脯氨酸存在下酪蛋白ACE抑制肽的Plastein反应修饰

利用枯草杆菌碱性蛋白酶水解酪蛋白制备ACE 抑制肽，其IC50 为47.1μg/mL；采用相同酶催化ACE 抑制肽和脯氨酸进行Plastein 反应，对ACE 抑制肽进一步修饰，并用响应面分析法优化反应条件。在ACE 抑制肽质量分数为35%，反应时间为6h，以反应体系游离氨基减少量为指标，得到适宜的反应条件为：温度为47.8℃、脯氨酸比例为0.54、酶添加量为9.5kU/g pro，此条件下体系游离氨基减少量约195.7μmol/g pro。在适宜条件下改变反应时间对酪蛋白ACE 抑制肽进行同程度的Plastein 反应修饰，制备出6 个修饰程度不同的多肽混合物并测定它们的ACE 抑制活性、计算其IC50 值。结果表明：修饰产物的ACE 抑制活性随修饰程度的增加不规则变化，当反应体系的游离氨基减少量为195.7μmol/g pro 时，修饰产物的IC50 降低至0.2μg/mL。

Plastein Reaction-based Modification of ACE Inhibitory Peptides Derived from Casein in the Presence of Proline

ACE inhibitory peptides with IC50 value of 47.1 μg/mL were prepared by hydrolyzing casein with alkaline protease from Bacillus subtilis and further modified by plastein reaction under the catalysis of the same protease in the presence of proline. The optimal conditions for plastein reaction were determined using response surface methodology at the fixed concentration of 35% (m/m) peptides and the reaction time of 6 h as follows: reaction temperature 47.8 ℃, molar ratio of proline to total free amino groups in ACE inhibitory peptides derived from casein 0.54, and addition level of alkaline protease 9.5 kU/g proteins. Under these optimal reaction conditions, a maximal decrease of free amino groups in reaction mixture of 195.7 μmol/g proteins was obtained. Six modified products with different modification degrees were prepared by adjusting reaction time. ACE inhibitory activities and IC50 values of these modified peptides were also analyzed. Results indicated that ACE inhibitory activity of modified products revealed irregular change with increasing modification degree. When the reaction mixture had a decrease of free amino groups of 195.7 μmol/g proteins, there was a decline of IC50 value of modified products to 0.2 μg/mL.