Biosynthesis, purification and characterization of β-fructofuranosidase from Bifidobacterium longum KN29.1

Abstract

Bifidobacterium longum KN29.1 exhibits greater β-fructofuranosidase activity than two Bifidobacterium animalis strains with highest activity observed when B. longum KN29.1 was grown on fructose. Properties of the native β-fructofuranosidase from B. longum KN29.1 and recombinant β-fructofuranosidase were identical. The molecular mass of the purified β-fructofuranosidase was approximately 67kDa as determined by SDS-PAGE. Its isoelectric point was 4.6. The enzyme was stable at pH 5.7–9.1 and the temperature up to 45°C. It was optimally active in Actilight® 950P and sucrose hydrolysis at pH 6.0–6.2 while the optimum temperature of these processes was 50°C and 37–45°C, respectively. In the presence of Cu2+, Ag+, Hg+ ions and p-chloromercuribenzoic acid (1mM) B. longum KN29.1 β-fructofuranosidase activity was completely inhibited, its slight stimulation was observed in solutions containing Mn2+ (1mM). This enzyme showed higher affinity for nystose (Km 1.2±0.15mM) and 1-kestose (Km 4.6±0.2mM) than for sucrose (Km 29.4±1.5mM). It catalyzed the hydrolysis of sucrose, 1-kestose and inulin at the relative activities of 100, 251.7±7.36 and 62.2±1.15%, respectively. TLC and HPLC analysis demonstrated that the enzyme released β-fructofuranose from the non-reducing end of inulin and fructooligosaccharides. The comparative analysis of known crystallographic structures from the glycoside hydrolase family 32 (GH32) available in databases was performed and a three-dimensional model of B. longum KN29.1 β-fructofuranosidase was proposed. The modeled structure was compared to the structure of B. longum KN29.1 β-fructofuranosidase which was solved by X-ray crystallography and published after the first version of this manuscript had been submitted. The described B. longum KN29.1 enzyme is a monomeric protein consisting of 2 domains: 5-bladed β-propeller catalytic domain and β-sandwich domain.