耐热碱性磷酸酯酶的功能结构域的定位

 为了确定耐热碱性磷酸酯酶 (TAPND2 7)发挥活性所必需的功能结构域 ,通过 PCR介导的诱变缺失 ,得到了 N端分别缺失 8、1 6、2 5个氨基酸的 3个缺失体 p TAPN8、p TAPN1 6和p TAPN2 5以及 C端分别缺失 1 0和 30个氨基酸的两个缺失体 p TAPC1 0和 p TAPC30 .经表达和活性测定 ,发现 p TAPN8和 TAPC1 0保持了较高的活性而其余 3个缺失体则失去酶活性 .据此 ,TAPND2 7的活性区域被定位在 8～ 465氨基酸之间 .在分离纯化的基础上测定了一些酶学性质 .发现 TAPN8和 TAPC1 0的比活没有大的改变 ,Tm 下降了 5.5℃ ;TAPN8的最适反应温度上升了1 0℃ .结果提示了 N端和 C端的这些氨基酸残基对热稳定性有一定的贡献 ,N端氨基酸残基还对酶的亲热性有贡献 .

Location on the Functional Domain of Thermostable Alkaline Phosphatase

In order to determine the functional domain of thermostable alkaline phosphatase(TAPND27),three truncators of pTAPN8,pTAPN16 and pTAPN25 with deletions of 8,16 and 25 amino acids at N|terminal and two truncators of pTAPC10 and pTAPC30 with deletions of 10 and 30 amino acids at C|terminal were obtained through PCR|mediated site|directed mutagenesis.It was found that TAPN8 and TAPC10 maintained their enzymatic activity meanwhile the other three lost their enzymatic activity by expression and enzymatic activity assaying.It was indicated that the essential region for activity of TAPND27 was located between amino acid residues 8 and 465.TAPN8 and TAPC10 were purified and some of their enzymatic characteristics were determined.It was found that their specific activity changed a little while their T m descended 5 5℃ and the optinal reaction temperature of TAPN8 ascended 10℃.The result implied that the amino acids at C| or N|terminal of TAPND27 partially contributed to its thermostability and those at N|terminal also devoted to its thermophilicity.