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Applied and Environmental Microbiology, September 2001, p. 4256-4263, Vol. 67, No. 9
Department of Food Science and Agricultural
Chemistry1 and Department of
Chemistry,2 McGill University,
Ste-Anne-de-Bellevue, Quebec H9X 3V9, and Food Research and
Development Center, Agriculture and Agri-Food Canada, Ste-Hyacinthe,
Quebec J2S 8E3,4 Canada, and
Department of Biochemistry, National Chung Hsing University,
Taichung, Taiwan 402273
Received 28 July 2000/Accepted 30 May 2001
Two genes encoding
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.9.4256-4263.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Molecular and Biochemical Analysis of Two
-Galactosidases from Bifidobacterium infantis
HL96
-galactosidase isoenzymes,
-galI and -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. -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 -galI.
Further upstream a partial sequence of an open reading frame revealed a
putative lactose permease gene transcribing divergently from
-galI. The -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
-GalI and -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
-GalI, and a possible acid-base site proposed for the LacG family
was located in -GalIII, which featured a glutamate at residue 160. The coding regions of the -galI and
-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. -GalI and -GalIII were specific for
-D-anomer-linked galactoside substrates. Both are more
active in response to ONPG
(o-nitrophenyl--D-galactopyranoside) than
in response to lactose, particularly -GalIII. The
galacto-oligosaccharide yield in the reaction catalyzed by -GalI at
37°C in 20% (wt/vol) lactose solution was 130 mg/ml, which is more
than six times higher than the maximum yield obtained with -GalIII.
The structure of the major trisaccharide produced by -GalI catalysis
was characterized as
O--D-galactopyranosyl-(1-3)-O--D-galactopyranosyl-(1-4)-D-glucopyranose (3'-galactosyl-lactose).
*
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:
blee{at}macdonald.mcgill.ca.
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