Synthesis of β-Galactooligosaccharide Using Bifidobacterial β-Galactosidase Purified from Recombinant Escherichia coli
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Galactooligosaccharides (GOSs) are known to be selectively utilized by Bifidobacterium, which can bring about healthy changes of the composition of intestinal microflora. In this study, β-GOS were synthesized using bifidobacterial β-galactosidase (G1) purified from recombinant E. coli with a high GOS yield and with high productivity and enhanced bifidogenic activity. The purified recombinant G1 showed maximum production of β-GOSs at pH 8.5 and 45oC. A matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the major peaks of the produced β-GOSs showed MW of 527 and 689, indicating the synthesis of β-GOSs at degrees of polymerization (DP) of 3 and DP4, respectively. The trisaccharides were identified as β-D-galactopyranosyl-(1→4)-O-β-D-galactopyranosyl-(1→4)-O-β-D-glucopyranose, and the tetrasaccharides were identified as β-D-galactopyranosyl-(1→4)-O-β-D-galactopyranosyl- (1→4)-O-β-D-galactopyranosyl-(1→4)-O-β-D-glucopyranose. The maximal production yield of GOSs was as high as 25.3% (w/v) using purified recombinant β-galactosidase and 36% (w/v) of lactose as a substrate at pH 8.5 and 45oC. After 140 min of the reaction under this condition, 268.3 g/l of GOSs was obtained. With regard to the prebiotic effect, all of the tested Bifidobacterium except for B. breve grew well in BHI medium containing β-GOS as a sole carbon source, whereas lactobacilli and Streptococcus thermophilus scarcely grew in the same medium. Only Bacteroides fragilis, Clostridium ramosum, and Enterobacter cloacae among the 17 pathogens tested grew in BHI medium containing β-GOS as a sole carbon source; the remaining pathogens did not grow in the same medium. Consequently, the β-GOS are expected to contribute to the beneficial change of intestinal microbial flora.
Abstract
Galactooligosaccharides (GOSs) are known to be selectively utilized by Bifidobacterium, which can bring about healthy changes of the composition of intestinal microflora. In this study, β-GOS were synthesized using bifidobacterial β-galactosidase (G1) purified from recombinant E. coli with a high GOS yield and with high productivity and enhanced bifidogenic activity. The purified recombinant G1 showed maximum production of β-GOSs at pH 8.5 and 45oC. A matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the major peaks of the produced β-GOSs showed MW of 527 and 689, indicating the synthesis of β-GOSs at degrees of polymerization (DP) of 3 and DP4, respectively. The trisaccharides were identified as β-D-galactopyranosyl-(1→4)-O-β-D-galactopyranosyl-(1→4)-O-β-D-glucopyranose, and the tetrasaccharides were identified as β-D-galactopyranosyl-(1→4)-O-β-D-galactopyranosyl- (1→4)-O-β-D-galactopyranosyl-(1→4)-O-β-D-glucopyranose. The maximal production yield of GOSs was as high as 25.3% (w/v) using purified recombinant β-galactosidase and 36% (w/v) of lactose as a substrate at pH 8.5 and 45oC. After 140 min of the reaction under this condition, 268.3 g/l of GOSs was obtained. With regard to the prebiotic effect, all of the tested Bifidobacterium except for B. breve grew well in BHI medium containing β-GOS as a sole carbon source, whereas lactobacilli and Streptococcus thermophilus scarcely grew in the same medium. Only Bacteroides fragilis, Clostridium ramosum, and Enterobacter cloacae among the 17 pathogens tested grew in BHI medium containing β-GOS as a sole carbon source; the remaining pathogens did not grow in the same medium. Consequently, the β-GOS are expected to contribute to the beneficial change of intestinal microbial flora.
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