Bekalu Zelalem Eshetu, Madsen Claus Krogh, Dionisio Giuseppe, Brinch-Pedersen Henrik
Department of Molecular Biology and Genetics, Research Center Flakkebjerg, Aarhus University, Slagelse, Denmark.
PLoS One. 2017 May 4;12(5):e0176838. doi: 10.1371/journal.pone.0176838. eCollection 2017.
In the current study, we report for the first time that grain components of barley, rice, wheat and maize can inhibit the activity of Aspergillus ficuum phytase. The phytase inhibition is dose dependent and varies significantly between cereal species, between cultivars of barley and cultivars of wheat and between Fusarium graminearum infected and non-infected wheat grains. The highest endpoint level of phytase activity inhibition was 90%, observed with grain protein extracts (GPE) from F. graminearum infected wheat. Wheat GPE from grains infected with F. graminearum inhibits phytase activity significantly more than GPE from non-infected grains. For four barley cultivars studied, the IC50 value ranged from 0.978 ± 0.271 to 3.616 ± 0.087 mg×ml-1. For two non-infected wheat cultivars investigated, the IC50 values were varying from 2.478 ± 0.114 to 3.038 ± 0.097 mg×ml-1. The maize and rice cultivars tested gaveIC50 values on 0.983 ± 0.205 and 1.972 ± 0.019 mg×ml-1, respectively. After purifying the inhibitor from barley grains via Superdex G200, an approximately 30-35 kDa protein was identified. No clear trend for the mechanism of inhibition could be identified via Michaelis-Menten kinetics and Lineweaver-Burk plots. However, testing of the purified phytase inhibitor together with the A. ficuum phytase and the specific protease inhibitors pepstatin A, E64, EDTA and PMSF revealed that pepstatin A repealed the phytase inhibition. This indicates that the observed inhibition of A. ficuum phytase by cereal grain extracts is caused by protease activity of the aspartic proteinase type.
在本研究中，我们首次报道大麦、水稻、小麦和玉米的谷物成分能够抑制泡盛曲霉植酸酶的活性。植酸酶抑制作用呈剂量依赖性，且在不同谷物种类之间、大麦品种之间、小麦品种之间以及禾谷镰刀菌感染和未感染的小麦籽粒之间存在显著差异。植酸酶活性抑制的最高终点水平为90%，这是在用禾谷镰刀菌感染的小麦的谷物蛋白提取物（GPE）观察到的。来自感染禾谷镰刀菌的籽粒的小麦GPE对植酸酶活性的抑制作用明显强于来自未感染籽粒的GPE。对于所研究的四个大麦品种，IC50值范围为0.978±0.271至3.616±0.087mg×ml-1。对于所研究的两个未感染的小麦品种，IC50值在2.478±0.114至3.038±0.097mg×ml-1之间变化。所测试的玉米和水稻品种的IC50值分别为0.983±0.205和1.972±0.019mg×ml-1。通过Superdex G200从大麦籽粒中纯化抑制剂后，鉴定出一种约30 - 35kDa的蛋白质。通过米氏动力学和林-贝氏图无法确定抑制机制的明显趋势。然而，将纯化的植酸酶抑制剂与泡盛曲霉植酸酶以及特定的蛋白酶抑制剂胃蛋白酶抑制剂A、E64、EDTA和PMSF一起测试发现，胃蛋白酶抑制剂A消除了植酸酶抑制作用。这表明谷物提取物对泡盛曲霉植酸酶的观察到的抑制作用是由天冬氨酸蛋白酶类型的蛋白酶活性引起的。
