Beta-galactosidase from Lactobacillus pentosus: purification, characterization and formation of galacto-oligosaccharides

• Published in: Biotechnology journal Vol. 5; no. 8; p. 838
• Main Authors: Maischberger, Thomas, Leitner, Elisabeth, Nitisinprasert, Sunee, Juajun, Onladda, Yamabhai, Montarop, Nguyen, Thu-Ha, Haltrich, Dietmar
• Format: Journal Article
• Language: English
• Published: Germany 01.08.2010
• Subjects: Ammonium Sulfate; Bacterial Proteins - chemistry; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; beta-Galactosidase - chemistry; beta-Galactosidase - isolation & purification; beta-Galactosidase - metabolism; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Food Technology; Galactans - biosynthesis; Galactans - chemistry; Galactans - metabolism; Galactose - metabolism; Glycosylation; Hydrogen-Ion Concentration; Kinetics; Lactobacillus - chemistry; Lactobacillus - enzymology; Lactose - chemistry; Lactose - metabolism; Nitrophenylgalactosides - chemistry; Nitrophenylgalactosides - metabolism; Substrate Specificity; Temperature
• ISSN: 1860-7314
• DOI: 10.1002/biot.201000126

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.