Identification of the Gene for β-Fructofuranosidase of Bifidobacterium lactis DSM10140^T and Characterization of the Enzyme Expressed in Escherichia coli

Abstract

Bifidobacterium lactis is a moderately oxygen-tolerant, saccharolytic bacterium often used in combination with fructooligosaccharides (FOS) as a probiotic supplement in diverse dairy products. This is the first report describing the gene structure and enzymatic properties of a β-fructofuranosidase [EC 3.2.1.26] from Bifidobacteria. BfrA was identified in Bifidobacterium lactis DSM 10140^T and heterologously expressed in Escherichia coli. The G+C content was identical with the G+C content as determined for the total genomic DNA (61.9 mol %). The gene codes for a 532-aa residue polypeptide of 59.4 kDa. Surprisingly, the deduced aa sequence revealed only minor similarity to other fructofuranosidases (18% to E. coli cscA). The enzyme was purified to homogeneity after incorporation of a C-terminal 6xHIS affinity tag. It hydrolased sucrose, 1-kestose, Raftilose®, Actilight®, inulin, and raffinose (100%, 91%, 84%, 80%, 37%, 4%). Fructose moieties were released in an exo-type fashion. Substrates with α-glycosidic linkages or residues other than fructose were not attacked. The kinetic parameters K _m and V _max for sucrose hydrolysis were 10.3 mM and 0.031 μM/min (pH 7.6; 37°C). The activity was abolished by Zn²⁺ (1 mM) and significantly inhibited by Fe²⁺ and Ni²⁺ (10 mM). The enzyme showed its maximal activity at 40°C.