Insights into the transglucosylation activity of α-glucosidase from Schwanniomyces occidentalis.

The α-glucosidase from Schwanniomyces occidentalis (GAM1p) was expressed in Komagataella phaffii to about 70 mg/L, and its transferase activity studied in detail. Several isomaltooligosaccharides (IMOS) were formed using 200 g/L maltose. The major production of IMOS (81.3 g/L) was obtained when 98% maltose was hydrolysed, of which 34.8 g/L corresponded to isomaltose, 26.9 g/L to isomaltotriose, and 19.6 g/L to panose. The addition of glucose shifted the IMOS synthesis towards products containing exclusively α(1 → 6)-linkages, increasing the production of isomaltose and isomaltotriose about 2-4 fold, enabling the formation of isomaltotetraose, and inhibiting that of panose to about 12 times. In addition, the potential of this enzyme to glycosylate 12 possible hydroxylated acceptors, including eight sugars and four phenolic compounds, was evaluated. Among them, only sucrose, xylose, and piceid (a monoglucosylated derivative of resveratrol) were glucosylated, and the main synthesised products were purified and characterised by MS and NMR. Theanderose, α(1 → 4)-D-glucosyl-xylose, and a mixture of piceid mono- and diglucoside were obtained with sucrose, xylose, and piceid as acceptors, respectively. Maximum production of theanderose reached 81.7 g/L and that of the glucosyl-xylose 26.5 g/L, whereas 3.4 g/L and only 1 g/L were produced of the piceid mono- and diglucoside respectively. KEY POINTS: • Overexpression of a yeast α-glucosidase producing novel molecules. • Yeast enzyme producing the heterooligosaccharides theanderose and glucosyl-xylose. • Glycosylation of the polyphenol piceid by a yeast α-glucosidase.

Structural Analysis of ß-Fructofuranosidase from Xanthophyllomyces dendrorhous Reveals Unique Features and the Crucial Role of N-Glycosylation in Oligomerization and Activity.

Xanthophyllomyces dendrorhousß-fructofuranosidase (XdINV)is a highly glycosylated dimeric enzyme that hydrolyzes sucrose and releases fructose from various fructooligosaccharides (FOS) and fructans. It also catalyzes the synthesis of FOS, prebiotics that stimulate the growth of beneficial bacteria in human gut. In contrast to most fructosylating enzymes, XdINV produces neo-FOS, which makes it an interesting biotechnology target. We present here its three-dimensional structure, which shows the expected bimodular arrangement and also a long extension of its C terminus that together with anN-linked glycan mediate the formation of an unusual dimer. The two active sites of the dimer are connected by a long crevice, which might indicate its potential ability to accommodate branched fructans. This arrangement could be representative of a group of GH32 yeast enzymes having the traits observed in XdINV. The inactive D80A mutant was used to obtain complexes with relevant substrates and products, with their crystals structures showing at least four binding subsites at each active site. Moreover, two different positions are observed from subsite +2 depending on the substrate, and thus, a flexible loop (Glu-334-His-343) is essential in binding sucrose and ß(2-1)-linked oligosaccharides. Conversely, ß(2-6) and neo-type substrates are accommodated mainly by stacking to Trp-105, explaining the production of neokestose and the efficient fructosylating activity of XdINV on α-glucosides. The role of relevant residues has been investigated by mutagenesis and kinetics measurements, and a model for the transfructosylating reaction has been proposed. The plasticity of its active site makes XdINV a valuable and flexible biocatalyst to produce novel bioconjugates.