Molecular and biochemical analysis of two β-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 -gall. 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 -Gall 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 -Gall, 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 -Gall 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 degreesC 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 -Gall catalysis was characterized as O-beta -D-galactopyranosyl-(1-3)-O-beta -D-galactopyranosyl-(1-4)-D-glucopyranose (3'-galactosyl-lactose).