Study on Milk-Clotting Mechanism of Rennet-Like Enzyme from Glutinous Rice Wine: Proteolytic Property and the Cleavage Site on κ-Casein

Chinese Royal cheese, an ancient and attractive dairy product now in China, is made from milk coagulated with glutinous rice wine. In this paper, it was mainly studied on the proteolytic property toward proteins of bovine milk including caseins (CN) and whey proteins and the cleavage bond on the κ-CN of rennet-like enzyme purified from glutinous rice wine by ion-exchange chromatography. Compared with whey protein, the rennet-like protease has substrate specificity toward CN but with different hydrolysis degrees among κ-, α-, and β-CN, and the α-CN was almost completely degraded, whereas κ- and β-CN partly showed hydrolysis in 12 h. The analysis for enzyme digestion by electrospray tandem mass spectrometry, Q-TOF2, and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry revealed that the cleavage of protease from glutinous rice wine on κ-CN mainly happens at the Thr94-Met95 bond, which is different from the most chymosin-sensitive bond, Phe105-Met106.