Three different types (A, B, and C) of bile salt hydrolase from different Bifidobacterium strains revealed during the purification study showed the type-specific characteristics in their electrophoretic migration and elution profiles from anion exchange and hydrophobic interaction chromatographic columns. The subunit molecular mass estimated by SDS-PAGE was around 35 kDa and the native molecular mass in all types of BSH was estimated to be between 130 and 150 kDa by gel filtration chromatography, indicating that all BSH enzymes have tetrameric structure. From the isoelectric focusing, pI value of 4.45 was obtained with type B, but type A and C BSHs showed similar pI values of around 4.65. While the N-terminal amino acid sequences of types A and B were highly homologous (19/20), six out of twenty amino acid residues were different in the N-terminal sequences of types A and C. / As the type A bsh gene was cloned from a strain of B. longum and the nucleotide sequence became available from the GenBank, our study has focused on the cloning and characterization of the type B and C bsh genes from Bifidobacterium strains. / The type B bsh gene was cloned from B. bifidum ATCC11863 and the DNA flanking the bsh gene was sequenced. The 951 by-long bsh gene encoded a 316-amino-acid protein with a molecular mass of 35 kDa and a pI of 4.48. For the first time in the genus Bifidobacterium, the transcriptional start point of the bsh gene was identified by primer extension analysis. Furthermore, Northern blot analysis revealed that B. bifidum bsh was transcribed as a monocistronic unit, contrary to that of B. longum bsh. Despite a high level of sequence similarity among the bsh genes, a BSH type-specific primer set based on the variable regions of bsh genes was designed in order to differentiate B. bifidum strains from the other species of Bifidobacterium commonly detected in the human gut. / The type C bsh gene was cloned from a bile salt tolerant strain of Bifidobacterium and the DNA flanking the bsh gene was further identified by a thermal asymmetric interlaced PCR (TAIL-PCR) technique. The 945 by-long bsh gene encoded a 314-amino-acid protein with a molecular mass of 35 kDa and a pI of 4.71. A predicted BSH promoter (Pbsh) sequence was experimentally verified and the transcriptional start point of the type C bsh gene was determined by primer extension analysis. An operonic structure including the type C bsh gene and two more ORFs, which were found within a complete set of a promoter and a transcription terminator, was identified in this study for the first time in the genus Bifidobacterium, and the polycistronic bsh transcript was revealed by RT-PCR and Northern blot analysis.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.84272 |
| Date | January 2004 |
| Creators | Kim, Geun-Bae, 1966- |
| Contributors | Lee, Byong H. (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Food Science and Agricultural Chemistry.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 002149981, proquestno: AAINQ98290, Theses scanned by UMI/ProQuest. |
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