Enhanced FOS synthesis by rational design of a novel β-fructofuranosidase from Trichoderma atroviride

A novel β-fructofuranosidase from Trichoderma atroviride (TaINV) of 74 kDa was heterologously expressed in Escherichia coli for the first time, purified and biochemically characterized. TaINV exhibited hydrolytic activity mainly towards sucrose and other substrates containing β-(2→1) linkages, with minor activity towards β-(2→6) bonds. In addition to hydrolysis, it catalyzed the synthesis of fructooligosaccharides (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 about 50.3% (w/w) of the total sugars in the reaction mixture, with 1-kestose as major product, representing about 85% of the total products synthesized. Structural analysis of TaINV using AlphaFold and docking analysis revealed conserved glycosyl hydrolase family 32 (GH32) motifs and key residues involved in the protein hydrolytic and transferase activities. Site-directed mutagenesis confirmed the essential role of the catalytic triad (Asp63, Asp201, Glu277) and highlighted key residues shaping transfructosylation specificity. Variants including substitutions W60Y and N62S displayed enhanced transferase activity, increasing total FOS production by 24.5% and 14.4%, respectively, reaching 62.7% and 57.4% (w/w) of total sugars, 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.