Optimized and automated protocols for high-throughput screening of amylosucrase libraries.
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.
Emond
Stéphane
S
Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés, Toulouse, France.
Potocki-Véronèse
Gabrielle
G
Mondon
Philippe
P
Bouayadi
Khalil
K
Kharrat
Hakim
H
Monsan
Pierre
P
Remaud-Simeon
Magali
M
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
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D012997:Solvents; D014867:Water; D005964:Glucosyltransferases; C019700:amylosucrase; D004121:Dimethyl Sulfoxide