Division of Applied Life Science (BK21 Plus), Graduate School, Gyeongsang National University, Jinju 660-701, Republic of Korea.
J Microbiol Biotechnol. 2014 Jul;24(7):969-78. doi: 10.4014/jmb.1401.01034.
The aprE2 gene with its prosequence from Bacillus subtilis CH3-5 was overexpressed in Escherichia coli BL21(DE3) by using plasmid pET26b(+). After IPTG induction, active and mature AprE2 was produced when cells were grown at 20°C, whereas inactive and insoluble enzyme was produced in a large amount when cells were grown at 37°C. The insoluble fraction was resuspended with 6 M guanidine-HCl and dialyzed against 2 M Tris-HCl (pH 7.0) or 0.5 M sodium acetate (pH 7.0) buffer. Then active AprE2 was regenerated and purified by a Ni-NTA column. Purified AprE2 from the soluble fraction had a specific activity of 1,069.4 ± 42.4 U/mg protein, higher than that from the renatured insoluble fraction. However, more active AprE2 was obtained by renaturation of the insoluble fraction. AprE2 was most stable at pH 7 and 40°C, respectively. The fibrinolytic activity of AprE2 was inhibited by PMSF, but not by EDTA and metal ions. AprE2 degraded Aα and Bβ chains of fibrinogen quickly, but not the γ-chain. AprE2 exhibited the highest specificity for N-succinyl-Ala-Ala-Pro-Phe-pNA. The Km and kcat/Km of AprE2 was 0.56 mM and 3.10 × 10(4) S(-1) M(-1), respectively.
AprE2 基因及其前导序列来自枯草芽孢杆菌 CH3-5，通过质粒 pET26b(+) 在大肠杆菌 BL21(DE3) 中过表达。当细胞在 20°C 下生长时，会产生有活性和成熟的 AprE2，而当细胞在 37°C 下生长时，会大量产生无活性和不溶性的酶。不溶性部分用 6 M 盐酸胍重悬，并在 2 M Tris-HCl(pH7.0) 或 0.5 M 乙酸钠(pH7.0) 缓冲液中透析。然后通过 Ni-NTA 柱再生和纯化有活性的 AprE2。从可溶部分纯化的 AprE2 的比活性为 1069.4±42.4 U/mg 蛋白，高于复性不溶部分的比活性。然而，通过复性不溶性部分获得了更多的有活性的 AprE2。AprE2 在 pH7 和 40°C 下分别最稳定。AprE2 的纤维蛋白溶解活性被 PMSF 抑制，但不受 EDTA 和金属离子的影响。AprE2 迅速降解纤维蛋白原的 Aα 和 Bβ 链，但不降解 γ 链。AprE2 对 N-琥珀酰-Ala-Ala-Pro-Phe-pNA 表现出最高的特异性。AprE2 的 Km 和 kcat/Km 分别为 0.56 mM 和 3.10×10(4) S(-1) M(-1)。
