State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University, Beijing, China.
J Microbiol Biotechnol. 2011 Mar;21(3):256-62.
A 20.9-kDa metalloprotease was isolated from dried fruiting bodies of the wild basidiomycete mushroom Lepista nuda. The N-terminal amino acid sequence of the protease was seen to be ATFVLTAATNTLFTA, thus displaying no similarity with the sequences of previously reported metalloproteases. The protease was purified using a procedure that entailed ion-exchange chromatography on CM-Cellulose, Q-Sepharose, and Mono S, and FPLC-gel filtration on Superdex 75. The protease functioned at an optimum pH of 7.0 and an optimum temperature of 50 degrees C. It was also noted that the protease demonstrated a proteolytic activity of 1,756 U/mg toward casein. The Km of the purified protease toward casein was 6.36 mg/ml at a pH of 7.0 and with a temperature of 37 degrees C, whereas the Vmax was 9.11 microgram ml(-1) min(-1). The activity of the protease was adversely affected by EDTA-2Na, suggesting that it is a metalloprotease. PMSF, EGTA, aprotinin, and leupeptin exerted no striking inhibitory effect. The activity of the protease was enhanced by Fe2+, but was curtailed by Cd2+, Cu2+, Hg2+, Pb2+, Zn2+, and Fe3+ ions. The protease also exhibited inhibitory activity against HIV-1 reverse transcriptase with an IC50 value of 4.00 micrometer. The IC50 values toward hepatoma Hep G2 and leukemia L1210 cells in vitro were 4.99 micrometer and 3.67 micrometer, respectively.
从野生担子菌蘑菇 Lepista nuda 的干燥子实体中分离出一种 20.9 kDa 的金属蛋白酶。该蛋白酶的 N 末端氨基酸序列为 ATFVLTAATNTLFTA，因此与先前报道的金属蛋白酶序列没有相似性。该蛋白酶通过 CM-Cellulose、Q-Sepharose 和 Mono S 的离子交换层析以及 Superdex 75 的 FPLC 凝胶过滤进行纯化。该蛋白酶在最佳 pH 值为 7.0 和最佳温度为 50°C 时发挥作用。还注意到该蛋白酶对酪蛋白具有 1,756 U/mg 的蛋白水解活性。在 pH 值为 7.0 和温度为 37°C 时，纯化的蛋白酶对酪蛋白的 Km 为 6.36 mg/ml，而 Vmax 为 9.11 microgram ml(-1) min(-1)。该蛋白酶的活性受到 EDTA-2Na 的不利影响，表明它是一种金属蛋白酶。PMSF、EGTA、抑肽酶和亮抑蛋白酶对其没有明显的抑制作用。Fe2+ 增强了该蛋白酶的活性，但 Cd2+、Cu2+、Hg2+、Pb2+、Zn2+ 和 Fe3+ 离子则抑制了其活性。该蛋白酶还对 HIV-1 逆转录酶表现出抑制活性，IC50 值为 4.00 微米。体外对肝癌 Hep G2 和白血病 L1210 细胞的 IC50 值分别为 4.99 微米和 3.67 微米。
