ABSTRACTEfficient enzymatic synthesis of tailor-made prebiotic fructo-oligosaccharides (FOS) used in functional food formulation is a relevant biotechnological objective. We have engineered theSaccharomyces cerevisiaeinvertase (Suc2) to improve its transferase activity and to identify the enzymatic determinants for product specificity. Amino acid replacement (W19Y, N21S, N24S) within a conserved motif (β-fructosidase) specifically increased the synthesis of 6-kestose up to 10-fold. Mutants with lower substrate (sucrose) affinity produced FOS with longer half-lives. A mutation (P205V) adjacent to another conserved motif (EC) caused a 6-fold increment in 6-kestose yield. Docking studies with a Suc2 modeled structure defined a putative acceptor substrate binding subsite constituted by Trp 291 and Asn 228. Mutagenesis studies confirmed the implication of Asn 228 in directing the orientation of the sucrose molecule for the specific synthesis of β(2,6) linkages.