The In vitro Directed Evolution of E.coli Alkaline Phosphatase

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 EAP. 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.