Recombinant expression and characterization of a reducing-end xylose-releasing exo-oligoxylanase from Bifidobacterium adolescentis.
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ABSTRACT:
SUBMITTER: Lagaert S
PROVIDER: S-EPMC1950967 | biostudies-literature | 2007 Aug
REPOSITORIES: biostudies-literature
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Recombinant expression and characterization of a reducing-end xylose-releasing exo-oligoxylanase from Bifidobacterium adolescentis.
Lagaert Stijn S Van Campenhout Steven S Pollet Annick A Bourgois Tine M TM Delcour Jan A JA Courtin Christophe M CM Volckaert Guido G
Applied and environmental microbiology 20070622 16
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. ...[more]
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Project description:BACKGROUND:We have previously reported on purification and characterization of beta-fructofuranosidase (beta-FFase) from Bifidobacterium adolescentis G1. This enzyme showed high activity of hydrolysis on fructo-oligosaccharides with a low degree of polymerization. Recently, genome sequences of B. longum NCC2705 and B. adolescentis ATCC 15703 were determined, and cscA gene in the both genome sequences encoding beta-FFase was predicted. Here, cloning of cscA gene encoding putative beta-FFase from B. adolescentis G1, its expression in E. coli and properties of the recombinant protein are described. RESULTS:Using the information of cscA gene from Bifidobacterium adolescentis ATCC 15703, cscA gene from B. adolescentis G1 was cloned and sequenced. The N-terminal amino acid sequence of purified beta-FFase from B. adolescentis G1 was identical to the deduced amino acid sequences of cscA gene from B. adolescentis G1. To confirm the translated product of the cscA gene, the recombinant protein was expressed in Escherichia coli. Molecular mass of the purified recombinant enzyme was estimated to be about 66,000 by SDS-PAGE and 60,300 by MALDI TOF-MS. The optimum pH of the enzyme was 5.7 and the enzyme was stable at pH 5.0-8.6. The thermostability of the enzyme was up to 50 degrees C. The K(m) (mM), Vmax (micromol/mg of protein/min), k0 (sec(-1)) and k0/K(m)(mM(-1) sec(-1)) for 1-kestose, neokestose, nystose, fructosylnystose, sucrose and inulin were 1.7, 107, 107.5, 63.2, and 1.7, 142, 142.7, 83.9, and 3.9, 152, 152.8, 39.2, and 2.2, 75, 75.4, 34.3, and 38, 79, 79.4, 2.1, and 25.9, 77, 77.4, 3.0, respectively. The hydrolytic activity was strongly inhibited by AgNO3, SDS, and HgCl2. CONCLUSION:The recombinant enzyme had similar specificity to the native enzyme, high affinity for 1-kestose, and low affinity for sucrose and inulin, although properties of the recombinant enzyme showed slight difference from those of the native one previously described.
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Project description:Xylose isomerase (EC 5.3.1.5) is a key enzyme in xylose metabolism which is industrially important for the transformation of glucose and xylose into fructose and xylulose, respectively. The Bifidobacterium adolescentis xylA gene (NC_008618.1) encoding xylose isomerase (XI) was cloned and the enzyme was overexpressed in Escherichia coli. Purified recombinant XI was crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol 3350 as the precipitating agent. A complete native data set was collected to 1.7?Å resolution using a synchrotron-radiation source. The crystals belonged to the orthorhombic space group P21212, with unit-cell parameters a = 88.78, b = 123.98, c = 78.63?Å.
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Project description:UnlabelledA GH8 family enzyme involved in xylan depolymerization has been characterized. The enzyme, Rex8A, is a reducing-end xylose-releasing exo-oligoxylanase (Rex) that efficiently hydrolyzes xylooligosaccharides and shows minor activity on polymeric xylan. Rex8A hydrolyzes xylooligomers of 3 to 6 xylose units to xylose and xylobiose in long-term incubations. Kinetic constants of Rex8A were determined on xylotriose, showing a Km of 1.64 ± 0.03 mM and a kcat value of 118.8 s(-1) Besides linear xylooligosaccharides, the enzyme hydrolyzed decorated xylooligomers. The catalytic activity on branched xylooligosaccharides, i.e., the release of xylose from the reducing end, is a newly described trait of xylose-releasing exo-oligoxylanases, as the exo-activity on these substrates has not been reported for the few of these enzymes characterized to date. Modeling of the three-dimensional (3D) structure of Rex8A shows an (α/α)6 barrel fold where the loops connecting the α-helices contour the active site. These loops, which show high sequence diversity among GH8 enzymes, shape a catalytic cleft with a -2 subsite that can accommodate methyl-glucuronic acid decorations. The hydrolytic ability of Rex8A on branched oligomers can be crucial for the complete depolymerization of highly substituted xylans, which is indispensable to accomplish biomass deconstruction and to generate efficient catalysts.ImportanceA GH8 family enzyme involved in xylan depolymerization has been characterized. The Rex8A enzyme from Paenibacillus barcinonensis is involved in depolymerization of glucuronoxylan, a major component of the lignocellulosic substrates. The study shows that Rex8A is a reducing-end xylose-releasing exo-oligoxylanase that efficiently hydrolyzes xylose from neutral and acidic xylooligosaccharides generated by the action of other xylanases also secreted by the strain. The activity of a Rex enzyme on branched xylooligosaccharides has not been described to date. This report provides original and useful information on the properties of a new example of the rarely studied Rex enzymes. Depolymerization of highly substituted xylans is crucial for biomass valorization as a platform for generation of biofuels, chemicals, and solvents.
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Project description:The gene encoding β-mannanase (EC 3.2.1.78) BaMan26A from the bacterium Bifidobacterium adolescentis (living in the human gut) was cloned and the gene product characterized. The enzyme was found to be modular and to contain a putative signal peptide. It possesses a catalytic module of the glycoside hydrolase family 26, a predicted immunoglobulin-like module, and two putative carbohydrate-binding modules (CBMs) of family 23. The enzyme is likely cell attached either by the sortase mechanism (LPXTG motif) or via a C-terminal transmembrane helix. The gene was expressed in Escherichia coli without the native signal peptide or the cell anchor. Two variants were made: one containing all four modules, designated BaMan26A-101K, and one truncated before the CBMs, designated BaMan26A-53K. BaMan26A-101K, which contains the CBMs, showed an affinity to carob galactomannan having a dissociation constant of 0.34 μM (8.8 mg/liter), whereas BaMan26A-53K did not bind, showing that at least one of the putative CBMs of family 23 is mannan binding. For BaMan26A-53K, k(cat) was determined to be 444 s(-1) and K(m) 21.3 g/liter using carob galactomannan as the substrate at the optimal pH of 5.3. Both of the enzyme variants hydrolyzed konjac glucomannan, as well as carob and guar gum galactomannans to a mixture of oligosaccharides. The dominant product from ivory nut mannan was found to be mannotriose. Mannobiose and mannotetraose were produced to a lesser extent, as shown by high-performance anion-exchange chromatography. Mannobiose was not hydrolyzed, and mannotriose was hydrolyzed at a significantly lower rate than the longer oligosaccharides.
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Project description:Bifidobacteria are considered one of the most beneficial probiotics and have been widely studied for their effects against specific pathogens. The present study investigated the antiviral activity of probiotics isolated from Koreans against Coxsackievirus B3 (CVB3). The effect of probiotic isolates against CVB3 was measured by the plaque assay and cellular toxicity of bifidobacteria in HeLa cells was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Among 13 probiotic isolates, 3 Bifidobacterium adolescentis, 2 Bifidobacterium longum and 1 Bifidobacterium pseudocatenulatum had an antiviral effect against CVB3, while the others did not show such effect. B. adolescentis SPM1605 showed the greatest inhibitory properties against CVB3. When the threshold cycle (CT) values for the treated B. adolescentis SPM1605 samples were compared to the results for the non-treated samples, it was shown that the amplified viral sequences from the CVB3 had their copy number lowered by B. adolescentis SPM1605. Moreover, the gene expression in infected HeLa cells was also inhibited by 50%. The results suggest that B. adolescentis SPM1605 suppresses CVB3 and could be used as an alternative therapy against infectious diseases caused by coxsackieviruses.
| S-EPMC4433936 | biostudies-other
Project description:Bifidobacteria have been described as a key component of the human gut microbiota, and recently significant efforts have been made to investigate their genome contents and assess the genetic variability at inter- and intra-species level. In the current work we investigated genome diversity among representatives of bifidobacterial species, i.e., Bifidobacterium adolescentis. These analyses were performed with comparative genomic hybridization (CGH) experiments and they revealed the existence of a strictly conserved set of 685 gene families. Furthermore, CGH analyses showed that genetic regions of diversity included mobile elements and putative genomic life-style adaptation islands, such as loci that encode pili and capsular polysaccharides, and genes involved in carbohydrate metabolism. CGH analysis was performed with microarrays that were based on the genome sequences of B. adolescentis ATCC15703 (NC_008618) . A total of 39,249 probes of 35 bp in length were designed using OligoArray 2.1 software. Oligos were synthesized in triplicate on a 2 × 40-k CombiMatrix array (CombiMatrix, Mulkiteo, USA). Replicates were distributed on the chip at random, non-adjacent positions. A set of 74 negative control probes designed on phage and plant sequences was also included on the chip. Seventeen micrograms of purified genomic DNA was labeled with Cy5-ULS using the Kreatech ULS array CGH Labeling kit (Kreatech Diagnostics) according to the supplier’s instructions. Hybridization of labeled test DNA to these microarrays was performed according to CombiMatrix protocols.
2013-02-06 | GSE38992 | GEO
Project description:The gut microbiota is an important contributor to both health and disease. While previous studies have reported on the beneficial influences of the gut microbiota and probiotic supplementation on bone health, their role in recovery from skeletal injury and resultant systemic sequelae remains unexplored. This study aimed to determine the extent to which probiotics could modulate bone repair by dampening fracture-induced systemic inflammation. Our findings demonstrate that femur fracture induced an increase in gut permeability lasting up to 7 days after trauma before returning to basal levels. Strikingly, dietary supplementation with Bifidobacterium adolescentis augmented the tightening of the intestinal barrier, dampened the systemic inflammatory response to fracture, accelerated fracture callus cartilage remodeling, and elicited enhanced protection of the intact skeleton following fracture. Together, these data outline a mechanism whereby dietary supplementation with beneficial bacteria can be therapeutically targeted to prevent the systemic pathologies induced by femur fracture.
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