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Screening β-Fructofuranosidases Mutant Libraries to Enhance the Transglycosylation Rates of β-(2→6) Fructooligosaccharides

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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.





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

Document Type: Research Article

Publication date: September 1, 2011

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  • Combinatorial Chemistry & High Throughput Screening publishes full length original research articles and reviews describing various topics in combinatorial chemistry (e.g. small molecules, peptide, nucleic acid or phage display libraries) and/or high throughput screening (e.g. developmental, practical or theoretical). Ancillary subjects of key importance, such as robotics and informatics, will also be covered by the journal. In these respective subject areas, Combinatorial Chemistry & High Throughput Screening is intended to function as the most comprehensive and up-to-date medium available. The journal should be of value to individuals engaged in the process of drug discoveryand development, in the settings of industry, academia or government.
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