MOE Key Laboratory of Arid and Grassland Ecology, School of Life Science, Lanzhou University, Lanzhou 730000, P. R. China.
J Dairy Sci. 2011 Aug;94(8):3928-35. doi: 10.3168/jds.2010-3752.
The experiment was conducted to investigate the exopeptidase classes in alfalfa (Medicago sativa L.) leaves, and to determine their contribution to the formation of nonprotein nitrogen (NPN) components during ensiling. Six classes of inhibitors that included bestatin (aminopeptidase inhibitor), potato carboxypeptidase inhibitor (PCI, carboxypeptidase inhibitor), 1,10-phenanthroline (dipeptidase inhibitor), diprotin A (dipeptidyl-peptidase inhibitor), butabindide (tripeptidyl-peptidase inhibitor), and dipeptide Phe-Arg (peptidyl-dipeptidase inhibitor) were used. To determine the contribution of each exopeptidase to the formation of NPN products, aqueous extracts of fresh alfalfa were fermented to imitate the proteolytic process of ensiled alfalfa and to ensure that each class of exopeptidase inhibitor would have immediate contact with the proteases in the alfalfa extract. Five classes of exopeptidases; namely, aminopeptidase, carboxypeptidase, dipeptidase, dipeptidyl-peptidase, and tripeptidyl-peptidase, were shown to be present in alfalfa leaves, each playing a different role in alfalfa protein degradation. Aminopeptidase, carboxypeptidase, and dipeptidase were the main exopeptidases contributing to the formation of NH(3)-N. Among the 5 exopeptidases, tripeptidyl-peptidase appeared to be the principal exopeptidase in hydrolyzing forage protein into peptides, whereas carboxypeptidase and dipeptidase appeared to be more important in contributing to the formation of amino acid-N. Dipeptidyl-peptidase and tripeptidyl-peptidase did not play a role in the formation of NH(3)-N or amino acid-N. Dipeptidase, carboxypeptidase, and tripeptidyl-peptidase were the principal exopeptidases for hydrolyzing forage protein into NPN during ensilage, and treatment with a mixture of the 5 inhibitors reduced the total NPN concentration in the fermented alfalfa extract to about 45% of that in the control after 21 d of fermentation.
该实验旨在研究紫花苜蓿（Medicago sativa L.）叶片中的外肽酶种类，并确定其在青贮过程中形成非蛋白氮（NPN）成分的贡献。实验使用了 6 种抑制剂，包括苯并噻二唑（氨基肽酶抑制剂）、马铃薯羧肽酶抑制剂（PC1，羧肽酶抑制剂）、1,10-邻菲咯啉（二肽酶抑制剂）、二肽基丙氨酸（二肽基肽酶抑制剂）、丁酰苯丙氨酸（三肽基肽酶抑制剂）和二肽基苯丙氨酸-精氨酸（肽基二肽酶抑制剂）。为了确定每种外肽酶对 NPN 产物形成的贡献，将新鲜紫花苜蓿的水提取物进行发酵，以模拟青贮紫花苜蓿的蛋白水解过程，并确保每种外肽酶抑制剂类都能与苜蓿提取物中的蛋白酶立即接触。结果表明，紫花苜蓿叶片中存在 5 种外肽酶，即氨基肽酶、羧肽酶、二肽酶、二肽基肽酶和三肽基肽酶，它们在苜蓿蛋白降解中发挥不同的作用。氨基肽酶、羧肽酶和二肽酶是导致 NH3-N 形成的主要外肽酶。在 5 种外肽酶中，三肽基肽酶似乎是将饲料蛋白水解成肽的主要外肽酶，而羧肽酶和二肽酶似乎在形成氨基酸-N 方面更为重要。二肽基肽酶和三肽基肽酶在 NH3-N 或氨基酸-N 的形成中不起作用。二肽酶、羧肽酶和三肽基肽酶是青贮过程中将饲料蛋白水解成 NPN 的主要外肽酶，用 5 种抑制剂的混合物处理后，发酵 21 天后，发酵的紫花苜蓿提取物中的总 NPN 浓度降低到对照的约 45%。
