Directed evolution of alpha-aspartyl dipeptidase from Salmonella typhimurium

Model-free approaches (error-prone PCR to introduce random mutations, DNA s huffling to combine positive mutations, and screening of the resultant muta nt libraries) have been used to enhance the catalytic activity and thermost ability of a-aspartyl dipeptidase from Salmonella typhimurium, which is uni quely able to hydrolyze Asp-X dipeptides (where X is any amino acid) and on e tripeptide (Asp-Gly-Gly). Under double selective pressures of activity an d thermostability, through two rounds of error-prone PCR and three sequenti al generations of DNA shuffling, coupled with screening, a mutant pepEM3074 with approximately 47-fold increased enzyme activity compared with its wil d-type parent was obtained. Moreover, the stability of pepEM3074 is increas ed significantly. Three amino acid substitutions (Asn89His, Gln153Glu, and Leu205Arg), two of them are near the active site and substrate binding pock et, were identified by sequencing the genes encoding this evolved enzyme. T he mechanism of the enhancement of activity and stability was analyzed in t his paper. (C) 2001 Academic Press.