从生姜中分离得到具有凝乳活力的生姜蛋白酶,采用Urea SDS-PAGE、RP-HPLC和MALDI-TOF/MS等方法分析生姜蛋白酶对酪蛋白单体和脱脂乳中酪蛋白的水解作用。结果表明,生姜蛋白酶水解κ-酪蛋白生成的主要产物比较稳定,不会被进一步水解。温度高于60℃时生成κ-CN(f 1-90)和κ-CN(f 1-102)两个末端疏水性肽段,其次为κ-CN(f 1-121),温度较低时,还生成大量分子量高于κ-CN(f 1-121)的产物。在脱脂乳体系中,生姜蛋白酶水解脱脂乳的κ-酪蛋白,而对αS1-、αS2-和β-酪蛋白没有显著的水解作用。主要裂解κ-酪蛋白的Thr121-Ile122键,生成疏水性N末端肽段κ-CN(f 1-121)。这一结果表明生姜蛋白酶凝乳的主要机理是水解κ-酪蛋白Thr121-Ile122肽键,破坏了酪蛋白微粒的稳定性,促使酪蛋白形成凝胶。

Worldwidely,substitution of plant chymosin for calf rennet has always been the hot topic of dairy science. Ginger protease with milk coagulating activity was isolated from ginger root. The hydrolysis of casein isolates and casein in skim milk was analyzed by Urea SDS-PAGE,RP-HPLC,and MALDI-TOF / MS. The results showed that the primary hydrolyzates of κ-casein proteolyzed by ginger protease were stable,and didn't undergo further proteolysis. At temperatures higher than 60℃,the main products were two N terminal hydrophobic peptides κ-CN( f 1-90) and κ-CN( f 1-102) with κ-CN( f 1-121) to a lesser amount. While at low temperatures,large amount of peptides with molecular weights higher than that of κ-CN( f 1-121) were formed. Ginger exhibited proteolysis on κ-casein,but didn't show significant hydrolysis on α S1-,α S2-,and β-casein in skim milk system. Proteolysis of κ-casein occurred at Thr121-Ile122,and generated a hydrophobic N terminal peptide κ-CN( f 1-121). The milk coagulating mechanism of ginger protease was demonstrated to be that the κ-casein was hydrolyzed at peptide bond of Thr121-Ile122,which destroyed the stability of casein micelle and formed gels.