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Applied and Environmental Microbiology, September 2004, p. 5603-5612, Vol. 70, No. 9
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.9.5603-5612.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Cloning and Characterization of the Bile Salt Hydrolase Genes (bsh) from Bifidobacterium bifidum Strains

Geun-Bae Kim,1 Carol M. Miyamoto,2 Edward A. Meighen,2 and Byong H. Lee1,3*

Department of Food Science and Agricultural Chemistry,1 Department of Biochemistry, McGill University, Ste-Anne-de-Bellevue,2 Food Research and Development Center, Agriculture and Agri-Food Canada, Ste-Hyacinthe, Quebec, Canada3

Received 19 December 2003/ Accepted 16 May 2004

Biochemical characterization of the purified bile salt hydrolase (BSH) from Bifidobacterium bifidum ATCC 11863 revealed some distinct characteristics not observed in other species of Bifidobacterium. The bsh gene was cloned from B. bifidum, and the DNA flanking the bsh gene was sequenced. Comparison of the deduced amino acid sequence of the cloned gene with previously known sequences revealed high homology with BSH enzymes from several microorganisms and penicillin V amidase (PVA) of Bacillus sphaericus. The proposed active sites of PVA were highly conserved, including that of the Cys-1 residue. The importance of the SH group in the N-terminal cysteine was confirmed by substitution of Cys with chemically and structurally similar residues, Ser or Thr, both of which resulted in an inactive enzyme. The transcriptional start point of the bsh gene has been determined by primer extension analysis. Unlike Bifidobacterium longum bsh, B. bifidum bsh was transcribed as a monocistronic unit, which was confirmed by Northern blot analysis. PCR amplification with the type-specific primer set revealed the high level of sequence homology in their bsh genes within the species of B. bifidum.

* Corresponding author. Mailing address: Department of Food Science and Agricultural Chemistry, McGill University, 21111 Lakeshore Rd., Ste-Anne-de-Bellevue, Quebec H9X 3V9, Canada. Phone: (514) 398-7979. Fax: (514) 398-7977. E-mail: byong.lee{at}mcgill.ca.

Applied and Environmental Microbiology, September 2004, p. 5603-5612, Vol. 70, No. 9
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.9.5603-5612.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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