无机磷酸盐对耐热碱性磷酸酶耐热性的影响

 为研究体外磷酸根离子对嗜热菌 (Thermussp .30 4 1)碱性磷酸酶TAPND2 7耐热性的影响 ,通过克隆于高表达质粒上编码TAPND2 7的基因在E .coli中的诱导表达和蛋白产物的纯化 ,获得了电泳纯的TAPND2 7.耐热性实验表明磷酸根离子能显著提高该酶的热稳定性 ,在 10mmol L磷酸根离子存在下 ,TAPND2 7的Tm 值从 91℃上升到了 97℃ ;酶的动力学实验证实无机磷酸盐 (Pi)对TAP ND2 7的酶活性有可逆竞争性抑制作用 ,Ki 值为 0 99× 10 -4 mol L .上述结果说明 ,TAPND2 7耐热性的提高是由于磷酸根离子对TAPND2 7活性位点的竞争性结合 ,这种结合 ,推测为Pi 与氨基酸残基间的非共价相互作用 ,减小了TAPND2 7的自由能 ,从而提高了它的热稳定性

Influence of Inorganic Phosphate on the Thermostability of Thermostable Alkaline Phosphatase

To understand how inorganic phosphate (P-i) influences the thermostability of alkaline phosphatase (TAPND27) from %Thermus% p.3041 in vitro , The gene coding for TAPND27 in high expression plasmid was induced and expressed in %E.coli,% and purified TAPND27 was obtained. Thermostability measurement suggested that P i could greatly improve the thermostability of TAPND27. In the presence of 10 mmol/L phosphate, The T m value of TAPND27 increased from 91℃ to 97℃. Enzymatic kinetic experiment verified that P i is a reversible competitive inhibitor, with a K i value 0^99×10 -4 mol/L. Those results proved that the improved thermostability of TAPND27 is due to reversible interaction between P-i and the enzyme, which was assumed to be weak noncovalent bond. This interaction decreases the free energy of TAPND27, thus results in improvement of thermostabililty.