Purification and Characterization of Two Novel β-Galactosidases from Lactobacillus reuteri

Abstract

The intracellular beta-galactosidase (beta-gal) enzymes from two strains of Lactobacillus reuteri, L103 and L461, were purified by ammonium sulfate fractionation, hydrophobic interaction, and affinity chromatography. Both enzymes are heterodimers with a molecular mass of 105 kDa, consisting of a 35 kDa subunit and a 72 kDa subunit. Active staining of L. reuteri L103 and L461 beta-gal with 4-methylumbelliferyl beta-d-galactoside showed that the intact enzymes as well as the larger subunits possess beta-galactosidase activity. The isoelectric points of L. reuteri L461 and L103 beta-gal were found to be in the range of 3.8-4.0 and 4.6-4.8, respectively. Both enzymes are most active in the pH range of 6-8; however, they are not stable at pH 8. The L. reuteri beta-galactosidases are activated by various mono- and divalent cations, including Na(+), K(+), and Mn(2+), and are moderately inhibited by their reaction products d-glucose and d-galactose. Because of their origin from beneficial and potentially probiotic lactobacilli, these enzymes could be of interest for the synthesis of prebiotic galacto-oligosaccharides.

Selection of lactic acid bacteria strain for simultaneous production of α- and β-galactosidases

“…On the other hand β-galactosidase showed broad range of optimal temperatures 40-50 °C. These results match previously reported results for L. Reuteri [53].…”

Causes of the Production of Multiple Forms of β-Galactosidase byBacillus circulans

“…These findings indicate that -Gal-A, -Gal-B, -Gal-C, and -Gal-D are undoubtedly monomeric proteins, although most bacterial -galactosidases reported to date are multimeric, except for that from Streptococcus mitis. 31) -Galactosidases from L. reuteri, 15) Enterobacter agglomerans B1, 20) and L. plantarum WCFS 30) are dimeric, those from B. adolescentis DSM20083, 14) B. infantis HL96, 28) and E. coli, 58) tetrameric, and that from B. bifidum NCIMB41171 22,25) is hexameric. Some of the biochemical characteristics determined for purified -Gal-A, -Gal-B, -Gal-C, and -Gal-D are summarized in Table 1.…”

“…GOS are important prebiotics that enhance the proliferation of intestinal bifidobacteria and lactobacilli in the colon, which in turn promote human health by protecting individuals against infection, reducing the accumulation of harmful bacteria/toxic compounds, and facilitating the normal function of the gut. [8][9][10][11] Because of this, a number of studies have reported attempts to isolate a -galactosidase with high transgalactosylation activity from various microorganisms, [4][5][6][7][8] including Bacillus circulans, 12,13) Bifidobacterium adolescentis, 14) Lactobacillus reuteri, 15,16) and others. [17][18][19][20] Furthermore, the genes that encode -galactosidases from bacteria, including B. circulans, 21) Bifidobacterium bifidum [22][23][24][25] / Bifidobacterium infantis, 22,[26][27][28] L. reuteri, 29) L. plantarum, 30) and others, 20,31) have been cloned and sequenced, and the enzymes have been overexpressed in heterologous cells.…”

Production of β-galactosidase from streptococcus thermophilus for galactooligosaccharides synthesis

“…The activity assay was carried out as described by Splechtna et al (2007) and Nguyen et al (2006) with some modifications. Th e chromoge nic substrate o-nitropheno l-β -Dgalactopyranoside (ONPG) (4 mg mL −1 ) was dissolved in 50 mM sodium phosphate buffer (pH 6.8).…”