title: Focused directed evolution of β-glucosidases: theoretical versus real effectiveness of a minimal working setup and simple robust screening.
abstract: Here we present an optimized procedure to generate amino acid variations at specific site(s) of proteins, followed by a simple one-step screen for mutants with the desired β-glucosidase activity. The procedure was evaluated by introducing sequence variation into a codon specifying a non-functional variant of the catalytic nucleophile (E401) of the maize β-glucosidase Zm-p60.1. Observed and theoretically expected frequencies of the four possible variants of the codon and the two possible phenotypes (functional and non-functional) were investigated. Deviations in codon and phenotype frequencies were expressed as a coefficient. This coefficient was then used to estimate the extent of oversampling, of the mutant library, which would be necessary to compensate for the underrepresentation of some sequences. This evaluation of the overall performance of the method allows experimentally derived parameters to be incorporated into mutant library design. This method combines the application of a well-defined distribution of variability with a reliable screening process. Thus, it facilitates the production of novel functional variants of β-glucosidases for either fundamental studies or potential biotechnological applications.
authors.affiliation: Department of Molecular Biology and Radiobiology, Mendel University in Brno, Zemědělská 1, CZ-61300 Brno and Institute of Biophysics AS CR, v.v.i., Královopolská 135, CZ-61265 Brno, Czech Republic. mazura@sci.muni.cz
authors.forename: Pavel
authors.initials: P
authors.lastname: Mazura
authors.forename: Tomáš
authors.initials: T
authors.lastname: Filipi
authors.forename: Přemysl
authors.initials: P
authors.lastname: Souček
authors.forename: Břetislav
authors.initials: B
authors.lastname: Brzobohatý
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D044602:Cellulases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003062:Codon / Q000737:chemistry / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D016296:Mutagenesis; D003313:Zea mays / Q000201:enzymology
chemical_list: D000596:Amino Acids; D003062:Codon; D044602:Cellulases