Identification of the gene for beta-fructofuranosidase of Bifidobacterium lactis DSM10140T and characterization of the enzyme expressed in Escherichia coli
2003
Ehrmann, M.A. | Korakli, M. | Vogel, R.F.
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 beta-fructofuranosidase [EC 3.2.1.26] from Bifidobacteria. BfrA was identified in Bifidobacterium lactis DSM 10140T 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 alpha-glycosidic linkages or residues other than fructose were not attacked. The kinetic parameters Km and Vmax for sucrose hydrolysis were 10.3 mM and 0.031 micromolar/min (pH 7.6; 37 degrees 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 degrees C.
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