Synthesis of 6‐Kestose using an Efficient β‐Fructofuranosidase Engineered by Directed Evolution

Abstract

AbstractThe β‐fructofuranosidase (Ffase) from Schwanniomyces occidentalis (Ffase‐Leu196 variant) was subjected to four cycles of directed evolution to enhance the transglycosylation activity for the synthesis of β‐(2→6) linked fructooligosaccharides (FOS). With a 5.5‐fold improvement in fructose transferase activity over the parental type and greater selectivity for the synthesis of 6‐kestose (up to 73% of the total FOS), the mutants doubled FOS synthesis to 168 g L.−1 Whilst the F523V and H510P mutations were located at the C‐terminus of the protein, mutations Q78L and I203L were associated with the acidic catalytic triad where they modified its interactions with the surrounding residues, in turn varying the hydrolase and transferase rates.