title: High-efficiency synthesis of oligosaccharides with a truncated beta-galactosidase from Bifidobacterium bifidum.
abstract: An exceptionally large beta-galactosidase, BIF3, with a subunit molecular mass of 188 kDa (1,752 amino acid residues) was recently isolated from Bifidobacterium bifidum DSM20215 [Møller et al. (2001) Appl Environ Microbiol 67:2276-2283]. The BIF3 polypeptide comprises a signal peptide followed by an N-terminal beta-galactosidase region and a C-terminal galactose-binding motif. We have investigated the functional importance of the C-terminal part of the BIF3 sequence by deletion mutagenesis and expression of truncated enzyme variants in Escherichia coli. Deletion of approximately 580 amino acid residues from the C-terminal end converted the enzyme from a normal, hydrolytic beta-galactosidase into a highly efficient, transgalactosylating enzyme. Quantitative analysis showed that the truncated beta-galactosidase utilised approximately 90% of the reacted lactose for the production of galacto-oligosaccharides, while hydrolysis constituted a 10% side reaction. This 9:1 ratio of transgalactosylation to hydrolysis was maintained at lactose concentrations ranging from 10% to 40%, implying that the truncated beta-galactosidase behaved as a "true" transgalactosylase even at low lactose concentrations.
authors.affiliation: Department of Enzyme Technology, Biotechnological Institute, Hørsholm, Denmark.
authors.forename: F
authors.initials: F
authors.lastname: Jørgensen
authors.forename: O C
authors.initials: OC
authors.lastname: Hansen
authors.forename: P
authors.initials: P
authors.lastname: Stougaard
mesh_terms: D001644:Bifidobacterium / Q000201:enzymology* / Q000235:genetics; D001709:Biotechnology; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D007785:Lactose / Q000378:metabolism; D008970:Molecular Weight; D009844:Oligosaccharides / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D009844:Oligosaccharides; D011994:Recombinant Proteins; D001616:beta-Galactosidase; D007785:Lactose