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

Bifidobacterium longum KN29.1 exhibits greater beta-fructofuranosidase activity than two Bifidobacterium animalis strains with highest activity observed when B. longum KN29.1 was grown on fructose. Properties of the native beta-fructofuranosidase from B. longum KN29.1 and recombinant beta-fructofuranosidase were identical. The molecular mass of the purified beta-fructofuranosidase was approximately 67 kDa 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 degrees C. It was optimally active in Actilight (R) 950P and sucrose hydrolysis at pH 6.0-6.2 while the optimum temperature of these processes was 50 degrees C and 37-45 degrees C, respectively. In the presence of Cu2+, Ag+, Hg+ ions and p-chloromercuribenzoic acid (1 mM) B. longum KN29.1 beta-fructofuranosidase activity was completely inhibited, its slight stimulation was observed in solutions containing Mn2+ (1 mM). This enzyme showed higher affinity for nystose (K-m 1.2 +/- 0.15 mM) and 1-kestose (K-m 4.6 +/- 0.2 mM) than for sucrose (K-m 29.4 +/- 1.5 mM). 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 beta-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 beta-fructofuranosidase was proposed. The modeled structure was compared to the structure of B. longum KN29.1 beta-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 beta-propeller catalytic domain and beta-sandwich domain. (C) 2011 Elsevier Ltd. All rights reserved.