Cloning, Expression, Purification, and Characterization of β-Galactosidase from Bifidobacterium longum and Bifidobacterium pseudocatenulatum

Du, Mingzhu; Yang, Shuanghong; Jiang, Tong; Liang, Tingting; Li, Ying; Cai, Shuzhen; Wu, Qingping; Zhang, Jumei; Chen, Wei; Xie, Xinqiang

doi:10.3390/molecules27144497

Cited by 3 publications

(2 citation statements)

References 45 publications

(47 reference statements)

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“…Common sources of obtaining this enzyme are plants, fungi and bacteria. Many strains of Bifidobacterium , including B. adolenscentis and B. longum , show the ability to produce it, which additionally increases their probiotic value [ 43 , 44 ]. Therefore, we decided to check whether the selected strains produce this enzyme, and if so, whether its production is influenced by the external environment.…”

Section: Discussionmentioning

confidence: 99%

What happens to Bifidobacterium adolescentis and Bifidobacterium longum ssp. longum in an experimental environment with eukaryotic cells?

Jakubczyk,

Leszczyńska,

Pacyga-Prus

et al. 2024

BMC Microbiol

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Background The impact of probiotic strains on host health is widely known. The available studies on the interaction between bacteria and the host are focused on the changes induced by bacteria in the host mainly. The studies determining the changes that occurred in the bacteria cells are in the minority. Within this paper, we determined what happens to the selected Bifidobacterium adolescentis and Bifidobacterium longum ssp. longum in an experimental environment with the intestinal epithelial layer. For this purpose, we tested the bacteria cells' viability, redox activity, membrane potential and enzymatic activity in different environments, including CaCo-2/HT-29 co-culture, cell culture medium, presence of inflammatory inductor (TNF-α) and oxygen. Results We indicated that the external milieu impacts the viability and vitality of bacteria. Bifidobacterium adolescentis decrease the size of the live population in the cell culture medium with and without TNF-α (p < 0.001 and p < 0.01 respectively). In contrast, Bifidobacterium longum ssp. longum significantly increased survivability in contact with the eukaryotic cells and cell culture medium (p < 0.001). Bifidobacterium adolescentis showed significant changes in membrane potential, which was decreased in the presence of eukaryotic cells (p < 0.01), eukaryotic cells in an inflammatory state (p < 0.01), cell culture medium (p < 0.01) and cell culture medium with TNF-α (p < 0.05). In contrast, Bifidobacterium longum ssp. longum did not modulate membrane potential. Instead, bacteria significantly decreased the redox activity in response to milieus such as eukaryotic cells presence, inflamed eukaryotic cells as well as the culture medium (p < 0.001). The redox activity was significantly different in the cells culture medium vs the presence of eukaryotic cells (p < 0.001). The ability to β-galactosidase production was different for selected strains: Bifidobacterium longum ssp. longum indicated 91.5% of positive cells, whereas Bifidobacterium adolescentis 4.34% only. Both strains significantly reduced the enzyme production in contact with the eukaryotic milieu but not in the cell culture media. Conclusion The environmental-induced changes may shape the probiotic properties of bacterial strains. It seems that the knowledge of the sensitivity of bacteria to the external environment may help to select the most promising probiotic strains, reduce research costs, and contribute to greater reproducibility of the obtained probiotic effects.

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Section: Discussionmentioning

confidence: 99%

What happens to Bifidobacterium adolescentis and Bifidobacterium longum ssp. longum in an experimental environment with eukaryotic cells?

Jakubczyk,

Leszczyńska,

Pacyga-Prus

et al. 2024

BMC Microbiol

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“…In addition to colorimetric detection methods, active β-galactosidases can be detected in vivo by fluorescence, bioluminescence, chemiluminescence, magnetic resonance (MR), single photoemission computed tomography or positron emission tomography (PET) [16]. Similar to in vivo detection, in vitro determination of β-galactosidase activity is often performed using chromogenic or fluorogenic labeled substrates [17][18][19]. The products are usually detected by a photometer or fluorimeter, respectively.…”

Section: Introductionmentioning

confidence: 99%

Expression and Characterization of a β-Galactosidase from the Pacific Oyster, Crassostrea gigas, and Evaluation of Strategies for Testing Substrate Specificity

Thoma,

Grabherr,

Staudacher

2023

IJMS

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β-Galactosidases (EC 3.2.1.23) are exoglycosidases that catalyze the cleavage of glycoconjugates with terminal β-D-galactose residues in β1,3-, β1,4- or β1,6-linkage. Although this family of exoglycosidases has been extensively studied in vertebrates, plants, yeast, and bacteria, little information is available for mollusks. Mollusks are a diverse and highly successful group of animals that play many different roles in their ecosystems, including filter feeders and detritivores. Here, the first β-galactosidase from the Pacific oyster, Crassostrea gigas was discovered, biochemically characterized, and compared to our previously characterized slug enzyme from Arion vulgaris (UniProt Ref. Nr.: A0A0B7AQJ9). Overall, the mussel enzyme showed similar biochemical parameters to the snail enzyme. The enzyme from C. gigas was most active in an acidic environment (pH 3.5) and at a reaction temperature of 50 °C. Optimal storage conditions were up to 37 °C. In contrast to the enzyme from A. vulgaris, the supplementation of cations (Ni2+, Co2+, Mn2+, Mg2+, Ca2+, Cu2+, Ba2+) increased the activity of the enzyme from C. gigas. Substrate specificity studies of the β-galactosidases from the mussel, C. gigas, and the slug, A. vulgaris, revealed activity towards terminal β1,3- and β1,4-linked galactose residues for both enzymes. Using the same substrates in labeled and unlabeled form, we were able to detect the effect of labeling on the β-galactosidase activity using MALDI-TOF MS, HPTLC, and HPLC. While lactose was cleaved by the enzymes in an unlabeled or labeled state, galacto-N-biose was not cleaved as soon as a 2-amino benzoic acid label was added. In this study we present the biochemical characterization of the first recombinantly expressed β-galactosidase from the Pacific oyster, C. gigas, and we compare different analytical methods for the determination of β-galactosidase activity using the enzyme from C. gigas and A. vulgaris.

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In Vitro Production of Galactooligosaccharides by a Novel β-Galactosidase of Lactobacillus bulgaricus

Arsov

Ivanov²,

Tsigoriyna³

et al. 2022

IJMS

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β-galactosidase is an enzyme with dual activity and important industrial application. As a hydrolase, the enzyme eliminates lactose in milk, while as a trans-galactosidase it produces prebiotic galactooligosaccharides (GOS) with various degrees of polymerization (DP). The aim of the present study is the molecular characterization of β-galactosidase from a Bulgarian isolate, Lactobacillus delbrueckii subsp. bulgaricus 43. The sequencing of the β-gal gene showed that it encodes a new enzyme with 21 amino acid replacements compared to all other β-galactosidases of this species. The molecular model revealed that the new β-galactosidase acts as a tetramer. The amino acids D207, H386, N464, E465, Y510, E532, H535, W562, N593, and W980 form the catalytic center and interact with Mg2+ ions and substrate. The β-gal gene was cloned into a vector allowing heterologous expression of E. coli BL21(DE3) with high efficiency, as the crude enzyme reached 3015 U/mL of the culture or 2011 U/mg of protein. The enzyme’s temperature optimum at 55 °C, a pH optimum of 6.5, and a positive influence of Mg2+, Mn2+, and Ca2+ on its activity were observed. From lactose, β-Gal produced a large amount of GOS with DP3 containing β-(1→3) and β-(1→4) linkages, as the latter bond is particularly atypical for the L. bulgaricus enzymes. DP3-GOS formation was positively affected by high lactose concentrations. The process of lactose conversion was rapid, with a 34% yield of DP3-GOS in 6 h, and complete degradation of 200 g/L of lactose for 12 h. On the other hand, the enzyme was quite stable at 55 °C and retained about 20% of its activity after 24 h of incubation at this temperature. These properties expand our horizons as regards the use of β-galactosidases in industrial processes for the production of lactose-free milk and GOS-enriched foods.

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Cloning, Expression, Purification, and Characterization of β-Galactosidase from Bifidobacterium longum and Bifidobacterium pseudocatenulatum

Cited by 3 publications

References 45 publications

What happens to Bifidobacterium adolescentis and Bifidobacterium longum ssp. longum in an experimental environment with eukaryotic cells?

What happens to Bifidobacterium adolescentis and Bifidobacterium longum ssp. longum in an experimental environment with eukaryotic cells?

Expression and Characterization of a β-Galactosidase from the Pacific Oyster, Crassostrea gigas, and Evaluation of Strategies for Testing Substrate Specificity

In Vitro Production of Galactooligosaccharides by a Novel β-Galactosidase of Lactobacillus bulgaricus

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