Abstract
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.
Highlights
The β-fructofuranosidase from Schwanniomyces occidentalis (Ffase) is a useful biotechnological tool for the fructosylation of different acceptors to produce fructooligosaccharides (FOS) and fructoconjugates
Previous work addressed the molecular basis of the existing Ffase transfructosylating mechanism for yielding 6-kestose as the major product, concluding two possible alternative binding modes for the donor s ucrose[2,4]
The second binding mode of the donor sucrose maintains the same polar interaction with Gln-228 at subsite + 1, but placing its terminal glucose or fructose unit in a different way stacking against the Trp-76 at subsite + 2, resulting in the synthesis of the secondary products 1-kestose and neokestose
Summary
The β-fructofuranosidase from Schwanniomyces occidentalis (Ffase) is a useful biotechnological tool for the fructosylation of different acceptors to produce fructooligosaccharides (FOS) and fructoconjugates. N254T variant enhanced the wild-type protein production of fructosylerythritol and FOS by ∼ 30% and 48%, respectively It produced neokestose, which represented ∼ 27% of total FOS, and yielded 31.8 g l−1 blastose by using glucose as exclusive fructosylacceptor. 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 pair Gln-228/Asn-254, structurally equivalent to the Asp-239/Lys-242 pair of plant invertases[5], was demonstrated by targeted mutation analysis to be significantly involved in the configuration of the acceptor-substrate binding site for transfructosylation and in the modulation of the enzymatic transfer specificity. The maltosylation of erythritol enhanced its sweetness without affecting its physical–chemical properties[20], the glucosylation of mannitol increased its solubility in w ater[21], and the galactosylation of sorbitol raised the microbiotic population of Bifidobacterium, Lactobacillus and Streptococcus species[22]
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