Presence Of Rhamnose Research Articles

Structural characteristics and functional properties of a fucose containing prebiotic exopolysaccharide from Bifidobacterium breve NCIM 5671.

To evaluate the structure and functions of capsular exopolysaccharide (CPS) from Bifidobacterium breve NCIM 5671. A CPS produced by the probiotic bacteria B. breve NCIM 5671 was isolated and subjected to characterization through GC analysis, which indicated the presence of rhamnose, fucose, galactose, and glucose in a molar ratio of 3:1:5:3. The average molecular weight of the CPS was determined to be ∼8.5×105 Da. Further, NMR analysis revealed the probable CPS structure to be composed of major branched tetra- and penta-saccharide units alternately repeating and having both α- and β-configuration sugar residues. CPS displayed an encouraging prebiotic score for some of the studied probiotic bacteria. Compared to standard inulin, CPS showed better resistance to digestibility against human GI tract in vitro. DPPH, total antioxidant, and ferric reducing assays carried out for CPS displayed decent antioxidant activity too. This study indicates that the CPS from B. breve NCIM 5671 has the potential to be utilized as a prebiotic food supplement. It is a high-molecular-weight (∼8.5×105 Da) capsular heteropolysaccharide containing rhamnose, fucose, galactose, and glucose.

Preparation and Characterization of Novel Green Seaweed Films from Ulva rigida.

Ulva rigida green seaweed is an abundant biomass consisting of polysaccharides and protein mixtures and a potential bioresource for bioplastic food packaging. This research prepared and characterized novel biodegradable films from Ulva rigida extracts. The water-soluble fraction of Ulva rigida was extracted and prepared into bioplastic films. 1H nuclear magnetic resonance indicated the presence of rhamnose, glucuronic and sulfate polysaccharides, while major amino acid components determined via high-performance liquid chromatography (HPLC) were aspartic acid, glutamic acid, alanine and glycine. Seaweed extracts were formulated with glycerol and triethyl citrate (20% and 30%) and prepared into films. Ulva rigida films showed non-homogeneous microstructures, as determined via scanning electron microscopy, due to immiscible crystalline component mixtures. X-ray diffraction also indicated modified crystalline morphology due to different plasticizers, while infrared spectra suggested interaction between plasticizers and Ulva rigida polymers via hydrogen bonding. The addition of glycerol decreased the glass transition temperature of the films from -36 °C for control films to -62 °C for films with 30% glycerol, indicating better plasticization. Water vapor and oxygen permeability were retained at up to 20% plasticizer content, and further addition of plasticizers increased the water permeability up to 6.5 g·mm/m2·day·KPa, while oxygen permeability decreased below 20 mL·mm/m2·day·atm when blending plasticizers at 30%. Adding glycerol efficiently improved tensile stress and strain by up to 4- and 3-fold, respectively. Glycerol-plasticized Ulva rigida extract films were produced as novel bio-based materials that supported sustainable food packaging.

Inability to Catabolize Rhamnose by Sinorhizobium meliloti Rm1021 Affects Competition for Nodule Occupancy.

Rhizobium leguminosarum strains unable to grow on rhamnose as a sole carbon source are less competitive for nodule occupancy. To determine if the ability to use rhamnose as a sole carbon source affects competition for nodule occupancy in Sinorhizobium meliloti, Tn5 mutants unable to use rhamnose as a sole carbon source were isolated. S. meliloti mutations affecting rhamnose utilization were found in two operons syntenous to those of R. leguminosarum. Although the S. meliloti Tn5 mutants were complemented using an R. leguminosarum cosmid that contains the entire wild-type rhamnose catabolic locus, complementation did not occur if the cosmids carried Tn5 insertions within the locus. Through a series of heterologous complementation experiments, enzyme assays, gene fusion, and transport experiments, we show that the S. meliloti regulator, RhaR, is dominant to its R. leguminosarum counterpart. In addition, the data support the hypothesis that the R. leguminosarum kinase is capable of directly phosphorylating rhamnose and rhamnulose, whereas the S. meliloti kinase does not possess rhamnose kinase activity. In nodule competition assays, S. meliloti mutants incapable of rhamnose transport were shown to be less competitive than the wild-type and had a decreased ability to bind plant roots in the presence of rhamnose. The data suggests that rhamnose catabolism is a general determinant in competition for nodule occupancy that spans across rhizobial species.

An acidic polysaccharide (AGC3) isolated from North American ginseng (Panax quinquefolius) suspension culture as a potential immunomodulatory nutraceutical

Polysaccharides isolated from Panax quinquefolius roots are widely used as nutraceuticals due to their immunomodulatory properties. Despite their popularity, several challenges exist in isolating ginseng root polysaccharides such as batch-to-batch structural inconsistencies and bacterial endotoxin contamination. A plant tissue culture-based platform offers a potential solution to isolate natural polysaccharide fractions with consistent chemical characteristics and reduced endotoxin content. In this study, an acidic polysaccharide fraction (AGC3) with immunomodulatory properties was isolated from Panax quinquefolius suspension cultures. The heterogeneous fraction (molecular weight: 4.81 and 32.14 kDa), purified by anion exchange chromatography, was predominantly composed of galactose (>60%) along with the presence of rhamnose, arabinose, glucose, glucuronic acid and galacturonic acid. The major glycosidic linkages were found to be t-Galp (47.7%), 4-Galp (15.6%), 2,4-Rhap (8.1%), 6-Galp (8.1%) and 4-GalAp (6.8%). Structural analyses indicated the presence of a pectic rhamnogalacturonan I polysaccharide in AGC3. AGC3 significantly (p < 0.05) stimulated RAW 264.7 murine macrophage cells and primary murine splenocytes by enhancing the production of several immunomodulatory mediators such as IL-6, TNF-α, GM-CSF and MCP-1. The results also indicated the putative roles of NF-κB (p65/RelA) and MAPK (p38) signaling pathways in the immunostimulatory response. Additionally, AGC3 induced murine splenocyte proliferation, another major indicator of immunostimulation. Overall, AGC3 has the potential to be used as an immunostimulatory nutraceutical.

Real-time monitoring of rhamnose induction effect on the expression of mpt64 gene fused with pelB signal peptide in Escherichia coli BL21 (DE3).

In this research, Escherichia coli BL21 (DE3) harboring an expression vector constructed with a rhamnose-inducible promoter and a pelB signal peptide was used as a host cell to produce MPT64 protein. The objective of this research was to figure out the optimum time of mpt64 gene expression through real-time monitoring of MPT64 protein production and distribution in host compartments. The mpt64 expression was regulated by the rhamnose presence at a concentration of 4 mM. The real-time isolated protein was monitored using polyacrylamide gel electrophoresis in denaturation condition. Based on real-time monitoring, the MPT64 protein (24 kDa) in the cytoplasm was optimum detected at 24 h after induction. For periplasmic fraction, the protein was detected at 4 h after induction but thinning at 15 h after induction. At 16 h after induction, the MPT64 protein band was found in the medium with increasing concentrations until 24 h. Thus, it can be concluded that the mpt64 gene expression was regulated in the presence of rhamnose as an inducer, and the proteins were shown to be translocated throughout the host cell compartment with different levels of protein accumulation at different times, according to the role of pelB as a signal peptide.

CHARACTERIZATION AND HYPOGLYCEMIC STUDY OF ISOLATED NATURAL POLYMER FROM THE STEM OF MANILKARA HEXANDRA (ROXB.) DUBARD

Objective: In the present study, the isolated natural polymer from the stem of Manilkara hexandra and their physiochemical parameters were investigated. It is further involved in hypoglycemic studies.&#x0D; Methods: The gum exudates were screened for phytochemicals, physicochemically analyzed for solubility, pH, total ash, moisture content, acid insoluble ash, water soluble ash, sulfated ash, flow property, and it is characterized by Fourier Transform Infrared analysis (FT-IR), Scanning Electron Microscope (SEM), X-ray diffraction analysis (XRD), Nuclear Magnetic Resonance (NMR) studies (13Carbon and 1Proton) and it was examined in vitro studies by hypoglycaemic activity.&#x0D; Results: The isolated gum extracted from the stem of Manilkara hexandra (Roxb.) Dubard ash values were found to be low. The gum is found to be hygroscopic in nature due to its high moisture content (0.9131±0.03). Fourier Transform Infrared (FT-IR) spectra show relevant functional groups for gum, which is further confirmed by resonance spectral studies. The X-ray diffraction (XRD) pattern shows that the gum is amorphous as well as crystalline in nature. Scanning Electron Microscope (SEM) image confirms that the gum particles have irregular size and shape. Sugar composition analysis by Thin Layer Chromatography indicated the presence of rhamnose, arabinose. The in vitro study of hypoglycemic activity shows the best report compared with the standard. The experimental evidence offers scope to use this natural polymer in the food and pharmaceutical industry.&#x0D; Conclusion: The isolated natural polymer shows good result in hypoglycemic studies compared with standard.

PHARMACOGNOSTIC STUDIES ON FLOWERS OF DREGEA VOLUBILIS: EVALUATION FOR AUTHENTICATION AND STANDARDIZATION

Objective: The various parts of Dregea volubilis (Family: Apocynaceae), locally known as Jukti (Bengali), are commonly used in Indian system of medicine to treat various ailments such as inflammation, piles, leukoderma, asthma, and tumors. Literature review suggested that there has been no detailed work on systemic pharmacognostic and phytochemical studies done on the flowers of the plant. The present study is aimed to lay down quality control parameters for D. volubilis flowers to confirm its identity, quality, and purity.&#x0D; Methods: The present work was designed to study detailed organoleptic, histological, quantitative standards, physicochemical, spectroscopic, and chromatographic characteristics of the flowers of D. volubilis. &#x0D; Results: The total ash, acid insoluble ash, water soluble ash, loss on drying, water, and alcohol soluble extractive values were found to be 11.767±0.130% (w/w), 1.287±0.106% (w/w), 9.140±0.344% (w/w), 14.110±0.061% (w/w), 21.600±0.133% (w/v), and 9.603±0.104% (w/v), respectively. Phytochemical screening of different extracts showed the presence of carbohydrates, proteins, amino acids, steroids, glycosides, alkaloids, flavonoids, tannins, and phenolics. The chromatographic study revealed the presence of rhamnose (103.229±4.994 μg/g), fructose (738.670±25.714 μg/g), glucose (285.532±24.465 μg/g), and maltose (49.082±5.206 μg/g).&#x0D; Conclusion: The characterization parameters of the present study may serve as a reference standard for proper authentication, identification and for distinguishing the plant from its adulterants.

Comparison of lipopolysaccharides composition of two different strains of Helicobacter pylori

BackgroundHelicobacter pylori (H. pylori) is a Gram-negative, microaerophilic bacterium that is recognized as a major cause of chronic gastritis, peptic ulcers, and gastric cancer. Comparable to other Gram-negative bacteria, lipopolysaccharides (LPS) are an important cellular component of the outer membrane of H. pylori. The LPS of this organism plays a key role in its colonization and persistence in the stomach. In addition, H. pylori LPS modulates pathogen-induced host inflammatory responses resulting in chronic inflammation within the gastrointestinal tract. Very little is known about the comparative LPS compositions of different strains of H. pylori with varied degree of virulence in human. Therefore, LPS was analyzed from two strains of H. pylori with differing potency in inducing inflammatory responses (SS1 and G27). LPS were extracted from aqueous and phenol layer of hot-phenol water extraction method and subjected for composition analysis by gas chromatography – mass spectrometry (GC-MS) to sugar and fatty acid compositions.ResultsThe major difference between the two strains of H. pylori is the presence of Rhamnose, Fucose and GalNAc in the SS1 strain, which was either not found or with low abundance in the G27 strain. On the other hand, high amount of Mannose was present in G27 in comparison to SS1. Fatty acid composition of lipid-A portion also showed considerable amount of differences between the two strains, phenol layer of SS1 had enhanced amount of 3 hydroxy decanoic acid (3-OH-C10:0) and 3-hydroxy dodecanoic acid (3-OH-C12:0) which were not present in G27, whereas myristic acid (C14:0) was present in G27 in relatively high amount.ConclusionThe composition analysis of H. pylori LPS, revealed differences in sugars and fatty acids composition between a mouse adapted strain SS1 and G27. This knowledge provides a novel way to dissect out their importance in host-pathogen interaction in further studies.

Conformational Dynamics of the Single Lipopolysaccharide O-Antigen in Solution.

The O-antigen is the most variable and highly immunogenic part of the lipopolysaccharide molecule that covers the surface of Gram-negative bacteria and makes up the first line of cellular defense. To provide insight into the details of the O-antigen arrangement on the membrane surface, we simulated its behavior in solution by molecular dynamics. We developed the energetically favorable O-antigen conformation by analyzing free-energy distributions for its disaccharide fragments. Starting from this conformation, we simulated the behavior of the O-antigen chain on long timescales. Depending on the force field and temperature, the single molecule can undergo reversible or irreversible coil-to-globule transitions. The mechanism of these transitions is related either to the rotation of the carbohydrate residues around O-glycosidic bonds or to flips of the pyranose rings. We found that the presence of rhamnose in the O-antigen chain crucially increases its conformational mobility.

Enzymatic Synthesis of Rhamnose Containing Chemicals by Reverse Hydrolysis.

Rhamnose containing chemicals (RCCs) are widely occurred in plants and bacteria and are known to possess important bioactivities. However, few of them were available using the enzymatic synthesis method because of the scarcity of the α-L-rhamnosidases with wide acceptor specificity. In this work, an α-L-rhamnosidase from Alternaria sp. L1 was expressed in Pichia pastroris strain GS115. The recombinant enzyme was purified and used to synthesize novel RCCs through reverse hydrolysis in the presence of rhamnose as donor and mannitol, fructose or esculin as acceptors. The effects of initial substrate concentrations, reaction time, and temperature on RCC yields were investigated in detail when using mannitol as the acceptor. The mannitol derivative achieved a maximal yield of 36.1% by incubation of the enzyme with 0.4 M L-rhamnose and 0.2 M mannitol in pH 6.5 buffers at 55°C for 48 h. In identical conditions except for the initial acceptor concentrations, the maximal yields of fructose and esculin derivatives reached 11.9% and 17.9% respectively. The structures of the three derivatives were identified to be α-L-rhamnopyranosyl-(1→6')-D-mannitol, α-L-rhamnopyranosyl-(1→1')-β-D-fructopyranose, and 6,7-dihydroxycoumarin α-L-rhamnopyranosyl-(1→6')-β-D-glucopyranoside by ESI-MS and NMR spectroscopy. The high glycosylation efficiency as well as the broad acceptor specificity of this enzyme makes it a powerful tool for the synthesis of novel rhamnosyl glycosides.

Pectic polysaccharide from corn (Zea mays L.) effectively inhibited multi-step mediated cancer cell growth and metastasis

Corn pectic polysaccharide (COPP) inhibited galectin-3 mediated hemagglutination at Minimum Inhibitory Concentration (MIC) of 4.08μg/mL as opposed to citrus pectin (25μg/mL), a well known galectin-3 inhibitor and lactose (4.16μg/mL) – sugar specific to galectin-3. COPP effectively (72%) inhibited invasion and metastasis in experimental animals. In vivo results were substantiated by modulation of cancer specific markers such as galectin-3, which is a key molecule for initiation of metastatic cascade, vascular endothelial growth factor (VEGF) that enhances angiogenesis, matrix metalloproteinases 2 and 9 that are required for invasion, NF-κB, a transcription factor for proliferative potency of tumor cells and a phosphoglucoisomerase (PGI), the activity of which favors cancer cell growth. Structural characterization studies indicate the active component (relatively less acidic, 0.05M ammonium carbonate, 160kDa fraction) which showed antimetastatic potency in vitro with MIC of 0.09μg/mL, and ∼45 fold increase in the activity when compared to that of COPP. Gas liquid chromatographic analysis indicated the presence of rhamnose (1%), arabinose (20%), xylose (3%), mannose (4%), galactose (54%) and uronic acid (10%) in different proportions. However, correlative data attributed galectin-3 inhibitory activity to enhanced levels of arabinose and galactose. FTIR, HPLC and NMR spectroscopic analysis further highlights that COPP is an arabinogalactan with methyl/ethyl esters. It is therefore suggested that the blockade of galectin-3 mediated lung metastasis appears to be a result of an inhibition of mixed functions induced during metastasis. The data signifies the importance of dietary carbohydrate as cancer-preventive agent. Although pectin digestibility and absorption are issues of concern, promising in vivo data provides evidence for the cancer preventive property of corn. The present study reveals for the first time a new component of corn, i.e., – corn pectin with cancer preventive activity apart from corn starch that has been in wide use for multipurpose health benefits.

Manilkara zapota (Linn.) Seeds: A Potential Source of Natural Gum.

Mucilage isolated from seeds of Manilkara zapota (Linn.) P. Royen syn. is a plant growing naturally in the forests of India. This mucilage is yet to be commercially exploited, and characterized as polymer. Various physicochemical methods like particle size analysis, scanning electron microscopy, thermal analysis, gel permeation chromatography, X-ray diffraction spectrometry, zeta potential, Fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy have been employed to characterize this gum in the present study. Particle size analyses suggest that mucilage has particle size in nanometer. Scanning electron microscopy analysis suggests that the mucilage has irregular particle size. The glass transition temperature of the gum was observed to be 138°C and 136°C by differential scanning calorimetry and differential thermal analysis, respectively. The thermogravimetric analysis suggested that mucilage had good thermal stability. The average molecular weight of mucilage was determined to be 379180, by gel permeation chromatography, while the viscosity of mucilage was observed to be 219.1 cP. The X-ray diffraction spectrometry pattern of the mucilage indicates a completely amorphous structure. Elemental analysis of the gum revealed the contents of carbon, hydrogen, nitrogen, and sulfur to be 80.9 (%), 10.1 (%), 1.58 (%), and 512 (mg/kg), respectively. Mucilage had specific content of calcium, magnesium, potassium, lower concentrations of aluminum, cadmium, cobalt, lead, and nickel. The major functional groups identified from FT-IR spectrum include 3441 cm−1 (–OH), 1660 cm−1 (Alkenyl C–H & C=C Stretch), 1632 cm−1 (–COO–), 1414 cm−1 (–COO–), and 1219 cm−1 (–CH3CO). Analysis of mucilage by paper chromatography and 1D NMR, indicated the presence of rhamnose, xylose, arabinose, mannose, and fructose.

A rhamnose-binding lectin from sea bass (Dicentrarchus labrax) plasma agglutinates and opsonizes pathogenic bacteria

The discovery of rhamnose-binding lectins (RBLs) in teleost fish eggs led to the identification of a novel lectin family characterized by a unique sequence motif and a structural fold, and initially proposed to modulate fertilization. Further studies of the RBL tissue localization and gene organization were also suggestive of role(s) in innate immunity. Here we describe the purification, and biochemical and functional characterization of a novel RBL (DlRBL) from sea bass (Dicentrarchus labrax) serum. The purified DlRBL had electrophoretic mobilities corresponding to 24kDa and 100kDa under reducing and non-reducing conditions, respectively, suggesting that in plasma the DlRBL is present as a physiological homotetramer. DlRBL subunit transcripts revealed an open reading frame encoding 212 amino acid residues that included two tandemly-arrayed carbohydrate-recognition domains, and an 18-residue signal sequence at the N-terminus. The deduced size of 24.1kDa for the mature protein was in good agreement with the subunit size of the isolated lectin. Binding activity of DlRBL for rabbit erythrocytes could be inhibited in the presence of rhamnose or galactose, did not require calcium, and was optimal at around 20°C and within the pH 6.5–8.0 range. DlRBL agglutinated Gram positive and Gram negative bacteria, and exposure of formalin-killed Escherichia coli to DlRBL enhanced their phagocytosis by D. labrax peritoneal macrophages relative to the unexposed controls. Taken together, the results suggest that plasma DlRBL may play a role in immune recognition of microbial pathogens and facilitate their clearance by phagocytosis.

Morphological, physico-chemical and structural characterization of mucilage isolated from the seeds of Buchanania lanzan Spreng

Context: Mucilage isolated from the seeds of Buchanania lanzan a wild Indian plant. This mucilage can be commercially exploited, by evaluating physico-chemical properties of this mucilage. Materials and Methods: Various physico-chemical parameter using scanning electron microscopy (SEM), molecular weight, X-ray diffraction (XRD) spectrometry, zeta potential (ZP), Fourier transform infrared (FT-IR) spectroscopy and 1D ( 1 H and 13 C) nuclear magnetic resonance (NMR) have been employed to characterize mucilage in the present study. Results: SEM analysis suggests that the mucilage has irregular particle size. Thermogravimetry analysis suggested that mucilage had good thermal stability with two stage decomposition. The weight-average molecular weight of mucilage was determined to be 4883, by gel permeation chromatography. The XRD pattern of the mucilage indicated a completely amorphous structure. The ZP was obtained 1.56 and −9.49 mV in water and 0.1 N NaCl respectively. The major functional groups identified from FT-IR spectrum include 3459/cm (-OH), 1667/cm (Alkenyl C-H and C = C stretch), 1407/cm (-COO-) and 1321/cm (-CH 3 CO). Analysis of mucilage by paper chromatography and 1D NMR indicated the presence of rhamnose, arabinose and fructose. Conclusion: The spectral and chromatography analysis of mucilage indicate that the mucilage is composed of basic sugar moiety such as (arabinose, rhamnose, fructose and mannose) as complex carbohydrates.

Low-pH rescue of acid-sensitive Salmonella enterica Serovar Typhi Strains by a Rhamnose-regulated arginine decarboxylase system.

For Salmonella, transient exposure to gastric pH prepares invading bacteria for the stresses of host-cell interactions. To resist the effects of low pH, wild-type Salmonella enterica uses the acid tolerance response and the arginine decarboxylase acid resistance system. However, arginine decarboxylase is typically repressed under routine culture conditions, and for many live attenuated Salmonella vaccine strains, the acid tolerance response is unable to provide the necessary protection. The objective of this study was to enhance survival of Salmonella enterica serovar Typhi vaccine strains at pHs 3.0 and 2.5 to compensate for the defects in the acid tolerance response imposed by mutations in rpoS, phoPQ, and fur. We placed the arginine decarboxylase system (adiA and adiC) under the control of the ParaBAD or PrhaBAD promoter to provide inducible acid resistance when cells are grown under routine culture conditions. The rhamnose-regulated promoter PrhaBAD was less sensitive to the presence of its cognate sugar than the arabinose-regulated promoter ParaBAD and provided tighter control over adiA expression. Increased survival at low pH was only observed when adiA and adiC were coregulated by rhamnose and depended on the presence of rhamnose in the culture medium and arginine in the challenge medium. Rhamnose-regulated acid resistance significantly improved the survival of ΔaroD and ΔphoPQ mutants at pHs 3 and 2.5 but only modestly improved the survival of a fur mutant. The construction of the rhamnose-regulated arginine decarboxylase system allowed us to render S. Typhi acid resistant (to pH 2.5) on demand, with survival levels approximately equivalent to that of the native arginine decarboxylase system.

RelE-Mediated Dormancy Is Enhanced at High Cell Density in Escherichia coli

Bacteria show remarkable adaptability under several stressful conditions by shifting themselves into a dormant state. Less is known, however, about the mechanism underlying the cell transition to dormancy. Here, we report that the transition to dormant states is mediated by one of the major toxin-antitoxin systems, RelEB, in a cell density-dependent manner in Escherichia coli K-12 MG1655. We constructed a strain, IKA121, which expresses the toxin RelE in the presence of rhamnose and lacks chromosomal relBE and rhaBAD. With this strain, we demonstrated that RelE-mediated dormancy is enhanced at high cell densities compared to that at low cell densities. The initiation of expression of the antitoxin RelB from a plasmid, pCA24N, reversed RelE-mediated dormancy in bacterial cultures. The activation of RelE increased the appearance of persister cells against β-lactams, quinolones, and aminoglycosides, and more persister cells appeared at high cell densities than at low cell densities. Further analysis indicated that amino acid starvation and an uncharacterized extracellular heat-labile substance promote RelE-mediated dormancy. This is a first report on the induction of RelE-mediated dormancy by high cell density. This work establishes a population-based dormancy mechanism to help explain E. coli survival in stressful environments.

Purification and characterization of a novel alkaline α-L-rhamnosidase produced by Acrostalagmus luteo albus

Rhamnosidases are enzymes that catalyze the hydrolysis of terminal nonreducing L-rhamnose for the bioconversion of natural or synthetic rhamnosides. They are of great significance in the current biotechnological area, with applications in food and pharmaceutical industrial processes. In this study we isolated and characterized a novel alkaline rhamnosidase from Acrostalagmus luteo albus, an alkali-tolerant soil fungus from Argentina. We also present an efficient, simple, and inexpensive method for purifying the A. luteo albus rhamnosidase and describe the characteristics of the purified enzyme. In the presence of rhamnose as the sole carbon source, this fungus produces a rhamnosidase with a molecular weight of 109 kDa and a pI value of 4.6, as determined by SDS-PAGE and analytical isoelectric focusing, respectively. This enzyme was purified to homogeneity by chromatographic and electrophoretic techniques. Using p-nitrofenil-α-L-rhamnopiranoside as substrate, the enzyme activity showed pH and temperature optima of 8.0 and 55°C, respectively. The enzyme exhibited Michaelis-Menten kinetics, with K (M) and V (max) values of 3.38 mmol l(-1) and 68.5 mmol l(-1) min(-1), respectively. Neither divalent cations such as Ca(2+), Mg(2+), Mn(2+), and Co(2+) nor reducing agents such as β-mercaptoethanol and dithiothreitol showed any effect on enzyme activity, whereas this activity was completely inhibited by Zn(2+) at a concentration of 0.2 mM. This enzyme showed the capacity to hydrolyze some natural rhamnoglucosides such as hesperidin, naringin and quercitrin under alkaline conditions. Based on these results, and mainly due to the high activity of the A. luteo albus rhamnosidase under alkaline conditions, this enzyme should be considered a potential new biocatalyst for industrial applications.

Development of a homologous expression system for rubber oxygenase RoxA from Xanthomonas sp.

Natural rubber (poly-[cis-1,4-isoprene]) can be cleaved into 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al by rubber oxygenase A (RoxA) isolated from Xanthomonas sp. RoxA is a novel type of dihaem dioxygenase with unknown cleavage mechanism of the rubber carbon backbone. Analysis of mutant RoxA after mutagenesis could be a way to investigate the function of selected amino acids of RoxA during catalysis. Unfortunately, expression of functional RoxA in recombinant Escherichia coli or in recombinant γ-Proteobacteria such as Pseudomonas putida was not possible in our hands. Therefore, expression of recombinant RoxA in the homologous host, Xanthomonas, was performed. A transformation system via electroporation was established, and a conjugation system was optimized for Xanthomonas sp. Inactivation of the chromosomal roxA gene by insertional mutagenesis resulted in inability of Xanthomonas sp. to produce active RoxA and to utilize rubber as a sole source of carbon and energy. When an intact copy of roxA was cloned under control of a rhamnose-inducible promoter in a broad host range vector and was transferred to Xanthomonas sp., high expression levels of functional RoxA in the presence of rhamnose were obtained. Purification of recombinantly expressed RoxA was simplified because of drastically shortened fermentation times and because separation of RoxA from remaining rubber latex particles was not necessary with rhamnose-induced cultures. About 6 mg purified RoxA were obtained from 1 l of cell-free culture fluid. Purified recombinant RoxA was highly active and revealed comparable spectral properties as RoxA purified from the wild type. The results of our study are the methodical basis for molecular biological manipulation in Xanthomonas sp. and will simplify investigation into the biochemical mechanisms by which rubber can be biodegraded in the environment by this novel extracellular dihaem dioxygenase RoxA.

Structure determination ofStreptococcus suisserotype 2 capsular polysaccharide

The capsular polysaccharide (CPS) of Streptococcus suis serotype 2 was isolated, purified, chemically modified, and characterized. Sugar and absolute configuration analyses of the CPS gave the following composition: D-Gal, 3; D-Glc, 1; D-GlcNAc, 1; D-Neu5Ac, 1; L-Rha, 1. Sialic acid was found to be terminal, and the CPS was quantitatively desialylated by mild acid hydrolysis. The CPS was also submitted to periodate oxidation followed by borohydride reduction and Smith degradation. Sugar and methylation analysis, 1H and 13C nuclear magnetic resonance, and mass spectrometry of the native CPS or of its specifically modified products allowed to determine the repeating unit sequence: [4)[Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)]Gal(beta1-4)[Gal(alpha1-3)]Rha(beta1-4)Glc(beta1-]n. The backbone sequence was found to be identical to that of Streptococcus agalactiae or group B Streptococcus (GBS) type VIII and Streptococcus pneumoniae type 23F. The S. suis CPS shares the sequence Neu5Ac-Gal-GlcNAc-Gal in common with GBS types Ia, Ib, II, III, and IV CPSs but differs from them by the presence of rhamnose and the fact that sialic acid is 2,6- rather than 2,3-linked to the following Gal. A correlation between the S. suis CPS sequence and genes of the serotype 2 cps locus encoding putative enzymes responsible for the biosynthesis of the repeating unit was tentatively established.

Overexpression of the rhamnose catabolism regulatory protein, RhaR: a novel mechanism for metronidazole resistance in Bacteroides thetaiotaomicron

ObjectivesThe aim of the investigation was to use in vitro transposon mutagenesis to generate metronidazole resistance in the obligately anaerobic pathogenic bacterium Bacteroides thetaiotaomicron, and to identify the genes involved to enable investigation of potential mechanisms for the generation of metronidazole resistance.MethodsThe genes affected by the transposon insertion were identified by plasmid rescue and sequencing. Expression levels of the relevant genes were determined by semi-quantitative RNA hybridization and catabolic activity by lactate dehydrogenase/pyruvate oxidoreductase assays.ResultsA metronidazole-resistant mutant was isolated and the transposon insertion site was identified in an intergenic region between the rhaO and rhaR genes of the gene cluster involved in the uptake and catabolism of rhamnose. Metronidazole resistance was observed during growth in defined medium containing either rhamnose or glucose. The metronidazole-resistant mutant showed improved growth in the presence of rhamnose as compared with the wild-type parent. There was increased transcription of all genes of the rhamnose gene cluster in the presence of rhamnose and glucose, likely due to the transposon providing an additional promoter for the rhaR gene, encoding the positive transcriptional regulator of the rhamnose operon. The B. thetaiotaomicron metronidazole resistance phenotype was recreated by overexpressing the rhaR gene in the B. thetaiotaomicron wild-type parent. Both the metronidazole-resistant transposon mutant and RhaR overexpression strains displayed a phenotype of higher lactate dehydrogenase and lower pyruvate oxidoreductase activity in comparison with the parent strain during growth in rhamnose.ConclusionsThese data indicate that overexpression of the rhaR gene generates metronidazole resistance in B. thetaiotaomicron