Label-free quantitative proteomic analysis of the biological functions of Moringa oleifera seed proteins provides insights regarding the milk-clotting proteases
Label-free quantitative proteomic analysis of the biological functions of Moringa oleifera seed proteins provides insights regarding the milk-clotting proteases
复制标题
对辣木种子蛋白生物功能的无标记定量蛋白质组学分析提供了有关凝乳蛋白酶的见解
DOI：
10.1016/j.ijbiomac.2019.12.070
复制
发表时间：
2019
影响因子：
8.2
通讯作者：
Aixiang Huang
中科院分区：
文献类型：
--
作者：
Xuefeng Wang;Yanan Shi;Rong He;Bing Li;Aixiang Huang
本研究利用无标记定量蛋白质组学技术对M. oleiferaseed蛋白质，导致牛奶凝固蛋白酶的鉴定。总共鉴定了921种蛋白质，并且分子量在30-50 kDa范围内的蛋白质是丰富的。所鉴定的蛋白质主要参与与碳水化合物和蛋白质代谢相关的催化活性和代谢过程，其中，所观察到的分子量范围内的蛋白酶可能是先前报道的凝乳活性的原因。纯化得到分子量为45，517 Da的天冬氨酸型内肽酶。使用硫酸铵沉淀、超滤和制备型高效液相色谱法纯化oleiferaseeds，并使用液相色谱-质谱（LC-MS）/MS进行表征。基因本体（GO）和京都基因和基因组百科全书（KEGG）分析揭示纯化的蛋白酶表现出水解酶活性并参与几种代谢途径，这进一步证实了蛋白质组学分析可以帮助凝乳蛋白酶的纯化。该酶的最适反应温度为60 °C，最适pH为5.0。该蛋白酶具有较高的热稳定性和较好的pH稳定性，可用于乳品工业。
In this study, label-free quantitative proteomics was used to investigate the biological functions ofM. oleiferaseed proteins, which resulted in the identification of milk-clotting proteases. In total, 921 proteins were identified, and proteins within the molecular weight range of 30–50 kDa were abundant. The identified proteins were mainly involved in catalytic activity and metabolic processes associated with carbohydrate and protein metabolism, among which, proteases in the observed molecular weight range could possibly be responsible for the previously reported milk-clotting activity. An aspartic-type endopeptidase with molecular mass of 45,517 Da was purified fromM. oleiferaseeds using ammonium sulfate precipitation, ultrafiltration, and preparative high performance liquid chromatography, and was characterized using liquid chromatography-mass spectrometry (LC-MS)/MS. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the purified protease exhibited hydrolase activity and was involved in several metabolic pathways, which further confirmed that proteomic analysis can assist in the purification of the milk-clotting protease. The optimal temperature and pH required for protease activity were 60 °C and 5.0, respectively. The high thermal stability and good pH stability of the protease indicated that it can be used in the dairy industry.