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

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.

Crystal Structure of a Sucrose-6-phosphate Hydrolase from Lactobacillus gasseri with Potential Applications in Fructan Production and the Food Industry

“…When comparing the oligomeric states of enzymes of the GH32 family from yeast, the Schwanniomyces occidentalis and Kluyveromyces marxianus present a dimeric assembly in their crystal structure, with a larger interface area than Lg As32, e.g. , an invertase So Inv (PDB ID 3KF3 and 3KF5), a β-fructofuranosidase So Ffase (PDB ID 6S1T and 6S2B), and an exoinulinase Km INU1 (PDB ID 6J0T) (data not published) with an interface area of 1991, 2025, and 2420 Å 2 , respectively (see Figure S4C–E). So Ffase is shown to be more stable with a Δ G diss of 45.8 kcal mol –1 and a considerable number of hydrogen bonds ( N HB : 37) and also salt bridges ( N SB : 2).…”

The Influence of Yarrowia lipolytica Glycosylation on the Biochemical Properties and Oligomerization of Heterologous Invertase

“…During this process, transient covalent sugar-enzyme intermediates are formed. Invertases have been widely studied in Saccharomyces cerevisiae [6][7][8], Schizosaccharomyces pombe [9][10][11], Rhodotorula glutinis [12,13], Candida utilis [14,15], Schwanniomyces occidentalis [4,16], and Fusarium solani [17]. In the wide range of microorganisms, invertases possess different biochemical properties, such as enzymatic activity at different pH or temperature.…”