Rhodotorula dairenensis β-fructofuranosidase is a highly glycosylated enzyme with broad substrate specificity that catalyzes the synthesis of 6-kestose and a mixture of the three series of fructooligosaccharides (FOS), fructosylating a variety of carbohydrates and other molecules as alditols. We report here its three-dimensional structure, showing the expected bimodular arrangement and also a unique long elongation at its N-terminus containing extensive O-glycosylation sites that form a peculiar arrangement with a protruding loop within the dimer. This region is not required for activity but could provide a molecular tool to target the dimeric protein to its receptor cellular compartment in the yeast. A truncated inactivated form was used to obtain complexes with fructose, sucrose and raffinose, and a Bis-Tris molecule was trapped, mimicking a putative acceptor substrate. The crystal structure of the complexes reveals the major traits of the active site, with Asn387 controlling the substrate binding mode. Relevant residues were selected for mutagenesis, the variants being biochemically characterized through their hydrolytic and transfructosylating activity. All changes decrease the hydrolytic efficiency against sucrose, proving their key role in the activity. Moreover, some of the generated variants exhibit redesigned transfructosylating specificity, which may be used for biotechnological purposes to produce novel fructosyl-derivatives.

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.

Here we report the first β-fructofuranosidase from the Trichoderma genus producing fructooligosaccharides (FOS). The novel enzyme from Trichoderma atroviride (TaINV) here characterized was heterologously expressed, purified, and biochemically analyzed. TaINV exhibited hydrolytic activity mainly toward sucrose and other substrates containing β-(2 → 1) linkages, with minor activity toward β-(2 → 6) bonds. In addition to hydrolysis, it catalyzed the synthesis of FOS of all three structural series ( ¹ F-FOS, ⁶ F-FOS, and ⁶ G-FOS). At the maximal production point, TaINV synthesized 252 g/L of total FOS, representing 50.3% (w/w) of the total sugars in the reaction mixture, with 1-kestose as the major product, representing ~ 85% of the total products synthesized. Structural analysis based on AlphaFold-predicted TaINV model and comparative superimposition with GH32-substrate complexes revealed conserved catalytic motifs and residues located in positions associated with substrate binding and specificity in characterized GH32 enzymes. Site-directed mutagenesis confirmed the essential role of the catalytic triad (Asp63, Asp201, Glu277) and identified additional residues shaping transfructosylation specificity. Variants including substitutions W60Y and N62S increased total FOS production, reaching 62.7% and 57.4% (w/w) of total sugars, respectively, which are comparable to yields obtained with commercial enzymes. Overall, TaINV represents a distinct intracellular fungal β-fructofuranosidase with strong transfructosylation capacity and preference for short-chain FOS. These findings expand the current knowledge of GH32 enzyme diversity and highlight TaINV as a promising biocatalyst for the efficient production of low-degree polymerization FOS with potential prebiotic applications. Key points: • Novel Trichoderma β-fructofuranosidase with high transfructosylation activity. • Catalytic residues defining FOS synthesis identified by structure–function analysis. • Engineered variant boosting FOS yield to 62% of total sugars. Graphical

β-Fructofuranosidases are key biocatalysts in the synthesis of fructooligosaccharides (FOS), prebiotics whose biological properties depend on the specific arrangement of β-(2 → 1) and/or β-(2 → 6) fructosyl linkages. Tailoring FOS structures therefore requires enzymes with adjustable specificities. In this work, we characterize the structural and functional features of the novel β-fructofuranosidase PlINV from Purpureocillium lilacinum, aiming to enhance its utility in the production of bioactive compounds. Purified PlINV exhibits broad substrate specificity, preferring sucrose but also acting on fructose-based polymers with diverse linkage types. It displays wide acceptor promiscuity in transfructosylation reactions, generating several trisaccharides from sucrose. The three-dimensional structure, solved at 2.1 Å, reveals a previously unreported tetrameric assembly within the GH32 family and a relatively occluded active site. Structures of various enzyme-substrate complexes identify residues governing substrate recognition and highlight substantial conformational plasticity, including numerous water-mediated interactions. Structure-guided mutagenesis within the active-site cleft generated variants with altered hydrolytic activity and distinct FOS profiles, confirming the functional importance of the identified residues. Substitutions Q259A and P207N increased overall FOS yield, whereas N289T selectively enriched neokestose within a broader product mixture. PlINV also efficiently fructosylated the polyphenols hydroxytyrosol and piceid-this latter reaction being described for the first time-validated by HPLC-MS analyses. Docking simulations suggest that polyphenol accommodation relies mainly on hydrophobic contacts, complemented by limited polar interactions. Collectively, these results provide a robust structural and mechanistic framework for engineering PlINV to fine-tune its biocatalytic properties, enabling the targeted production of specific FOS and other bioactive compounds for biotechnological applications.