Characterization of recombinant β-fructofuranosidase from Bifidobacterium adolescentis G1

Using the information of cscA gene from Bifidobacterium adolescentis ATCC 15703, cscA gene from B. adolescentis G1 was cloned and sequenced. The N-terminal amino acid sequence of purified β-FFase from B. adolescentis G1 was identical to the deduced amino acid sequences of cscA gene from B. adolescentis G1. To confirm the translated product of the cscA gene, the recombinant protein was expressed in Escherichia coli. Molecular mass of the purified recombinant enzyme was estimated to be about 66,000 by SDS-PAGE and 60,300 by MALDI TOF-MS. The optimum pH of the enzyme was 5.7 and the enzyme was stable at pH 5.0-8.6. The thermostability of the enzyme was up to 50°C. The Km (mM), Vmax (μmol/mg of protein/min), k0 (sec-1) and k0/Km(mM-1 sec-1) for 1-kestose, neokestose, nystose, fructosylnystose, sucrose and inulin were 1.7, 107, 107.5, 63.2, and 1.7, 142, 142.7, 83.9, and 3.9, 152, 152.8, 39.2, and 2.2, 75, 75.4, 34.3, and 38, 79, 79.4, 2.1, and 25.9, 77, 77.4, 3.0, respectively. The hydrolytic activity was strongly inhibited by AgNO3, SDS, and HgCl2.The recombinant enzyme had similar specificity to the native enzyme, high affinity for 1-kestose, and low affinity for sucrose and inulin, although properties of the recombinant enzyme showed slight difference from those of the native one previously described.Bifidobacteria are saccharolytic anaerobes generally present in human intestine. Growth of bifidobacteria is selectively promoted by prebiotics [1].Fructo-oligosaccharides, such as 1-kestose, nystose and fructosylnystose, consist of β-2,1-linked fructose to sucrose, and they are naturally contained in artichoke tubers [2], chicory roots [3] and burdock roots [4,5]. These saccharides have been produced and commercially manufactured from sucrose with bacterial fructosyltransferase [6] and β-fructofuransidases (β-FFases) [7-9], and have been on the market as prebiotics. Fructo-oligosaccharides are not hydrolyzed by digestive enzymes of mammalian origin, so they are able to