The β-fructofuranosidase Ffase from the yeast Schwanniomyces occidentalis produces potential prebiotic fructooligosaccharides with health-promoting properties, making it of biotechnological interest. Ffase is one of the highest and more selective known producers of 6-kestose by transfructosylation of sucrose. In this work, production of 6-kestose was simplified by directly using cultures of S. occidentalis and Saccharomyces cerevisiae expressing both the wild-type enzyme and a mutated Ffase variant including the Ser196Leu substitution (Ffase-Leu196). Best results were obtained using yeast cultures supplemented with sucrose and expressing the Ffase-Leu196, which after only 4 h produced ~ 116 g/L of 6-kestose, twice the amount obtained with the corresponding purified enzyme. The Ser196Leu substitution skewed production of 6-kestose and neofructooligosaccharides resulting in an increase of ~ 2.2- and 1.5-fold, respectively. Modeling neokestose and blastose into the Ffase-active site revealed the molecular basis explaining the peculiar specificity of this enzyme.
Ref.: D. Rodrigo-Frutos, D. Piedrabuena, J. Sanz-Aparicio, M. Fernández-Lobato. "Yeast cultures expressing the Ffase from Schwanniomyces occidentalis, a simple system to produce the potential prebiotic sugar 6-kestose". Applied Microbiology and Biotechnology (2018), doi:10.1007/s00253-018-9446-y