Expression and Characterization of Mutant Forms of Penicillin Acylase from Alcaligenesfaecalis

摘要

The sequencing of six plasmids carrying a gene of penicillin acylase from AlcaligenesfaecalisVKM B1518 (AfPA) revealed the presence of random mutations in the gene; they occurred during a polymerase chain reaction. Six mutant AfPAs and a wild-type enzyme were expressed in E. coli cells. The activity assay of mutant AfPAs in E. coli cells indicated that several amino acid substitutions affect the expression level of the AfPA gene and the rate of cell growth. Four mutant AfPAs were purified; their catalytic properties and thermal stability were studied. It is shown that the amino acid substitutions under study do not affect the catalytic efficiency value. Within the experimental error, the (3Q133R and PK184E (the AfPA M2 mutant) substitutions had no effect on the thermal stability of the enzyme; in the case of mutants AfPA M4 (pY90H), M5 (aD132G, pR97C), and M6 (aV5E, aN183S, and pE439G), the inactivation rate constant increased 2.4, 2.75, and 8.3 times, respectively, as compared to that of the wild-type enzyme.