New insights into the molecular mechanism behind mannitol and erythritol fructosylation by ß-fructofuranosidase from Schwanniomyces occidentalis.

The ß-fructofuranosidase from Schwanniomyces occidentalis (Ffase) is a useful biotechnological tool for the fructosylation of different acceptors to produce fructooligosaccharides (FOS) and fructo-conjugates. In this work, the structural determinants of Ffase involved in the transfructosylating reaction of the alditols mannitol and erythritol have been studied in detail. Complexes with fructosyl-erythritol or sucrose were analyzed by crystallography and the effect of mutational changes in positions Gln-176, Gln-228, and Asn-254 studied to explore their role in modulating this biocatalytic process. Interestingly, N254T variant enhanced the wild-type protein production of fructosyl-erythritol and FOS by [Formula: see text] 30% and 48%, respectively. Moreover, it produced neokestose, which represented [Formula: see text] 27% of total FOS, and yielded 31.8 g l-1 blastose by using glucose as exclusive fructosyl-acceptor. Noteworthy, N254D and Q176E replacements turned the specificity of Ffase transferase activity towards the synthesis of the fructosylated polyols at the expense of FOS production, but without increasing the total reaction efficiency. The results presented here highlight the relevance of the pair Gln-228/Asn-254 for Ffase donor-sucrose binding and opens new windows of opportunity for optimizing the generation of fructosyl-derivatives by this enzyme enhancing its biotechnological applicability.

Yeast cultures expressing the Ffase from Schwanniomyces occidentalis, a simple system to produce the potential prebiotic sugar 6-kestose.

The ß-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. 6-Kestose represented ~ 70% of the products synthesized. 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.

Enzymatic Synthesis of a Novel Pterostilbene α-Glucoside by the Combination of Cyclodextrin Glucanotransferase and Amyloglucosidase.

The synthesis of a novel α-glucosylated derivative of pterostilbene was performed by a transglycosylation reaction using starch as glucosyl donor, catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. The reaction was carried out in a buffer containing 20% (v/v) DMSO to enhance the solubility of pterostilbene. Due to the formation of several polyglucosylated products with CGTase, the yield of monoglucoside was increased by the treatment with a recombinant amyloglucosidase (STA1) from Saccharomyces cerevisiae (var. diastaticus). This enzyme was not able to hydrolyze the linkage between the glucose and pterostilbene. The monoglucoside was isolated and characterized by combining ESI-MS and 2D-NMR methods. Pterostilbene α-d-glucopyranoside is a novel compound. The α-glucosylation of pterostilbene enhanced its solubility in water to approximately 0.1 g/L. The α-glucosylation caused a slight loss of antioxidant activity towards ABTS˙⁺ radicals. Pterostilbene α-d-glucopyranoside was less toxic than pterostilbene for human SH-S5Y5 neurons, MRC5 fibroblasts and HT-29 colon cancer cells, and similar for RAW 264.7 macrophages.