title: Directed evolution of formate dehydrogenase from Candida boidinii for improved stability during entrapment in polyacrylamide.
abstract: In two cycles of an error-prone PCR process, variants of formate dehydrogenase from Candida boidinii were created which revealed an up to 4.4-fold (440%) higher residual activity after entrapment in polyacrylamide gels than the wild-type enzyme. These were identified in an assay using single precursor molecules of polyacrylamide instead of the complete gel for selection. The stabilization resulted from an exchange of distinct lysine, glutamic acid, and cysteine residues remote from the active site, which did not affect the kinetics of the catalyzed reaction. Thermal stability increased at the exchange of lysine and glutamic acid, but decreased due the exchange of cysteine. Overall, the variants reveal very suitable properties for application in a technical synthetic process, enabling use of entrapment in polyacrylamide as an economic and versatile immobilization method.
authors.affiliation: Department of Biotechnology, RWTH Aachen University, Germany. m.ansorge@biotec.rwth-aachen.de
authors.forename: Marion B
authors.initials: MB
authors.lastname: Ansorge-Schumacher
authors.forename: Heike
authors.initials: H
authors.lastname: Slusarczyk
authors.forename: Julia
authors.initials: J
authors.lastname: Schümers
authors.forename: Dennis
authors.initials: D
authors.lastname: Hirtz
mesh_terms: D000180:Acrylic Resins; D019943:Amino Acid Substitution / Q000235:genetics; D002175:Candida / Q000201:enzymology* / Q000235:genetics; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D005560:Formate Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012996:Solutions
chemical_list: D000180:Acrylic Resins; D012996:Solutions; C016680:polyacrylamide gels; D005560:Formate Dehydrogenases