proteases are closely related to their structural characteristics, especially in regions adjacent to the substrate-binding pocket. 16,21 Many crystal structures of milk-clotting aspartic proteases have been recorded in the PDB database (https:// www.rcsb.org/), including mammalian chymosin (4CMS and 4AA9), plant chymosin (1B5F and 5NFG), and fungal chymosin (3APR, 1MMP, and 2ASI). These structures are fundamental in defining the coagulating mechanism.…”