Molecular and biochemical analysis of two beta-galactosidases from Bifidobacterium infantis HL96.
Two genes encoding beta-galactosidase isoenzymes, beta-galI and beta-galIII, from Bifidobacterium infantis HL96 were revealed on 3.6- and 2.4-kb DNA fragments, respectively, by nucleotide sequence analysis of the two fragments. beta-galI (3,069 bp) encodes a 1,022-amino-acid (aa) polypeptide with a predicted molecular mass of 113 kDa. A putative ribosome binding site and a promoter sequence were recognized at the 5' flanking region of beta-galI. Further upstream a partial sequence of an open reading frame revealed a putative lactose permease gene transcribing divergently from beta-galI. The beta-galIII gene (2,076 bp) encodes a 691-aa polypeptide with a calculated molecular mass 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 are homologous to those found in the LacZ and the 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, which featured a glutamate at residue 160. The coding regions of the beta-galI and beta-galIII genes were each cloned downstream of a T7 promoter for overexpression in Escherichia coli. The molecular masses of the overexpressed proteins, as estimated by polyacrylamide gel electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, agree with their predicted molecular weights. beta-GalI and beta-GalIII were specific for beta-D-anomer-linked galactoside substrates. Both are more active in response to ONPG (o-nitrophenyl-beta-D-galactopyranoside) than in response to lactose, particularly beta-GalIII. The galacto-oligosaccharide yield in the reaction catalyzed by beta-GalI at 37 degrees C in 20% (wt/vol) lactose solution was 130 mg/ml, which is more than six times higher than the maximum yield obtained with beta-GalIII. The structure of the major trisaccharide produced by beta-GalI catalysis was characterized as O-beta-D-galactopyranosyl-(1-3)-O-beta-D-galactopyranosyl-(1-4)-D-glucopyranose (3'-galactosyl-lactose).
Hung
M N
MN
Department of Food Science and Agricultural Chemistry, McGill University, Ste-Anne-de-Bellevue, Quebec H9X 3V9, Canada.
Xia
Z
Z
Hu
N T
NT
Lee
B H
BH
D000595:Amino Acid Sequence; D001483:Base Sequence; D001644:Bifidobacterium / Q000201:enzymology* / Q000235:genetics; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D005690:Galactose / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D009844:Oligosaccharides; D001616:beta-Galactosidase; D005690:Galactose