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1087057107301978v1
12/5/715    most recent
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This version was published on August 1, 2007
Journal of Biomolecular Screening, Vol. 12, No. 5, 715-723 (2007)
DOI: 10.1177/1087057107301978

Optimized and Automated Protocols for High-Throughput Screening of Amylosucrase Libraries

Stéphane Emond

Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés, Toulouse, France, MilleGen SA, Bâtiment Prologue Biotech, Labège cedex, France

Gabrielle Potocki-Véronèse

Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés, Toulouse, France

Philippe Mondon

MilleGen SA, Bâtiment Prologue Biotech, Labège cedex, France

Khalil Bouayadi

MilleGen SA, Bâtiment Prologue Biotech, Labège cedex, France

Hakim Kharrat

MilleGen SA, Bâtiment Prologue Biotech, Labège cedex, France

Pierre Monsan

Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés, Toulouse, France

Magali Remaud-Simeon

Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés, Toulouse, France, magali.remaud{at}insa-toulouse.fr

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 °C compared to the parental wild-type enzyme. (Journal of Biomolecular Screening 2007:715-723)

Key Words: amylosucrase • directed evolution • high-throughput screening • thermostability • organic solvents


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S. Emond, I. Andre, K. Jaziri, G. Potocki-Veronese, P. Mondon, K. Bouayadi, H. Kharrat, P. Monsan, and M. Remaud-Simeon
Combinatorial engineering to enhance thermostability of amylosucrase
Protein Sci., June 1, 2008; 17(6): 967 - 976.
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