title: Characterization of a heterodimeric GH2 β-galactosidase from Lactobacillus sakei Lb790 and formation of prebiotic galacto-oligosaccharides.
abstract: The lacLM genes from Lactobacillus sakei Lb790, encoding a heterodimeric β-galactosidase that belongs to glycoside hydrolase family GH2, were cloned and heterologously expressed in Escherichia coli . Subsequently, the recombinant β-galactosidase LacLM was purified to apparent homogeneity and characterized. The enzyme is a β-galactosidase with narrow substrate specificity because o-nitrophenyl-β-D-galactopyranoside (oNPG) was efficiently hydrolyzed, whereas various structurally related oNP analogues were not. The K(m) and k(cat) values for oNPG and lactose were 0.6 mM and 180 s(-1) and 20 mM and 43 s(-1), respectively. The enzyme is inhibited competitively by its two end-products D-galactose and D-glucose (K(i) values of 180 and 475 mM, respectively). As judged by the ratio of the inhibition constant to the Michaelis constant, K(i)/K(m), this inhibition is only very moderate and much less pronounced than for other microbial β-galactosidases. β-Galactosidase from L. sakei possesses high transgalactosylation activity and was used for the synthesis of galacto-oligosaccharides (GalOS), employing lactose at a concentration of 215 g/L. The maximum GalOS yield was 41% (w/w) of total sugars at 77% lactose conversion and contained mainly non-lactose disaccharides, trisaccharides, and tetrasaccharides with approximately 38, 57, and 5% of total GalOS formed, respectively. The enzyme showed a strong preference for the formation of β-(1→6)-linked transgalactosylation products, whereas β-(1→3)-linked compounds were formed to a lesser extent and β-(1→4)-linked reaction products could not be detected.
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Sanaullah
authors.initials: S
authors.lastname: Iqbal
authors.forename: Thu-Ha
authors.initials: TH
authors.lastname: Nguyen
authors.forename: Hoang Anh
authors.initials: HA
authors.lastname: Nguyen
authors.forename: Tien Thanh
authors.initials: TT
authors.lastname: Nguyen
authors.forename: Thomas
authors.initials: T
authors.lastname: Maischberger
authors.forename: Roman
authors.initials: R
authors.lastname: Kittl
authors.forename: Dietmar
authors.initials: D
authors.lastname: Haltrich
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology*; D009844:Oligosaccharides / Q000096:biosynthesis*; D056692:Prebiotics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D001616:beta-Galactosidase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D009844:Oligosaccharides; D056692:Prebiotics; D011994:Recombinant Proteins; D001616:beta-Galactosidase