The transgalactosylation activity of Kluyveromyces lactis cells was studied in detail. Cells were permeabilized with ethanol and further lyophilized to facilitate the transit of substrates and products. The resulting biocatalyst was assayed for the synthesis of galacto-oligosaccharides (GOS) and compared with two soluble β-galactosidases from K. lactis (Lactozym 3000 L HP G and Maxilact LGX 5000). Using 400 g/L lactose, the maximum GOS yield, measured by HPAEC-PAD analysis, was 177 g/L (44% w/w of total carbohydrates). The major products synthesized were the disaccharides 6-galactobiose [Gal-β(1→6)-Gal] and allolactose [Gal-β(1→6)-Glc], as well as the trisaccharide 6-galactosyl-lactose [Gal-β(1→6)-Gal-β(1→4)-Glc], which was characterized by MS and 2D NMR. Structural characterization of another synthesized disaccharide, Gal-β(1→3)-Glc, was carried out. GOS yield obtained with soluble β-galactosidases was slightly lower (160 g/L for Lactozym 3000 L HP G and 154 g/L for Maxilact LGX 5000); however, the typical profile with a maximum GOS concentration followed by partial hydrolysis of the newly formed oligosaccharides was not observed with the soluble enzymes. Results were correlated with the higher stability of β-galactosidase when permeabilized whole cells were used.

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.