Kurosawa N, Fukuda K, Itoh Y H, Horiuchi T
Department of Bioengineering, Faculty of Engineering, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, Japan.
Curr Microbiol. 2000 Jan;40(1):57-60. doi: 10.1007/s002849910011.
Thermostable acid phosphatase (APase) from thermoacidophilic archaeon Sulfolobus acidocaldarius was isolated, partially purified, and characterized. The optimum pH and temperature of the enzyme for p-nitrophenylphosphate (pNPP) as a substrate were 5.0 and 70 degrees C, respectively. The apparent K(m) value was 1.9 mM. This APase showed a native molecular mass of 20 kDa on a gel filtration chromatography. Of the APase activity, 60% remained after 60 min of heat treatment at 75 degrees C. To confirm whether the APase is active in the monomeric form, we attempted to elute the enzyme from SDS-polyacrylamide gels with Disk electrophoresis apparatus and renature the enzyme. The APase activity was recovered up to 50% in the 14- to 35-kDa range, and maximum around 25 kDa. These results suggest that this APase is monomeric protein.
从嗜热嗜酸古菌嗜酸硫化叶菌中分离、部分纯化并表征了热稳定酸性磷酸酶（APase）。以对硝基苯磷酸酯（pNPP）为底物时，该酶的最适pH和温度分别为5.0和70℃。表观K(m)值为1.9 mM。在凝胶过滤色谱中，这种APase的天然分子量为20 kDa。在75℃热处理60分钟后，APase活性仍保留60%。为了确认APase是否以单体形式具有活性，我们尝试用圆盘电泳装置从SDS-聚丙烯酰胺凝胶中洗脱该酶并使其复性。在14至35 kDa范围内，APase活性恢复至50%，在约25 kDa处达到最大值。这些结果表明这种APase是单体蛋白。
