Identification of the gene for beta-fructofuranosidase of Bifidobacterium lactis DSM10140(T) and characterization of the enzyme expressed in Escherichia coli.
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 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 6 x HIS 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 K(m) and V(max) for sucrose hydrolysis were 10.3 m M and 0.031 microM/min (pH 7.6; 37 degrees C). The activity was abolished by Zn(2+) (1 m M) and significantly inhibited by Fe(2+) and Ni(2+) (10 m M). The enzyme showed its maximal activity at 40 degrees C.
Ehrmann
Matthias A
MA
Lehrstuhl für Technische Mikrobiologie, Technische Universität München, 85350 Freising-Weihenstephan, Germany. M.ehrmann@lrz.tu-muenchen
Korakli
Maher
M
Vogel
Rudi F
RF
D000595:Amino Acid Sequence; D001482:Base Composition; D001483:Base Sequence; D001644:Bifidobacterium / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism; D043324:beta-Fructofuranosidase
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D013395:Sucrose; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase