Mutant β-fructofuranosidase synthesizing blastose [β-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 β-Fruf-(2→1)-Fruf linkages, including 1-kestose [β-Fruf-(2→1)-β-Fruf-(2↔1)-α-Glcp] and nystose [β-Fruf-(2→1)-β-Fruf-(2→1)-β-Fruf-(2↔1)-α-Glcp]. However, the properties of other types of FOS-levan-type FOS with β-Fruf-(2→6)-Fruf linkages and neo-type FOS with β-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 β-fructofuranosidase from Zymomonas mobilis NBRC13756, we successfully obtained a mutant β-fructofuranosidase that specifically produces neo-type FOS. The H79G enzyme variant loses the native β-Fruf-(2→1)-Fru-transfer ability (which produces 1-kestose), and instead has β-Fruf-(2→6)-Glc-transfer ability and produces neokestose. Its hydrolytic activity specific to the β-Fruf-(2↔1)-α-Glcp bond of neokestose then yields blastose [β-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.

Keywords: blastose; fructooligosaccharide; glycoside hydrolase family 68; neokestose; site saturation mutation; β-fructofuranosidase.