辣木凝乳酶水解酪蛋白位点及其酪蛋白磷酸肽、酪蛋白糖巨肽

基于十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，液相色谱-串联质谱法研究辣木凝乳酶对酪蛋白的酶切位点和水解特性，并分析该凝乳酶水解产生的酪蛋白糖巨肽和酪蛋白磷酸肽。结果表明，辣木凝乳酶酶切κ-酪蛋白的Arg 93-His 94位点导致凝乳，区别于其他已报道的凝乳酶，具有明显特异性。酶作用下κ-酪蛋白的米氏常数Km=0.49 mg/mL，最大反应速率Vm=45.2 U/min，有效促进酪蛋白胶束的聚集和凝乳。该酶对α-、β-、κ-酪蛋白产生不同程度的水解，对α-酪蛋白的水解速度最快，说明其适用于软质奶酪的加工。以酪蛋白为底物，通过钡-乙醇沉淀法得到酪蛋白磷酸肽（casein phosphopeptides，CPPs），产率为15.87%，得到的CPPs可以使钙沉淀推迟23 min，有利于钙离子被肠道有效吸收。以水牛乳为底物时，乳凝固后析出的乳清中含酪蛋白糖巨肽6.61 mg/mL，糖基化程度477.527 μg/mg。研究为新型植物凝乳酶的研发，及其在奶酪、酪蛋白活性肽等产品的应用提供科学依据。

Hydrolysis Site of Casein by Milk-Clotting Protease from Moringa oleifera Lam. and Preparation and Evaluation of Casein Phosphopeptide and Casein Glycomacropeptide

In this study, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to explore the cleavage site and hydrolysis characteristics of casein by a milk-clotting protease from Moringa oleifera Lam. Then, casein phosphopeptide and casein glycomacropeptide in the resulting hydrolysate were analyzed. The results showed that the protease could specifically cleave κ-casein at the Arg93-His94 site leading to milk clotting, which is distinct from other reported chymosins. The Michaelis constant (Km) and maximum reaction rate (Vm) of the milk-clotting protease were 0.49 mg/ml and 45.2 U/min, respectively, and it could promote the aggregation and coagulation of casein micelles. The enzyme could hydrolyze α, β and κ-casein to different extents and exhibit the fastest hydrolysis rate toward α-casein, indicating that it was suitable for the processing of soft cheese. Casein phosphopeptide (CPP) was obtained by barium-ethanol precipitation method with casein as the substrate, with a yield of 15.87%. CPP could delay the precipitation of calcium by 23 min, which was beneficial to the intestinal absorption of calcium. When buffalo milk was used as the substrate, the concentration of casein glycomacropeptide (CGMP) was 6.61 mg/mL in whey, and the degree of glycosylation was 477.527 μg/mg. This study provides an important theoretical basis for the development and application of new plant chymosins in cheese and casein-derived bioactive peptides.