依赖新辅基PQQ甲醇脱氢酶的研究

依赖新辅基PQQ甲醇脱氢酶的研究 Studies on a New Prosthetic Group PQQ Dependent Methanol Dehydrogenase 赵永芳 ZHAO Yong fang 1 1 2 3 first-author 成汇 CHENG Hui 2 1 2 3 张经纬 ZHANG Jing wei 1 1 2 3 王银善 WANG Yin shan 2 1 2 3 武汉大学生命科学学院 武汉大学生命科学学院 1 College of Life Sciences, Wuhan University 1 College of Life Sciences, Wuhan University 中国科学院武汉病毒研究所 中国科学院武汉病毒研究所 2 Wuhan Institute of Virology, Academia Sinica 2 Wuhan Institute of Virology, Academia Sinica 武汉大学生命科学学院中国科学院武汉病毒研究所 武汉大学生命科学学院中国科学院武汉病毒研究所 生长在以甲醇作唯一碳源的甲基营养菌（Ｍｅｔｈｙｌｏｔｒｏｐｈｉｃｂａｃｔｅｒｉａ）Ｎｏ．２细胞，经超声波破碎，缓冲液抽提，ＤＥＡＥ－和ＣＭ－纤维素柱层析等纯化步骤，可得到纯化的甲醇脱氢酶（ＭＤＨ），其比活力为５．７ｕ／ｍｇ．该酶进行不连续聚丙烯酰胺凝胶电泳后，用相应的试剂分别染色，其蛋白质，酶活性和醌蛋白谱带均呈现一条迁移距离相同的主带．该酶由两个β亚基（６０ｋｂ）和两个α亚基（１０ｋｂ）构成，每个α亚基束缚一个新辅基ＰＱＱ（吡咯喹啉醌）．光谱分析显示，２９０ｎｍ有一肩峰，３４５ｎｍ有一特征峰，４００ｎｍ有一平台．这些数据表明，ＭＤＨ是一种依赖ＰＱＱ的醌酶 Growing on methanol as sole carbon source,the cells of methylotrophic bacteria NO.2 were passed following puricative steps:ultrasonic disruption,buffer solution extraction,column chromatographys on DEAE cellulose and on CM cellulose and so on.We could get purified methanol dehydrogenase（MDH） which specific activitywas 5.7 u/mg protein.Discontinuous polyacrylamide gel electrophoresis of the purified enzymes all showed one major band for protein,enzyme activity and quinoprotein which were stained by related reagents.The enzyme was composed of two α subunits（60 ku） and two β subunits（10 ku）.Every a subunits was bound to a new prothetic group PQQ（pyrroloquinoline quinone）.The spectrum analysis indicated that there was a shoulder peak at 290 nm,a featured at 345 nm,a platform at 400 nm.These data showed MDH was a PQQ dependent quinoenzyme. 甲醇脱氢酶 吡咯喹啉醌 甲基营养菌 methanol dehydrogenase pyrroloquinoline quinone methylotrophic bacteria Q554.9 国家自然科学基金(61672102) 1999-02-01 2021-04-01 2