大肠杆菌碱性磷酸酶的体外定向进化研究

The In vitro Directed Evolution of E.coli Alkaline Phosphatase

大肠杆菌碱性磷酸酶 (E .colialkalinephosphatase,EAP ,EC 3 1 3 1)是一个非特异性二聚体磷酸单酯酶 .采用易错聚合酶链反应 (errorpronePCR)的方法 ,在原有高活力突变株的基础上 ,对EAP远离活性中心催化三联体的区域进行定向进化 ,经两轮errorpronePCR ,获得催化活力较亲本D10 1S突变株提高 3倍、较野生型酶提高 35倍的进化酶 4 186 ,并对该酶的催化动力学特征进行了分析 .进化酶基因的DNA测序表明 4 186含两个有义氨基酸置换 :K16 7R和S374C ,二者既不位于底物结合位点 ,也不位于酶的金属离子结合位点 .

The evolution of phoA gene fragment distant from the Asp101-Ser102-Ala103 encoding region to increase the catalytic activity of EAP with a single mutant D101S as parent was directed. Through two cycles of error prone PCR, coupled with a sensitive screening method, an evolved variant 4-186 was obtained. Its catalytic activity was 3-fold higher than that of D101S parent and 35-fold more active than wild-type EAR The kinetic analysis indicated that the evolved enzyme exhibits a higher substrate binding ability and a higher catalytic efficiency than the D101S parent enzyme. DNA sequence revealed that 4-186 contains two amino acid substitutions, K167R and S374C, both of which locate neither the substrate-binding sites nor the metal-binding sites of EAP.