Crystal structure of sucrose phosphorylase from Bifidobacterium adolescentis

Around 80 enzymes are implicated in the generic starch and sucrose pathways. One of these enzymes is sucrose phosphorylase, which reversibly catalyzes the conversion of sucrose and orthophosphate to d-Fructose and alpha-d-glucose 1-phosphate. Here, we present the crystal structure of sucrose phosphorylase from Bifidobacterium adolescentis (BiSP) refined at 1.77 A resolution. It represents the first 3D structure of a sucrose phosphorylase and is the first structure of a phosphate-dependent enzyme from the glycoside hydrolase family 13. The structure of BiSP is composed of the four domains A, B, B', and C. Domain A comprises the (beta/alpha)(8)-barrel common to family 13. The catalytic active-site residues (Asp192 and Glu232) are located at the tips of beta-sheets 4 and 5 in the (beta/alpha)(8)-barrel, as required for family 13 members. The topology of the B' domain disfavors oligosaccharide binding and reduces the size of the substrate access channel compared to other family 13 members, underlining the role of this domain in modulating the function of these enzymes. It is remarkable that the fold of the C domain is not observed in any other known hydrolases of family 13. BiSP was found as a homodimer in the crystal, and a dimer contact surface area of 960 A(2) per monomer was calculated. The majority of the interactions are confined to the two B domains, but interactions between the loop 8 regions of the two barrels are also observed. This results in a large cavity in the dimer, including the entrance to the two active sites.     

Note: Purification of amylase secreted from Bifidobacterium adolescentis

Bifidobacterium adolescentis Int-57 isolated from human faeces produced extracellular amylase. The enzyme was purified from the culture supernatant fluids by ammonium sulphate precipitation, gel-filtration chromatography (Sephadex-G-75), ion-exchange chromatography (CM-cellulose) and FPLC. SDS-PAGE of the purified enzyme revealed a major band with an apparent molecular weight of 66 kDa. The pI was 5·2. Enzyme activity was optimal at 50°C, and at pH 5·5. The enzyme was stable at 20–40°C, and at pH 5–6 with a K _m value of 2·4 g l⁻¹ soluble starch. The activation energy was 42·3 kJ mol⁻¹. The enzyme was significantly inhibited by maltose (10%), glucose (10%), Cu²+ (5 mmol l⁻¹), Zn²+ (5 mmol l⁻¹), N- bromosuccinimide (5 mmol l⁻¹), EDTA (5 mmol l⁻¹), I₂ (1 mmol l⁻¹) and activated by β-mercaptoethanol (10 mmol l⁻¹).     

Production of Cell-Wall-Bound Proteinases in Bifidobacterium adolescentis 94-BIM

Bifidobacterium adolescentis 94-BIM was found to produce cell-wall-bound proteolytic enzymes active at acidic, neutral, and alkaline pH values. The solubilization of proteinases with 0.5% Triton X-100 substantially improved the yield of the enzymes. The most active accumulation of cell-bound proteinases was observed in the third hour of cultivation at rates of 156.7, 179.5, and 111.1 U/(mg h), measured at pH 2.5, 7.0, and 9.0, respectively. It is suggested that the cell-wall-bound proteinases of B. adolescentis 94-BIM are the precursors of the enzymes secreted into the medium.     

Functional properties of anti-inflammatory substances from quercetin-treated Bifidobacterium adolescentis

Abstract

The genus Bifidobacterium is well known to have beneficial health effects. We discovered that quercetin and related polyphenols enhanced the secretion of anti-inflammatory substances by Bifidobacterium adolescentis. This study investigated characteristics of the anti-inflammatory substances secreted by B. adolescentis. The culture supernatant of B. adolescentis with quercetin reduced the levels of inflammatory mediators in activated macrophages. Spontaneous quercetin degradant failed to increase anti-inflammatory activity, while the enhancement of anti-inflammatory activity by quercetin was sustained after washout of quercetin. Physicochemical treatment of the culture supernatant indicated that its bioactive substances may be heat-stable, non-phenolic, and acidic biomolecules with molecular weights less than 3 kDa. Acetate and lactate have little or no effect on nitric oxide production. Taken together, the anti-inflammatory substances secreted by B. adolescentis may be small molecules but not short chain fatty acids. In agreement with these findings, stearic acid was tentatively identified as a bioactive candidate compound.     

Characterization of the Cell-Bound Polysaccharides of Bifidobacterium adolescentis 94 BIM

The cell-bound polysaccharides (PSs) of Bifidobacterium adolescentis 94 BIM were stripped from the cell surface with 2% sodium dodecyl sulfate (SDS), 1.5% Cetavlon, and 1% Triton X-100 and purified by precipitation with 5 volumes of ethanol. According to the extraction conditions used, the polysaccharide preparations were designated as PS-SDS-6°C, PS-SDS-100°C, PS-Cet, and PS-Trit. The gel-permeation chromatography of the first two preparations with the use of a Bio-Gel P-10 column and 1% acetic acid as the eluant yielded two peaks, F1 and F2, which contained carbohydrates and no phosphorus. All polysaccharides were primarily composed of glucose and galactose. The polysaccharides PS-Cet and PS-Trit were found to be branched and contain glucose residues at the terminal position, position 4, and position 6, and galactose residue at position 3. PS-SDS-6°C has a glucose residue at position 4.     

Antioxidative properties and inhibitory effect of Bifidobacterium adolescentis on melanogenesis

Melanin is a dark pigment produced by melanocytes. Tyrosinase is a key enzyme which catalyzes the rate-limiting step of melanogenesis. However, accumulation of melanin leads to various skin hyperpigmentation disorders. To find a novel skin-whitening agent, the antioxidant capacity of Bifidobacterium adolescentis culture filtrate and inhibitory effect on melanogenesis were investigated. The antioxidant effects of B. adolescentis culture filtrate include 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging capacity, 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid)(ABTS) radical cation scavenging activity and reducing power were measured spectrophotometrically. The reducing power is a useful index for the evaluation of potential antioxidants which carry out reduction of ferricyanide to ferrocyanide. Furthermore, the inhibitory effects of the bacterial culture filtrate on mushroom tyrosinase, B16F10 intracellular tyrosinase activity and melanin content were also determined. The results revealed that B. adolescentis culture filtrate (2.5, 5.0 and 7.5?%; v/v) effectively scavenged DPPH and ABTS radicals, and lower concentrations of the bacterial culture filtrates (0.5, 1.0 and 1.5?%; v/v) showed potent reducing power in a dose-dependent pattern. Additionally, the bacterial culture filtrate suppressed murine tyrosinase activity and decreased the amount of melanin in a dose-dependent manner. Our results demonstrated that B. adolescentis culture filtrate decreases the melanogenesis process of melanoma cells by inhibiting tyrosinase activity, which we suggest may be mediated through its antioxidant activity.     

Characterization of the cell-bound polysaccharides of Bifidobacterium adolescentis 94 BIM

The cell-bound polysaccharides (PSs) of Bifidobacterium adolescentis 94 BIM were stripped from the cell surface with 2% sodium dodecyl sulfate (SDS), 1.5% Cetavlon, and 1% Triton X-100 and purified by precipitation with 5 volumes of ethanol. According to the extraction conditions used, the polysaccharide preparations were designated as PS-SDS-6 degrees C, PS-SDS-100 degrees C, PS-Cet, and PS-Trit. The gel-permeation chromatography of the first two preparations with the use of a Bio-Gel P-10 column and 1% acetic acid as the eluant yielded two peaks, F1 and F2, which contained carbohydrates and no phosphorus. All polysaccharides were primarily composed of glucose and galactose. The polysaccharides PS-Cet and PS-Trit were found to be branched and contain glucose residues at the terminal position, position 4, and position 6, and galactose residue at position 3. PS-SDS-6 degrees C has a glucose residue at position 4.     

Structural rearrangements of sucrose phosphorylase from Bifidobacterium adolescentis during sucrose conversion

The reaction mechanism of sucrose phosphorylase from Bifidobacterium adolescentis (BiSP) was studied by site-directed mutagenesis and x-ray crystallography. An inactive mutant of BiSP (E232Q) was co-crystallized with sucrose. The structure revealed a substrate-binding mode comparable with that seen in other related sucrose-acting enzymes. Wild-type BiSP was also crystallized in the presence of sucrose. In the dimeric structure, a covalent glucosyl intermediate was formed in one molecule of the BiSP dimer, and after hydrolysis of the glucosyl intermediate, a beta-D-glucose product complex was formed in the other molecule. Although the overall structure of the BiSP-glucosyl intermediate complex is similar to that of the BiSP(E232Q)-sucrose complex, the glucose complex discloses major differences in loop conformations. Two loops (residues 336-344 and 132-137) in the proximity of the active site move up to 16 and 4 A, respectively. On the basis of these findings, we have suggested a reaction cycle that takes into account the large movements in the active-site entrance loops.     

Production of proteinases associated with Bifidobacterium adolescentis 94-BIM cell wall

Bifidobacterium adolescentis 94-BIM was found to produce cell-wall bound proteolytic enzymes active at acidic, neutral, and alkaline pH values. The solubilization of proteinases with 0.5% Triton X-100 substantially improved the yield of the enzymes. The most active accumulation of cell-bound proteinases was observed in the third hour of cultivation at rates of 156.7, 179.5, and 111.1 U/(mg h), measured at pH 2.5, 7.0, and 9.0, respectively. It is suggested that the cell-wall bound proteinases of B. adolescentis 94-BIM are the precursors of the enzymes secreted into the medium.     

Allergic Patients with Long-Term Asthma Display Low Levels of Bifidobacterium adolescentis

Accumulated evidence suggests a relationship between specific allergic processes, such as atopic eczema in children, and an aberrant fecal microbiota. However, little is known about the complete microbiota profile of adult individuals suffering from asthma. We determined the fecal microbiota in 21 adult patients suffering allergic asthma (age 39.43 ± 10.98 years old) and compare it with the fecal microbiota of 22 healthy controls (age 39.29 ± 9.21 years old) using culture independent techniques. An Ion-Torrent 16S rRNA gene-based amplification and sequencing protocol was used to determine the fecal microbiota profile of the individuals. Sequence microbiota analysis showed that the microbial alpha-diversity was not significantly different between healthy and allergic individuals and no clear clustering of the samples was obtained using an unsupervised principal component analysis. However, the analysis of specific bacterial groups allowed us to detect significantly lower levels of bifidobacteria in patients with long-term asthma. Also, in allergic individuals the Bifidobacterium adolescentis species prevailed within the bifidobacterial population. The reduction in the levels on bifidobacteria in patients with long-term asthma suggests a new target in allergy research and opens possibilities for the therapeutic modulation of the gut microbiota in this group of patients.     

Structure of 6-deoxytalose-containing polysaccharide from the cell wall of Bifidobacterium adolescentis

The isolation and analysis of the cell wall and the polysaccharide-glycopeptide complexes of Bifidobacterium adolescentis YIT4011 are presented. Polysaccharide-glycopeptide complexes, PS-GP1 and PS-GP2, were solubilized from the cell wall by treatment with N-acetylmuramidase. PS-GP1 and PS-GP2 were found to be composed of glucose, 6-deoxytalose and a small amount of glycopeptide. The products of Smith degradation of the PS-GPs had no glucose-containing fraction, but were composed of 1,2/1,3-linked 6-deoxytalose. Furthermore, a second Smith degradation of this fraction yielded trisaccharide-glyceraldehyde. These results and methylation analysis led to the conclusion that PS-GP1 or 2 has a repeating unit of----3)6dTal(beta 1----3)6dTal(beta 1----3)6dTal(beta 1----2)-6dTal(alpha 1----2)6dTal(alpha 1----2)6dTal(alpha 1-, and that glucose residues are linked to position C-3 of the 2-O-substituted 6-deoxytalose residues.     

Recombinant expression and characterization of a reducing-end xylose-releasing exo-oligoxylanase from Bifidobacterium adolescentis

The family 8 glycoside hydrolase (RexA) from Bifidobacterium adolescentis was expressed in Escherichia coli. The recombinant enzyme was characterized as a reducing-end xylose-releasing exo-oligoxylanase. Apart from giving insights into this new class of enzymes, knowledge of the RexA enzyme helps to postulate a mechanism for the B. adolescentis breakdown of prebiotic xylooligosaccharides.     

青春双歧杆菌对1型糖尿病小鼠胰岛β细胞的保护作用

目的 探讨青春双歧杆菌对1型糖尿病小鼠胰岛β细胞的保护作用.方法 给予1型糖尿病小鼠口服青春双歧杆菌,观察实验组和对照组体重的变化及糖尿病发病率.ELISA法测定血清、脾细胞上清IL-4和IFN-γ浓度,电镜观察胰岛超微结构变化.结果 15周龄时实验组体重(23.41±1.64)g, 而对照组为(22.31±1.32)g(P<0.05);实验组发病率低于对照组(P＜0.05);实验组血清、脾细胞上清IL-4浓度明显高于对照组,而IFN-γ低于对照组(P<0.05);对照组残存胰岛的β细胞数目稀少,有核膜和内浆网扩张、核糖体脱颗粒和分泌颗粒空泡样变等超微结构异常,而实验组胰岛β细胞数显著多于对照组(P<0.05),且无上述超微异常.结论 青春双歧杆菌通过上调IL-4水平,降低IFN-γ水平,促使免疫平衡向Th2方向偏移,且有保护胰岛β细胞超微结构的作用,在一定程度上可预防和延缓NOD鼠糖尿病的发生.     

Bifidobacterium adolescentis protects from the development of nonalcoholic steatohepatitis in a mouse model

To investigate the hypothesis that an oral supplementation of Bifidobacterium adolescentis protects against a diet-induced nonalcoholic steatohepatitis in a mouse model, C57BL/6 mice were fed either a Western-style or a control diet±tap water fortified with B. adolescentis (5×10⁷ cfu/ml) ad libitum for 12 weeks. Mice fed a Western-style diet gained significantly more weight than mice fed a control diet and developed a mild steatohepatitis. Western-style diet fed groups concomitantly treated with B. adolescentis had significantly decreased liver damage, whereas portal endotoxin levels and toll-like receptor-4 protein levels as well as myeloid differentiation factor 88 mRNA were increased in livers of both Western-style diet fed groups. The protective effects of the B. adolescentis were associated with a significant attenuation of the formation of reactive oxygen species, activation of nuclear factor κB (NFκB) and induction of markers of inflammation in the liver. Taken together, our data suggest that an oral supplementation of the B. adolescentis attenuates diet-induced steatohepatitis, and this effect is associated with prevention from lipid peroxidation, NFκB activation and finally inflammation in the liver.     

Characterization of a novel beta-galactosidase from Bifidobacterium adolescentis DSM 20083 active towards transgalactooligosaccharides

This paper reports on the effects of both reducing and nonreducing transgalactooligosaccharides (TOS) comprising 2 to 8 residues on the growth of Bifidobacterium adolescentis DSM 20083 and on the production of a novel beta-galactosidase (beta-Gal II). In cells grown on TOS, in addition to the lactose-degrading beta-Gal (beta-Gal I), another beta-Gal (beta-Gal II) was detected and it showed activity towards TOS but not towards lactose. beta-Gal II activity was at least 20-fold higher when cells were grown on TOS than when cells were grown on galactose, glucose, and lactose. Subsequently, the enzyme was purified from the cell extract of TOS-grown B. adolescentis by anion-exchange chromatography, adsorption chromatography, and size-exclusion chromatography. Beta-Gal II has apparent molecular masses of 350 and 89 kDa as judged by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, indicating that the enzyme is active in vivo as a tetramer. Beta-Gal II had an optimal activity at pH 6 and was not active below pH 5. Its optimum temperature was 35 degrees C. The enzyme showed highest V(max) values towards galactooligosaccharides with a low degree of polymerization. This result is in agreement with the observation that during fermentation of TOS, the di- and trisaccharides were fermented first. Beta-Gal II was active towards beta-galactosyl residues that were 1-->4, 1-->6, 1-->3, and 1 <--> 1 linked, signifying its role in the metabolism of galactooligosaccharides by B. adolescentis.     

青春双歧杆菌对NOD小鼠1型糖尿病的免疫调节作用

目的 探讨早期应用青春双歧杆菌对NOD小鼠1型糖尿病发病的影响.方法 给予NOD小鼠口服青春双歧杆菌,观察实验组和对照组(PBS)糖尿病发病率,HE染色观察胰岛炎,免疫组化检测胰岛Bcl-2和Bax的表达,RT-PCR测定TNF-α、IFN-γ和IL-10 mRNA表达.结果 实验组胰岛炎程度较对照组明显减轻(P＜0.01),胰岛Bcl-2的表达高于对照组,而Bax的表达低于对照组(P＜0.05);胰腺TNF-α、IFN-γ mRNA表达实验组明显低于对照组(P＜0.05),而IL-10 mRNA表达差异无显著性;实验组发病率低于对照组(P＜0.05).结论 青春双歧杆菌对NOD小鼠1型糖尿病有预防作用,其机制可能与调节Th1/Th2型细胞因子的免疫失衡有关.     

Binding of human plasminogen to Bifidobacterium

Bifidobacteria constitute up to 3% of the total microbiota and represent one of the most important health-promoting bacterial groups of the human intestinal microflora. The presence of Bifidobacterium in the human gastrointestinal tract has been directly related to several health-promoting activities; however, to date, no information about the specific mechanisms of interaction with the host is available. In order to provide some insight into the molecular mechanisms involved in the interaction with the host, we investigated whether Bifidobacterium was able to capture human plasminogen on the cell surface. By using flow cytometry, we demonstrated a dose-dependent human plasminogen-binding activity for four strains belonging to three bifidobacterial species: Bifidobacterium lactis, B. bifidum, and B. longum. The binding of human plasminogen to Bifidobacterium was dependent on lysine residues of surface protein receptors. By using a proteomic approach, we identified five putative plasminogen-binding proteins in the cell wall fraction of the model strain B. lactis BI07. The data suggest that plasminogen binding to B. lactis is due to the concerted action of a number of proteins located on the bacterial cell surface, some of which are highly conserved cytoplasmic proteins which have other essential cellular functions. Our findings represent a step forward in understanding the mechanisms involved in the Bifidobacterium-host interaction.     

Intra-Species Diversity between Seven Bifidobacterium adolescentis Strains Identified by Genome-Wide Tiling Array Analysis

Six Bifidobacterium adolescentis strains, JCM1275, JCM1251, JCM7044, JCM7045, JCM7046, and 9-124, were analyzed using a tiling array designed according to the full genome sequence of ATCC15703. The results demonstrated deletion clusters along with single-gene mutations and deletions. Most deletions concerned genes involved in polysaccharide and cell-surface biogenesis. A dendrogram illustrating the deletions and mutations is presented and the evolution of B. adolescentis is discussed.     

Increasing the transglycosylation activity of alpha-galactosidase from Bifidobacterium adolescentis DSM 20083 by site-directed mutagenesis

The alpha-galactosidase (AGA) from Bifidobacterium adolescentis DSM 20083 has a high transglycosylation activity. The optimal conditions for this activity are pH 8, and 37 degrees C. At high melibiose concentration (600 mM), approximately 64% of the enzyme-substrate encounters resulted in transglycosylation. Examination of the acceptor specificity showed that AGA required a hydroxyl group at C-6 for transglycosylation. Pentoses, hexuronic acids, deoxyhexoses, and alditols did not serve as acceptor molecules. Disaccharides were found to be good acceptors. A putative 3D-structure of the catalytic site of AGA was obtained by homology modeling. Based on this structure and amino acid sequence alignments, site-directed mutagenesis was performed to increase the transglycosylation efficiency of the enzyme, which resulted in four positive mutants. The positive single mutations were combined, resulting in six double mutants. The mutant H497M had an increase in transglycosylation of 16%, whereas most of the single mutations showed an increase of 2%-5% compared to the wild-type AGA. The double mutants G382C-Y500L, and H497M-Y500L had an increase in transglycosylation activity of 10%-16%, compared to the wild-type enzyme, whereas the increase for the other double mutants was low (4%-7%). The results show that with a single mutation (H497M) the transglycosylation efficiency can be increased from 64% to 75% of all enzyme-substrate encounters. Combining successful single mutants in double mutations did not necessarily result in an extra increase in transglycosylation efficiency. The donor and acceptor specificity did not change in the mutants, whereas the thermostability of the mutants with G382C decreased drastically.