Two β-galactosidases from the human isolate Bifidobacterium breve DSM 20213: molecular cloning and expression, biochemical characterization and synthesis of galacto-oligosaccharides

• Published in: PloS one Vol. 9; no. 8; p. e104056
• Main Authors: Arreola, Sheryl Lozel, Intanon, Montira, Suljic, Jasmina, Kittl, Roman, Pham, Ngoc Hung, Kosma, Paul, Haltrich, Dietmar, Nguyen, Thu-Ha
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 04.08.2014; Public Library of Science (PLoS)
• Subjects: Bacteria; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; beta-Galactosidase - genetics; beta-Galactosidase - metabolism; Bifidobacterium - enzymology; Bifidobacterium - genetics; Bifidobacterium - isolation & purification; Bifidobacterium breve; Biology and Life Sciences; Biotechnology; Chaperones; Chaperonin 10 - genetics; Chaperonin 10 - metabolism; Chaperonin 60 - genetics; Chaperonin 60 - metabolism; Chemical synthesis; Cloning; Cloning, Molecular; D-Galactose; E coli; Enzymes; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Food science; Galactose; Galactose - metabolism; Gene Expression; Glucose; Glucose - metabolism; Glycoside hydrolase; Homogeneity; Humans; Hydrogen ions; Hydrogen-Ion Concentration; Hydrolase; Hydrolysis; Infant; Intestine; Intestines - microbiology; Isoenzymes - genetics; Isoenzymes - metabolism; Kinetics; Laboratories; Lactobacillus; Lactose; Lactose - metabolism; Life sciences; Microbiota; Molecular Weight; Natural resources; Oligosaccharides; Oligosaccharides - biosynthesis; pH effects; Prebiotics; Probiotics; Protein Multimerization; Proteins; Reaction products; Substrates; Sugar; Temperature; Austria; Philippines; Germany
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0104056

Two β-galactosidases, β-gal I and β-gal II, from Bifidobacterium breve DSM 20213, which was isolated from the intestine of an infant, were overexpressed in Escherichia coli with co-expression of the chaperones GroEL/GroES, purified to electrophoretic homogeneity and biochemically characterized. Both β-gal I and β-gal II belong to glycoside hydrolase family 2 and are homodimers with native molecular masses of 220 and 211 kDa, respectively. The optimum pH and temperature for hydrolysis of the two substrates o-nitrophenyl-β-D-galactopyranoside (oNPG) and lactose were determined at pH 7.0 and 50°C for β-gal I, and at pH 6.5 and 55°C for β-gal II, respectively. The kcat/Km values for oNPG and lactose hydrolysis are 722 and 7.4 mM-1s-1 for β-gal I, and 543 and 25 mM-1s-1 for β-gal II. Both β-gal I and β-gal II are only moderately inhibited by their reaction products D-galactose and D-glucose. Both enzymes were found to be very well suited for the production of galacto-oligosaccharides with total GOS yields of 33% and 44% of total sugars obtained with β-gal I and β-gal II, respectively. The predominant transgalactosylation products are β-D-Galp-(1→6)-D-Glc (allolactose) and β-D-Galp-(1→3)-D-Lac, accounting together for more than 75% and 65% of the GOS formed by transgalactosylation by β-gal I and β-gal II, respectively, indicating that both enzymes have a propensity to synthesize β-(1→6) and β-(1→3)-linked GOS. The resulting GOS mixtures contained relatively high fractions of allolactose, which results from the fact that glucose is a far better acceptor for galactosyl transfer than galactose and lactose, and intramolecular transgalactosylation contributes significantly to the formation of this disaccharide.