Wang H X, Ng T B
Department of Microbiology, China Agricultural University, and State Key Laboratory of Agrobiotechnology, Beijing, China.
Biochem Biophys Res Commun. 2004 Mar 5;315(2):450-4. doi: 10.1016/j.bbrc.2004.01.064.
A laccase with a novel N-terminal sequence, a low molecular mass of 43 kDa smaller than those of previously reported laccases, a pH optimum of 4, and a temperature optimum at 70 degrees C was isolated from fresh fruiting bodies of the mushroom Tricholoma giganteum. The activity of the enzyme rose steadily from 20 to 50 degrees C, increased very slowly from 50 to 70 degrees C, and fell slightly when the temperature was further increased to 80 degrees C. The activity of the laccase underwent little changes over the pH range 3.0-5.0. However, the enzyme activity dwindled to nothing after exposure to 100 degrees C for 10 min and when the ambient pH was 7 or above. The procedure used for purifying the enzyme included ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-cellulose, and FPLC-gel filtration on Superdex 75. The laccase was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and CM-cellulose. It inhibited HIV-1 reverse transcriptase with an IC(50) of 2.2 microM.
从巨大口蘑的新鲜子实体中分离出一种漆酶，其具有新颖的N端序列，分子量低至43 kDa，比先前报道的漆酶小，最适pH为4，最适温度为70℃。该酶的活性在20至50℃时稳步上升，在50至70℃时增加非常缓慢，当温度进一步升至80℃时略有下降。漆酶的活性在pH 3.0至5.0范围内变化不大。然而，在100℃下暴露10分钟以及环境pH为7或更高时，酶活性会降至零。纯化该酶的步骤包括在DEAE-纤维素上进行离子交换色谱、在Affi-凝胶蓝凝胶上进行亲和色谱、在CM-纤维素上进行离子交换色谱以及在Superdex 75上进行快速蛋白质液相色谱-凝胶过滤。该漆酶不吸附在DEAE-纤维素上，而是吸附在Affi-凝胶蓝凝胶和CM-纤维素上。它对HIV-1逆转录酶具有抑制作用，IC(50)为2.2 microM。
