Characterization of recombinant β-fructofuranosidase from Bifidobacterium adolescentis G1

Background: We have previously reported on purification and characterization of beta-fructofuranosidase (beta-FFase) from Bifidobacterium adolescentis G1. This enzyme showed high activity of hydrolysis on fructo-oligosaccharides with a low degree of polymerization. Recently, genome sequences of B. longum NCC2705 and B. adolescentis ATCC 15703 were determined, and cscA gene in the both genome sequences encoding beta-FFase was predicted. Here, cloning of cscA gene encoding putative beta-FFase from B. adolescentis G1, its expression in E. coli and properties of the recombinant protein are described. Results: 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 beta-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 degrees C. The K-m (mM), V-max (mu mol/mg of protein/min), k(0) (sec(-1)) and k(0)/K-m(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. Conclusion: 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.