Beta-galactosidase from Lactobacillus pentosus: purification, characterization and formation of galacto-oligosaccharides.
A novel heterodimeric beta-galactosidase with a molecular mass of 105 kDa was purified from crude cell extracts of the soil isolate Lactobacillus pentosus KUB-ST10-1 using ammonium sulphate fractionation followed by hydrophobic interaction and affinity chromatography. The electrophoretically homogenous enzyme has a specific activity of 97 U(oNPG)/mg protein. The K(m), k(cat) and k(cat)/K(m) values for lactose and o-nitrophenyl-beta-D-galactopyranoside (oNPG) were 38 mM, 20 s(-1), 530 M(-1).s(-1) and 1.67 mM, 540 s(-1), 325 000 M(-1).s(-1), respectively. The temperature optimum of beta-galactosidase activity was 60-65 degrees C for a 10-min assay, which is considerably higher than the values reported for other lactobacillal beta-galactosidases. Mg(2+) ions enhanced both activity and stability significantly. L. pentosus beta-galactosidase was used for the production of prebiotic galacto-oligosaccharides (GOS) from lactose. A maximum yield of 31% GOS of total sugars was obtained at 78% lactose conversion. The enzyme showed a strong preference for the formation of beta-(1-->3) and beta-(1-->6) linkages, and the main transgalactosylation products identified were the disaccharides beta-D-Galp-(1-->6)-D-Glc, beta-D-Galp-(1-->3)-D-Glc, beta-D-Galp-(1-->6)-D-Gal, beta-D-Galp-(1-->3)-D-Gal, and the trisaccharides beta-D-Galp-(1-->3)-D-Lac, beta-D-Galp-(1-->6)-D-Lac.
Maischberger
Thomas
T
BOKU University of Natural Resources and Applied Life Sciences, Vienna, Austria.
Leitner
Elisabeth
E
Nitisinprasert
Sunee
S
Juajun
Onladda
O
Yamabhai
Montarop
M
Nguyen
Thu-Ha
TH
Haltrich
Dietmar
D
D000645:Ammonium Sulfate; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005524:Food Technology; D005685:Galactans / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007778:Lactobacillus / Q000737:chemistry / Q000201:enzymology*; D007785:Lactose / Q000737:chemistry / Q000378:metabolism; D009598:Nitrophenylgalactosides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D001616:beta-Galactosidase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D005685:Galactans; C055012:2-nitrophenylgalactoside; D009598:Nitrophenylgalactosides; D001616:beta-Galactosidase; D007785:Lactose; D000645:Ammonium Sulfate; D005690:Galactose