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.
XU Hui-Fang, ZHANG Xian-En, ZHANG Zhi-Ping, ZHANG Yong-Mei, A. E. G. CASS. The In vitro Directed Evolution of E. coli Alkaline Phosphatase[J]. Progress in Biochemistry and Biophysics,2003,30(1):89-94
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