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Applied and Environmental Microbiology, July 2005, p. 3475-3482, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3475-3482.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Transcriptional Regulation and Characterization of a Novel ß-Fructofuranosidase-Encoding Gene from Bifidobacterium breve UCC2003

Sinéad M. Ryan,1,2* Gerald F. Fitzgerald,1,2,3 and Douwe van Sinderen1,2

Alimentary Pharmabiotic Centre,1 Departments of Microbiology,2 Food and Nutritional Sciences, Bioscience Institute, National University of Ireland, Cork, Western Road, Cork, Ireland3

Received 11 November 2004/ Accepted 9 January 2005

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 ß-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 ß-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°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 µmol min–1 mg–1, respectively. Interestingly, the enzyme was shown to specifically catalyze cleavage of the ß(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 ß(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.

* Corresponding author. Mailing address: Department of Microbiology, National University of Ireland Cork, Western Road, Cork, Ireland. Phone: 353 21 4901364. Fax: 353 21 4903101. E-mail: sineadmryan{at}hotmail.com.

Applied and Environmental Microbiology, July 2005, p. 3475-3482, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3475-3482.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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