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Biochemical and genomic analysis of -galactosidases from Bifidobacterium infantis HL96

Among 29 strains of bifidobacteria studied as sources of beta-galactosidase enzyme, Bifidobacterium infantis HL96 showed the highest hydrolytic and transgalactosylic activities. This strain grew well in a MRS medium containing various sugars including lactose, and produced three beta-galactosidases (termed beta-Gal I, II, III). / Two genes, beta-galI and beta-galIII, located on 4.6 and 4.4 kb DNA fragments respectively, were cloned into E. coli, and the nucleotide sequences were determined. The 3,069 by-long beta-galI, encoded a polypeptide with a Mr of 113 kDa. A putative ribosome-binding site and a promoter sequence were recognized at the 5' flanking region of beta-galI. A partial sequence of an ORF transcribing divergently from beta-galI resembled a lactose permease gene. The beta-galIII gene, which is 2,076 bp long, encoded a polypeptide with a Mr of 76 kDa. A rho-independent, transcription terminator-like sequence was found 25 bp downstream of the termination codon. / The amino acid sequences of beta-GalI and beta-GalIII were homologous to those in the LacZ and LacG families, respectively. The acid-base, nucleophilic, and substrate recognition sites conserved in the LacZ family were found in beta-GalI, and a possible acid-base site proposed for the LacG family was located in beta-GalIII, containing a glutamate at residue 160. beta-GalI and beta-GalIII were over-expressed 35 and 96 times respectively in E. coli by using a pET expression system. / Both beta-GalI and beta-GalIII were specific for beta-D -anomeric linked galactosides, but beta-GalI showed more hydrolytic and synthetic activities toward lactose than beta-GalIII. The galacto-oligosaccharides (GaOS) production mediated by beta-GalI at 37°C in 20% (w/v) lactose was 130 mg/ml, which is six times higher than that of beta-GalIII. The yield of GaOS further increased to 190 mg/ml in 30% (w/v) lactose. A major tri-saccharide produced by beta-GalI was characterized as O-beta- D-galactopyranosyl-(1-3)-O-beta-D-galactopyranosyl-(1-4)- D-glucopyranose. / beta-GalI was purified by ammonium sulphate precipitation, and anion-exchange (Mono-Q) and gel filtration (Superose 12) chromatographic steps. The enzyme appeared to be a tetramer, with a Mr of 470 kDa as estimated by native PAGE and gel-filtration chromatography. The optimum temperature and pH for ONPG and lactose as substrates were 60°C, pH 7.5, and 50°C, pH 7.5, respectively. The enzyme was stable over the pH range of 5~8.5, and was particularly active at 50°C for more than 80 min. The enzyme was significantly activated by reducing agents, especially glutathione, as well as by Na+ and K+ cations. Maximal activity required both Na+ and K+ at a concentration of 10 mM. The enzyme was strongly inhibited by p-chloromercuribenzoic acid, and by most bivalent metal ions. Hydrolytic activity using 20 mM lactose as substrate was significantly inhibited by 10 mM galactose. The Km and Vmax values for ONPG and lactose were 2.6 mM, 262 U/mg, and 73.8 mM, 1.28 U/mg, respectively. / The objectives of this research were to characterize beta-galactosidases of B. infantis HL96 at the molecular and biochemical levels, and to over-express the enzymes in Escherichia coli. Two beta-galactosidase isoenzymes with unique properties were genetically characterized for the first time. beta-GalI properties included a neutral pH optimum, relatively higher temperature stability and a high transgalactosylic activity that makes it very competitive for GaOS synthesis. The results were also important for the advancement of knowledge on the catalytic mechanism and the evolutionary aspect of this enzyme.

Identiferoai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.36953
Date January 2001
CreatorsHung, Ming-Ni, 1962-
ContributorsLee, Byong H. (advisor)
PublisherMcGill University
Source SetsLibrary and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada
LanguageEnglish
Detected LanguageEnglish
TypeElectronic Thesis or Dissertation
Formatapplication/pdf
CoverageDoctor of Philosophy (Department of Food Science and Agricultural Chemistry.)
RightsAll items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated.
Relationalephsysno: 001809512, proquestno: NQ70044, Theses scanned by UMI/ProQuest.

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