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.
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-(26)-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.
The β-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 $$\sim$$ ∼ 30% and 48%, respectively. Moreover, it produced neokestose, which represented $$\sim$$ ∼ 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.
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