title: Stability and activity improvement of cephalosporin esterase EstB from Burkholderia gladioli by directed evolution and structural interpretation of muteins.
abstract: Esterase EstB from Burkholderia gladioli, which belongs to a family of esterases related to beta-lactamases and DD-peptidases was evolved for increased stability and simultaneously maintaining high cephalosporin C deacetylation activity. Random mutagenesis PCR was used to generate up to 5 aa substitutions per gene. A newly designed colony filter-screening assay, which was based on pH change after deacetylation of cephalosporin C in presence of DMF was established. In a first evolution round employing random mutagenesis, which included about 10(6) mutants, a set of interesting mutants was isolated. Distinct mutations identified as significant for stability were combined by a rational recombination step and the resulting recombinant was further evolved by an additional random mutagenesis round. After screening an additional 10(5) clones, it was possible to isolate a variant of EstB having more than 100-fold better activity in reactions containing 35% DMF. This mutant also showed a high increase in temperature stability (T(m) was raised by 13 degrees C) and retained high activity towards cephalosporin C under standard assay conditions. The molecular effects of mutations found in random mutants are discussed in view of the three-dimensional structure of wild-type EstB.
authors.affiliation: Institut für Molekulare Biotechnologie, Technische Universität Graz, Petersgasse 14, A-8010 Graz, Austria.
authors.forename: Goran
authors.initials: G
authors.lastname: Valinger
authors.forename: Manuela
authors.initials: M
authors.lastname: Hermann
authors.forename: Ulrike G
authors.initials: UG
authors.lastname: Wagner
authors.forename: Helmut
authors.initials: H
authors.lastname: Schwab
mesh_terms: D000595:Amino Acid Sequence; D042725:Burkholderia gladioli / Q000201:enzymology*; D002152:Calorimetry, Differential Scanning; D019020:Directed Molecular Evolution / Q000379:methods*; D004795:Enzyme Stability; D004950:Esterases / Q000378:metabolism*; D006207:Half-Life; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D050505:Mutant Proteins / Q000737:chemistry* / Q000302:isolation & purification; D009154:Mutation / Q000235:genetics; D017433:Protein Structure, Secondary; D020539:Sequence Analysis, Protein; D044366:Transition Temperature
chemical_list: D050505:Mutant Proteins; D004950:Esterases