Mutant ß-fructofuranosidase synthesizing blastose [ß-d-Fruf-(2→6)-d-Glcp].

ABSTRACT

Fructooligosaccharides (FOS) are leading prebiotics that help keep the gut healthy and aid wellness by stimulating the growth and activity of beneficial intestinal bacteria. The best-studied FOS are inulin-type FOS, mainly oligosaccharides with ß-Fruf-(2→1)-Fruf linkages, including 1-kestose [ß-Fruf-(2→1)-ß-Fruf-(2↔1)-α-Glcp] and nystose [ß-Fruf-(2→1)-ß-Fruf-(2→1)-ß-Fruf-(2↔1)-α-Glcp]. However, the properties of other types of FOS-levan-type FOS with ß-Fruf-(2→6)-Fruf linkages and neo-type FOS with ß-Fruf-(2→6)-Glcp linkages-remain ambiguous because efficient methods have not been established for their synthesis. Here, using site-saturation mutation of residue His79 of ß-fructofuranosidase from Zymomonas mobilis NBRC13756, we successfully obtained a mutant ß-fructofuranosidase that specifically produces neo-type FOS. The H79G enzyme variant loses the native ß-Fruf-(2→1)-Fru-transfer ability (which produces 1-kestose), and instead has ß-Fruf-(2→6)-Glc-transfer ability and produces neokestose. Its hydrolytic activity specific to the ß-Fruf-(2↔1)-α-Glcp bond of neokestose then yields blastose [ß-Fruf-(2→6)-Glcp]. The enzyme produces 0.4 M blastose from 1.0 M sucrose (80 % of the theoretical yield). The production system for blastose established here will contribute to the elucidation of the physiological functions of this disaccharide.