Department of Biotechnology, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany.
PLoS One. 2013 Jul 19;8(7):e70055. doi: 10.1371/journal.pone.0070055. Print 2013.
The proline-specific X-prolyl dipeptidyl aminopeptidase (PepX; EC 3.4.14.11) and the general aminopeptidase N (PepN; EC 3.4.11.2) from Lactobacillus helveticus ATCC 12046 were produced recombinantly in E. coli BL21(DE3) via bioreactor cultivation. The maximum enzymatic activity obtained for PepX was 800 µkat(H-Ala-Pro-pNA) L(-1), which is approx. 195-fold higher than values published previously. To the best of our knowledge, PepN was expressed in E. coli at high levels for the first time. The PepN activity reached 1,000 µkat(H-Ala-pNA) L(-1). After an automated chromatographic purification, both peptidases were biochemically and kinetically characterized in detail. Substrate inhibition of PepN and product inhibition of both PepX and PepN were discovered for the first time. An apo-enzyme of the Zn(2+)-dependent PepN was generated, which could be reactivated by several metal ions in the order of Co(2+)>Zn(2+)>Mn(2+)>Ca(2+)>Mg(2+). PepX and PepN exhibited a clear synergistic effect in casein hydrolysis studies. Here, the relative degree of hydrolysis (rDH) was increased by approx. 132%. Due to the remarkable temperature stability at 50°C and the complementary substrate specificities of both peptidases, a future application in food protein hydrolysis might be possible.
瑞士乳杆菌 ATCC 12046 的脯氨酰特异性 X-脯氨酰二肽基氨基肽酶（PepX；EC 3.4.14.11）和一般氨基肽酶 N（PepN；EC 3.4.11.2）通过生物反应器培养在大肠杆菌 BL21(DE3)中被重组生产。获得的 PepX 的最大酶活为 800 µkat(H-Ala-Pro-pNA) L(-1)，比以前发表的值高约 195 倍。据我们所知，PepN 是首次在大肠杆菌中高水平表达的。PepN 的活性达到 1,000 µkat(H-Ala-pNA) L(-1)。经过自动色谱纯化后，详细地对两种肽酶进行了生化和动力学表征。首次发现 PepN 的底物抑制和两种肽酶的产物抑制。生成了依赖 Zn(2+)的 PepN 的脱辅基酶，该酶可以被 Co(2+)>Zn(2+)>Mn(2+)>Ca(2+)>Mg(2+)的顺序中的几种金属离子重新激活。在酪蛋白水解研究中，PepX 和 PepN 表现出明显的协同作用。在此，相对水解度（rDH）增加了约 132%。由于在 50°C 时具有显著的热稳定性和两种肽酶互补的底物特异性，因此它们未来可能在食品蛋白水解中得到应用。
