Substrate Specificity and Subsite Affinities of β-Fructofuranosidase from Bifidobacterium adolescentis G1

Abstract

The substrate specificity of β-fructofuranosidase from Bifidobacterium adolescentis G1 for fructooligosaccharides [1^F (1-β-D-fructofuranosyl)_n-1 sucrose, GF_n, (n=3-8)] andinulooligosaccharides [1^F (1-β-D-fructofuranosyl)_n-1 fructose, F_n, (n=2-7)] were investigated. The K_m (mM) and k₀ (s^-1) values were : GF₃, 1.1 and 155 ; GF₄, 1.6 and 154 ; GF₅, 3.2 and 252 ; GF₆, 4.2 and 186 ; GF₇, 7.1 and 260 ; GF₈, 7.0 and 180 ; F₂, 4.9 and 213 ; F₃, 1.5 and 423 ; F₄, 2.4 and 311 ; F₅, 1.9 and 458 ; F₆, 8.7 and 369 ; F₇, 8.1 and 323, respectively. The enzyme preferred oligosaccharides to inulin and sucrose as the substrate. The enzyme hardly catalyzed transfructosylation from GF₂. The dependence of rate parameters on the degree of polymerization differed from that of Penicillium trzebinskii exo-inulinase. The subsite atfinities in the active site were 0.93, 4.33, 1.15, -0.48, 0.38, -1.07, and -0.04 kcal/mol for subsites 1, 2, 3, 4, 5, 6, and 7, respectively.