The β-fructofuranosidase from the yeast Schwanniomyces occidentalis (Ffase) produces the prebiotic sugars 6-kestose and 1-kestose by transfructosylation of sucrose, which makes it of biotechnological interest. In this study, the hydrolase and transferase activity of this enzyme was kinetically characterized and its potential to synthesize new fructosylated products explored. A total of 40 hydroxylated compounds were used as potential fructosyl-acceptor alternatives to sucrose. Only 17 of them, including some monosaccharides, disaccharides, and oligosaccharides as well as alditols and glycosides were fructosylated. The best alternative acceptors were the alditols. The major transfer product of the reaction including mannitol was purified and characterized as 1-O-β-D-fructofuranosyl-D-mannitol, whose maximum concentration reached 44 g/L, representing about 7.3 % of total compounds in the mixture and 89 % of all products generated by transfructosylation. The reactions including erythritol produced 35 g/L of an isomer mixture comprising 1- and 4-O-β-D-fructofuranosyl-D-erythritol. In addition, Ffase produced 24 g/L of the disaccharide blastose by direct fructosylation of glucose, which makes it the first enzyme characterized from yeast showing this ability. Thus, novel fructosylated compounds with potential applications in food and pharmaceutical industries can be obtained due to the Ffase fructosyl-acceptor promiscuity.

The β-fructofuranosidase Ffase from the yeast Schwanniomyces occidentalis produces 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. A Ser196Leu substitution enhanced transferase activity of the Ffase by ~2.6-fold. In this work, production of 6-kestose was simplified by directly using cultures of Sw. occidentalis and Saccharomyces cerevisiae expressing both the wild-type enzyme and the mutated variant Ffase-Leu196. Best results were obtained using cultures supplemented with sucrose and expressing the mutated protein variant, which after only 4 h doubled the amount of 6-kestose obtained with the corresponding purified enzyme. 6-Kestose represented ~70% of the products synthesised. In addition, a small amount of 1-kestose and the neofructoligosaccharides neokestose and blastose were also produced. The Ser196Leu substitution skewed production of 6-kestose and neofructooligosaccharides resulting in an increase of ~2.2 and 1.5-fold respectively, without affecting production of 1-kestose. Supplementing yeast cultures with glucose clearly showed that blastose originates from direct fructosylation of glucose, a property that has not been described for other similar proteins from yeasts. Modeling neokestose and blastose into the Ffase active site revealed the molecular basis explaining the peculiar specificity of this enzyme. IMPORTANCEThe β-fructofuranosidase Ffase from the yeast Sw. occidentalis produced prebiotic sugars by transfructosylation of sucrose and showed high fructosylacceptor promiscuity, making it of biotechnological interest. A simplified process to produce prebiotic sugars in flask using yeast cultures expressing this enzyme has been developed and its effectiveness compared with that of the purified protein. Best results were obtained by using S. cerevisiae cultures expressing a mutated protein variant, which 3 also skewed the production profile towards synthesis of improved prebiotic sugars containing β-(26)-bonds. The unveiled promiscuity of the enzyme together with the bias in production of products, demonstrated with the selected mutant, make the system a most valuable tool in generating new bioactive compounds in a fast and simple way.

Glycerol, as a good acceptor for Ffase, produces novel fructosylated derivatives with biotechnological potential.