Transcriptional regulation and characterization of a novel beta-fructofuranosidase-encoding gene from Bifidobacterium breve UCC2003.
An operon involved in fructooligosaccharide breakdown was identified in the genome of Bifidobacterium breve UCC2003. This 2.6-kb transcriptional unit was comprised of three genes that encoded a putative permease, a conserved hypothetical protein, and a beta-fructofuranosidase. Active transcription of the operon was observed when B. breve UCC2003 was grown on sucrose or Actilight, while transcription appeared to be repressed when the organism was grown on glucose, fructose, a combination of glucose and sucrose, or a combination of fructose and sucrose. The beta-fructofuranosidase encoded by this operon was purified and biochemically characterized. The optimum pH and temperature for catalytic activity were determined to be pH 6.0 and 37 degrees C, respectively, and there was a dependence on bivalent cations, particularly manganese. The Km and Vmax values for sucrose hydrolysis were determined to be 25 +/- 2 mM and 24 +/- 3 micromol min(-1) mg(-1), respectively. Interestingly, the enzyme was shown to specifically catalyze cleavage of the beta(2-1) glycosidic bond between glucose and its neighboring fructose moiety in sucrose and other fructooligosaccharides with a relatively low degree of polymerization, and there was no detectable activity towards the beta(2-1) glycosidic bond between two fructose moieties within the same substrate. To our knowledge, such an enzymatic activity has not previously been described in bifidobacteria or other gram-positive bacteria.
Ryan
Sinéad M
SM
Alimentary Pharmabiotic Centre, National University of Ireland Cork, Western Road, Cork, Ireland. sineadmryan@hotmail.com.
Fitzgerald
Gerald F
GF
van Sinderen
Douwe
D
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001644:Bifidobacterium / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D003001:Cloning, Molecular; D005632:Fructose / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D009876:Operon; D017422:Sequence Analysis, DNA; D013395:Sucrose / Q000378:metabolism; D014158:Transcription, Genetic; D043324:beta-Fructofuranosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D001426:Bacterial Proteins; D005632:Fructose; D013395:Sucrose; D043324:beta-Fructofuranosidase