Zhao Shuang, Rong Cheng-Bo, Kong Chang, Liu Yu, Xu Feng, Miao Qian-Jiang, Wang Shou-Xian, Wang He-Xiang, Zhang Guo-Qing
Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing Engineering Research Center for Edible Mushroom, Beijing 100097, China.
State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University, Beijing 100193, China.
Biomed Res Int. 2014;2014:417461. doi: 10.1155/2014/417461. Epub 2014 Aug 28.
A novel laccase was isolated and purified from fermentation mycelia of mushroom Coprinus comatus with an isolation procedure including three ion-exchange chromatography steps on DEAE-cellulose, CM-cellulose, and Q-Sepharose and one gel-filtration step by fast protein liquid chromatography on Superdex 75. The purified enzyme was a monomeric protein with a molecular weight of 64 kDa. It possessed a unique N-terminal amino acid sequence of AIGPVADLKV, which has considerably high sequence similarity with that of other fungal laccases, but is different from that of C. comatus laccases reported. The enzyme manifested an optimal pH value of 2.0 and an optimal temperature of 60°C using 2,2'-azinobis(3-ethylbenzothiazolone-6-sulfonic acid) diammonium salt (ABTS) as the substrate. The laccase displayed, at pH 2.0 and 37°C, K(m) values of 1.59 mM towards ABTS. It potently suppressed proliferation of tumor cell lines HepG2 and MCF7, and inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) with an IC50 value of 3.46 μM, 4.95 μM, and 5.85 μM, respectively, signifying that it is an antipathogenic protein.
从毛头鬼伞发酵菌丝体中分离纯化出一种新型漆酶，分离过程包括在DEAE - 纤维素、CM - 纤维素和Q - 琼脂糖上进行三步离子交换色谱，以及通过Superdex 75快速蛋白质液相色谱进行一步凝胶过滤。纯化后的酶是一种分子量为64 kDa的单体蛋白。它具有独特的N端氨基酸序列AIGPVADLKV，与其他真菌漆酶的序列相似度相当高，但与已报道的毛头鬼伞漆酶不同。以2,2'-联氮双(3-乙基苯并噻唑啉-6-磺酸)二铵盐(ABTS)为底物时，该酶的最适pH值为2.0，最适温度为60°C。在pH 2.0和37°C条件下，该漆酶对ABTS的K(m)值为1.59 mM。它能有效抑制肿瘤细胞系HepG2和MCF7的增殖，并抑制人类免疫缺陷病毒1型(HIV-1)逆转录酶(RT)，IC50值分别为3.46 μM、4.95 μM和5.85 μM，表明它是一种抗病原体蛋白。
