title: Directed evolution of alpha-aspartyl dipeptidase from Salmonella typhimurium.
abstract: Model-free approaches (error-prone PCR to introduce random mutations, DNA shuffling to combine positive mutations, and screening of the resultant mutant libraries) have been used to enhance the catalytic activity and thermostability of alpha-aspartyl dipeptidase from Salmonella typhimurium, which is uniquely able to hydrolyze Asp-X dipeptides (where X is any amino acid) and one tripeptide (Asp-Gly-Gly). Under double selective pressures of activity and thermostability, through two rounds of error-prone PCR and three sequential generations of DNA shuffling, coupled with screening, a mutant pepEM3074 with approximately 47-fold increased enzyme activity compared with its wild-type parent was obtained. Moreover, the stability of pepEM3074 is increased significantly. Three amino acid substitutions (Asn89His, Gln153Glu, and Leu205Arg), two of them are near the active site and substrate binding pocket, were identified by sequencing the genes encoding this evolved enzyme. The mechanism of the enhancement of activity and stability was analyzed in this paper.
authors.affiliation: Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130023, People's Republic of China.
authors.forename: X
authors.initials: X
authors.lastname: Kong
authors.forename: Y
authors.initials: Y
authors.lastname: Liu
authors.forename: X
authors.initials: X
authors.lastname: Gou
authors.forename: S
authors.initials: S
authors.lastname: Zhu
authors.forename: H
authors.initials: H
authors.lastname: Zhang
authors.forename: X
authors.initials: X
authors.lastname: Wang
authors.forename: J
authors.initials: J
authors.lastname: Zhang
mesh_terms: D019943:Amino Acid Substitution; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004150:Dipeptidases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics*
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D004150:Dipeptidases; C042430:aspartyl peptidase E