化学修饰法表征BacillussmithiiT7产耐热菊粉酶催化活性中心的必需氨基酸残基.pdfVIP

第 30 卷 第 7 期 催 化 学 报 2009 年 7 月 Vol. 30 No. 7 Chinese Journal of Catalysis July 2009 文章编号: 0253-9837(2009)07-0673-06 研究论文: 673~678 化学修饰法表征 Bacillus smithii T7 产耐热菊粉酶 催化活性中心的必需氨基酸残基 刘 彬, 王静云, 包永明, 张 帆, 安利佳 大连理工大学环境与生命学院生物科学与工程系, 辽宁大连 116024 摘要：采用化学修饰法研究了史氏芽胞杆菌 Bacillus smithii T7 产耐热菊粉酶活性中心氨基酸残基, 发现该酶活性中心存在一个 组氨酸残基和一个谷氨酸 (或天冬氨酸) 残基. 修饰前后的酶动力学参数变化表明组氨酸残基参与了底物的结合和催化过程, 而 谷氨酸 (或天冬氨酸) 的羧基亲核攻击促使底物分解. 邹氏作图法证明酶活性中心存在两个必需的色氨酸残基, 荧光和圆二色光 谱研究表明色氨酸残基在酶的催化和酶的耐热性方面起重要作用. 关键词：菊粉酶; 化学修饰; 活性中心; 氨基酸残基; 催化 中图分类号：O643/Q55 文献标识码：A Characterization of Key Amino Acid Residues in Active Sites of Inulinase from Bacillus smithii T7 by Chemical Modification * LIU Bin, WANG Jingyun , BAO Yongming, ZHANG Fan, AN Lijia Department of Bioscience and Biotechnology, School of Environmental and Biological Science and Technology, Dalian University of Technology, Dalian 116024, Liaoning, China Abstract: The amino acid residues participating in the active site of the extracellular thermostable endo-inulinase from Bacillus smithii T7 were investigated by chemical modification. The results show that there are one histidine residue and one carboxylate residue in the active site. The kinetic study of the enzyme before and after modification by diethylpyrocarbonate and carbodiimide showed that one histidine resi- due might play a key role in substrate binding and catalysis, and one carboxylate residue located in the active site may act as a nucleophile base for the cleavage of substrate. The T