We report the first β-fructofuranosidase from the Trichoderma genus capable of producing fructooligosaccharides (FOS). The enzyme, identified in Trichoderma atroviride (TaINV), was heterologously expressed, purified, and biochemically characterized. TaINV mainly hydrolyzes sucrose and substrates containing β-(2→1) linkages and, importantly, also catalyzes the synthesis of FOS from all three structural series (1F-FOS, 6F-FOS, and 6G-FOS). Under optimal conditions, TaINV produced up to 252 g/L of total FOS, representing 50.3% of total sugars, with 1-kestose as the dominant product (~85%). Structural analysis using an AlphaFold model revealed conserved catalytic motifs typical of GH32 enzymes. Site-directed mutagenesis confirmed the role of the catalytic triad (Asp63, Asp201, Glu277) and identified residues influencing transfructosylation specificity. Notably, variants W60Y and N62S increased FOS production to 62.7% and 57.4%, respectively, comparable to commercial enzymes.
Overall, TaINV is a distinctive intracellular fungal β-fructofuranosidase with strong transfructosylation activity and a preference for short-chain FOS, highlighting its potential as a biocatalyst for producing prebiotic fructooligosaccharides.
Ref: Narmontaite, E., Plou, F.J. & Fernández-Lobato, M. Enhanced fructooligosaccharides synthesis by engineered Trichoderma atroviride β-fructofuranosidase. Appl Microbiol Biotechnol 110, 96 (2026). https://doi.org/10.1007/s00253-026-13748-7
