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Applied and Environmental Microbiology, May 2001, p. 2276-2283, Vol. 67, No. 5
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.5.2276-2283.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Intra- and Extracellular beta -Galactosidases from Bifidobacterium bifidum and B. infantis: Molecular Cloning, Heterologous Expression, and Comparative Characterization

Peter L. Møller,dagger Flemming Jørgensen, Ole C. Hansen, Søren M. Madsen, and Peter Stougaard*

Biotechnological Institute, DK-2970 Hørsholm, Denmark

Received 30 October 2000/Accepted 27 February 2001

Three beta -galactosidase genes from Bifidobacterium bifidum DSM20215 and one beta -galactosidase gene from Bifidobacterium infantis DSM20088 were isolated and characterized. The three B. bifidum beta -galactosidases exhibited a low degree of amino acid sequence similarity to each other and to previously published beta -galactosidases classified as family 2 glycosyl hydrolases. Likewise, the B. infantis beta -galactosidase was distantly related to enzymes classified as family 42 glycosyl hydrolases. One of the enzymes from B. bifidum, termed BIF3, is most probably an extracellular enzyme, since it contained a signal sequence which was cleaved off during heterologous expression of the enzyme in Escherichia coli. Other exceptional features of the BIF3 beta -galactosidase were (i) the monomeric structure of the active enzyme, comprising 1,752 amino acid residues (188 kDa) and (ii) the molecular organization into an N-terminal beta -galactosidase domain and a C-terminal galactose binding domain. The other two B. bifidum beta -galactosidases and the enzyme from B. infantis were multimeric, intracellular enzymes with molecular masses similar to typical family 2 and family 42 glycosyl hydrolases, respectively. Despite the differences in size, molecular composition, and amino acid sequence, all four beta -galactosidases were highly specific for hydrolysis of beta -D-galactosidic linkages, and all four enzymes were able to transgalactosylate with lactose as a substrate.

* Corresponding author. Mailing address: Biotechnological Institute, Kogle Allé 2, DK-2970 Hørsholm, Denmark. Phone: 45 45160444. Fax: 45 45160455. E-mail: pst{at}bioteknologisk.dk.

dagger Present address: Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, DK-1958 Frederiksberg C, Denmark.

Applied and Environmental Microbiology, May 2001, p. 2276-2283, Vol. 67, No. 5
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.5.2276-2283.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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