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

Author(s): Miguel A. de Abreu, Miguel Alvaro-Benito, Francisco J. Plou, Maria Fernandez-Lobato, Miguel Alcalde

Journal Name: Combinatorial Chemistry & High Throughput Screening
Accelerated Technologies for Biotechnology, Bioassays, Medicinal Chemistry and Natural Products Research

Volume 14 , Issue 8 , 2011

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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.

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

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Article Details

Year: 2011
Page: [730 - 738]
Pages: 9
DOI: 10.2174/138620711796504370
Price: $65

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