耐热碱性磷酸酶功能结构域的进一步定位  

Further Location of the Functional Domain on Thermostable Alkaline Phosphatase

作　　者：郁锋[1] 许晓风[1] 

机构地区：[1]南京师范大学生命科学学院,南京210097

出　　处：《中国生物化学与分子生物学报》2004年第1期89-94,共6页Chinese Journal of Biochemistry and Molecular Biology

摘　　要：为了进一步精确定位耐热碱性磷酸酶 (TAP)的功能结构域和建立酶功能结构域定位的有效方法 ,在对TAP进行序列相容性比对及二级结构统计预测的基础上 ,以二级结构单元的完整性为依据对其功能结构域进行定位 .TAP的功能结构域在 35～ 4 76氨基酸之间 ,记为TAPN3 4C2 5(TAP的N端去掉 34个氨基酸而C端去掉 2 5个氨基酸 ) ,比盛小禹等定位的少 15个氨基酸 .克隆、表达实验结果证明了TAPN3 4C2 5的酶学活性 ,其最适反应温度为 72℃ ,比TAPND2 7(N端去除了 2 7个信号肽氨基酸的TAP)上升了 7℃ ,而最高耐受温度为 83℃ ,比TAPND2 7下降了 16℃ .实验结果表明 ,以二级结构预测结果进行酶功能结构域定位是一种更为精确的方法 .To locate the functional domain of the thermostable alkaline phosphatase (TAP) more precisely and to explore a new efficient location method, the secondary structure was predicted on Network Protein Sequence Analysis (NPS@) server and its functional domain was determined as TAP N34C25 (with 34 amino acids deleted at N terminal and 25 at C terminal) according to the intactness of its two terminal secondary structures. TAP N34C25 was 15 amino acid residues shorter than the functional domain TAPND27 determined by Sheng Xiao-yu et al. The cloning and expression have proved that TAP N34C25 did possess the expected enzymatic activity. Its optimal reaction temperature was 72℃, with 7℃ higher than TAPND27, and its highest tolerable temperature 83℃, with 16℃ lower than TAPND27. A more exact TAP’s functional domain was obtained.The results showed that two terminal secondary structure-determined method was a more efficient for the functional domain location of the thermostable enzyme.

关 键 词：耐热碱性磷酸酶 功能结构域 序列相容性比对 二级结构统计预测 

分 类 号：Q55[生物学—生物化学]