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.

Enzymatic synthesis of novel fructosylated compounds by Ffase from Schwanniomyces occidentalis in green solvents.

The ß-fructofuranosidase from the yeast Schwanniomyces occidentalis (Ffase) produces potential prebiotic fructooligosaccharides (FOS) by self-transfructosylation of sucrose, being one of the highest known producers of 6-kestose. The use of Green Solvents (GS) in biocatalysis has emerged as a sustainable alternative to conventional organic media for improving product yields and generating new molecules. In this work, the Ffase hydrolytic and transfructosylating activity was analysed using different GS, including biosolvents and ionic liquids. Among them, 11 were compatible for the net synthesis of FOS. Besides, two glycerol derivatives improved the yield of total FOS. Interestingly, polyols ethylene glycol and glycerol were found to be efficient alternative fructosyl-acceptors, both substantially decreasing the sucrose fructosylation. The main transfer product of the reaction with glycerol was a 62 g L-1 isomeric mixture of 1-O and 2-O-ß-d-fructofuranosylglycerol, representing 95% of all chemicals generated by transfructosylation. Unexpectedly, the non-terminal 2-O fructo-conjugate was the major molecule catalysed during the process, while the 1-O isomer was the minor one. This fact made Ffase the first known enzyme from yeast showing this catalytic ability. Thus, novel fructosylated compounds with potential applications in food, cosmetics, and pharmaceutical fields have been obtained in this work, increasing the biotechnological interest of Ffase with innocuous GS.

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.

Exploring the transferase activity of Ffase from Schwanniomyces occidentalis, a ß-fructofuranosidase showing high fructosyl-acceptor promiscuity.

The ß-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-ß-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-ß-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.