Identification of the gene for beta-fructofuranosidase of Bifidobacterium lactis DSM10140(T) and characterization of the enzyme expressed in Escherichia coli

Curr Microbiol. 2003 Jun;46(6):391-7. doi: 10.1007/s00284-002-3908-1.


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] 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.

MeSH terms

  • Amino Acid Sequence
  • Base Composition
  • Base Sequence
  • Bifidobacterium / enzymology
  • Bifidobacterium / genetics*
  • Bifidobacterium / metabolism
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics
  • Escherichia coli / genetics
  • Glycoside Hydrolases / genetics*
  • Glycoside Hydrolases / metabolism
  • Hydrogen-Ion Concentration
  • Kinetics
  • Molecular Sequence Data
  • Molecular Weight
  • Polymerase Chain Reaction
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Sequence Alignment
  • Sequence Analysis, DNA
  • Substrate Specificity
  • Sucrose / metabolism
  • beta-Fructofuranosidase


  • DNA, Bacterial
  • Recombinant Proteins
  • Sucrose
  • Glycoside Hydrolases
  • beta-Fructofuranosidase