title: Optimized and automated protocols for high-throughput screening of amylosucrase libraries.
abstract: This article describes the design and validation of a general procedure for the high-throughput isolation of amylosucrase variants displaying higher thermostability or increased resistance to organic solvents. This procedure consists of 2 successive steps: an in vivo selection that eliminates inactive variants followed by automated screening of active variants to isolate mutants displaying enhanced features. The authors chose an Escherichia coli expression vector, allowing a high production rate of the recombinant enzyme in miniaturized culture conditions. The screening assay was validated by minimizing variability for various parameters of the protocol, especially bacterial growth and protein production in cultures in 96-well microplates. Recombinant amylosucrase production was normalized by decreasing the coefficient of variance from 27% to 12.5%. Selective screening conditions were defined to select variants displaying higher thermostability or increased resistance to organic solvents. A first-generation amylosucrase variant library, constructed by random mutagenesis, was subjected to this procedure, yielding a mutant displaying a 25-fold increased stability at 50 degrees C compared to the parental wild-type enzyme.
authors.affiliation: Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés, Toulouse, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Emond
authors.forename: Gabrielle
authors.initials: G
authors.lastname: Potocki-Véronèse
authors.forename: Philippe
authors.initials: P
authors.lastname: Mondon
authors.forename: Khalil
authors.initials: K
authors.lastname: Bouayadi
authors.forename: Hakim
authors.initials: H
authors.lastname: Kharrat
authors.forename: Pierre
authors.initials: P
authors.lastname: Monsan
authors.forename: Magali
authors.initials: M
authors.lastname: Remaud-Simeon
mesh_terms: D001331:Automation; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D019020:Directed Molecular Evolution; D004353:Drug Evaluation, Preclinical; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005798:Genes, Bacterial; D014644:Genetic Variation; D005822:Genetic Vectors; D005964:Glucosyltransferases / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006358:Hot Temperature; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015203:Reproducibility of Results; D012641:Selection, Genetic; D012680:Sensitivity and Specificity; D012997:Solvents / Q000737:chemistry; D013997:Time Factors; D014170:Transformation, Genetic; D014867:Water / Q000737:chemistry
chemical_list: D004269:DNA, Bacterial; D011994:Recombinant Proteins; D012997:Solvents; D014867:Water; D005964:Glucosyltransferases; C019700:amylosucrase; D004121:Dimethyl Sulfoxide