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RevistaCombinatorial Chemistry & High Throughput Screening

Screening beta-fructofuranosidases mutant libraries to enhance the transglycosylation rates of beta(2-6) fructooligosaccharides

Autores:Miguel A. de Abreu , Miguel Alvaro-Benito , Francisco José Plou Gasca, Maria Fernandez-Lobato , Miguel Alcalde Galeote
Grupos de investigación:Biocatálisis Aplicada

β-Fructofuranosidases can divert their hydrolytic activity towards transglycosylation for the synthesis of high value-added products, including prebiotic fructooligosaccharides (FOS). A directed evolution strategy has been employed to enhance the transferase rate of the β-fructofuranosidase (SoINV) from the Schwanniomyces occidentalis yeast for the production of β-(2→6)-linked FOS. To screen for transferase activity of the SoINV functionally expressed in Saccharomyces cerevisiae, a high-throughput screening protocol based on two colorimetric assays was validated (with coefficient of variance below 11%). Mutagenic libraries were constructed by error-prone PCR and clones showing higher glucose:fructose ratio with respect to the parental type were identified. Further analysis by anion-exchange chromatography coupled with pulsed amperometric detection helped to identify mutants with improved yields for the synthesis of β-(2→6) fructooligosaccharides. Selected mutants displayed transferase initial rates enhanced ∼2-fold over parent type, reaching production levels up to 47 g/L after 48 h of reaction for the formation of 6-kestose.

Palabras clave:β-(2→6) fructooligosaccharides; transglycosylation; Schwanniomyces occidentalis; prebiotics; directed evolution; hydrolytic activity; transglycosylation; high-throughput screening; PCR; anion-exchange chromatography; amperometric detection; sucrose
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