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1.
Polymers (Basel) ; 14(8)2022 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-35458250

RESUMO

Biodegradable and potentially biobased polyesteramide oligomers (PEA-Pro), obtained from melt condensation of sebacic acid and 3-amino-1-propanol, were characterized by nuclear magnetic resonance (NMR), matrix assisted laser desorption/ionization-time of flight/time of flight-mass spectrometry/mass spectrometry (MALDI-TOF/TOF-MS/MS), thermogravimetric analysis (TGA), and pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS). NMR analysis showed the presence of hydroxyl and amino terminal groups as well as carboxylic groups of the sebacate moiety. Hydroxyl and carboxyl termination had the same abundance, while the amine termination was 2.7-times less frequent. Information regarding the fragmentation pathways and ester/amide bond sequences was obtained by MALDI-TOF/TOF-MS/MS analysis performed on sodiated adducts of cyclic species and linear oligomers. Different end groups did not influence the observed fragmentation. Three fragmentation pathways were recognized. The ß-hydrogen-transfer rearrangement, which leads to the selective scission of the -O-CH2- bonds, was the main mechanism. Abundant product ions originating from -CH2-CH2- (ß-γ) bond cleavage in the sebacate moiety and less abundant ions formed by -O-CO- cleavages were also detected. TGA showed a major weight loss (74%) at 381 °C and a second degradation step (22% weight loss) at 447 °C. Py-GC/MS performed in the temperature range of 350-400 °C displayed partial similarity between the degradation products and the main fragments detected in the MALDI-TOF/TOF-MS/MS experiments. Degradation products derived from amide bonds were related to the formation of CN groups, in agreement with the literature.

2.
Materials (Basel) ; 12(6)2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30875886

RESUMO

A biodegradable poly(3-R-hydroxyalkanoate) synthesized by Pseudomonas mediterranea was investigated as a biomaterial to obtain colloidal drug delivery systems. Using a nanoprecipitation method, nanoparticles with a mean size of 155 nm and a negative surface charge were formed. They can be freeze-dried by adding hydroxypropyl-ß-cyclodextrin as a cryoprotectant, and they have been shown to efficiently load both a hydrophilic (calcein) and a lipophilic (Nile red) model probe. Since this polymer contains terminal double bonds in the side chains, cross-linking conditions were tested. In particular, under the action of UV rays or irradiation with an incandescent yellow lamp, this polymer tended to cross-link.

3.
Int J Biol Macromol ; 108: 608-614, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29223758

RESUMO

Pseudomonas aeruginosa ATCC 27853 was cultured on media containing long odd-chain fatty acids. Heptadecanoic, nonadecanoic, and heneicosanoic acids sustained cell growth and resulted in polyhydroxyalkanoate (PHA) accumulation when culturing was conducted under nitrogen starvation conditions. No PHA was produced using a complete or magnesium-deprived medium. The isolated polyesters were characterized by gas chromatography and liquid chromatography-electrospray ionization mass spectrometry (ESI-MS) of methanolyzed samples, 1H and 13C NMR spectroscopy, gel permeation chromatography, ESI MS of partially pyrolyzed samples, and differential scanning calorimetry. These PHAs are composed of seven different odd-chain repeating units starting from 3-hydroxyvalerate, with the highest species being the, to date, unreported constituent 3-hydroxyheptadecanoate, and minor amounts of 2 or 3 even-chain comonomers. The PHAs are soft, sticky, rubber-like materials having glass transition temperatures between -45 and -39°C, melting temperatures between 48 and 52°C, enthalpies of melting around 11J/g, and molar masses ranging from 77 to 188kg/mol. Statistical analysis of the ESI mass spectra of the products of their partial pyrolysis showed that they are pure copolymers and not a blend of copolymers or homopolymers.


Assuntos
Ácidos Graxos/química , Poli-Hidroxialcanoatos/biossíntese , Poli-Hidroxialcanoatos/química , Pseudomonas aeruginosa/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Espectrometria de Massas por Ionização por Electrospray , Termodinâmica
4.
N Biotechnol ; 39(Pt A): 36-41, 2017 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-28587884

RESUMO

Pseudomonas strains produce rhamnolipid mixtures (RLs) that generally consist of one or two molecules of rhamnose linked to one or two molecules of 3-hydroxyalkanoic acid. This study evaluates carbon source effects (glycerol, glucose, myristic acid, and Brassica carinata oil) on the synthesis of monorhamnolipids (mono-RLs) versus dirhamnolipids (di-RLs) in a human isolate of Pseudomonas aeruginosa PAL05. Spectrophotometry, an emulsifying index (E24) test, and an orcinol assay confirmed the production of RLs by PAL05. Purified RLs were characterized by 1H NMR analysis. PAL05 primarily produces mono-RLs when provided carbon sources containing long chain fatty acids (FAs) (myristic acid and B. carinata oil) and di-RLs when provided glycerol or glucose. qRT-PCR analysis showed that delayed expression of rhlC occurred when B. carinata oil was used, but not glycerol, glucose, or myristic acid. Our data show that the carbon source influenced the transcriptional expression of the rhlC gene and, consequently, the predominance of mono-RLs or di-RLs in PAL05 cultures.


Assuntos
Carbono/farmacologia , Decanoatos/metabolismo , Glicolipídeos/metabolismo , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/metabolismo , Sistema Respiratório/microbiologia , Ramnose/análogos & derivados , Emulsões/química , Glicolipídeos/isolamento & purificação , Humanos , Espectroscopia de Prótons por Ressonância Magnética , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Ramnose/metabolismo
5.
Rapid Commun Mass Spectrom ; 29(9): 811-20, 2015 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-26377009

RESUMO

RATIONALE: Bacterial poly(3-hydroxyalkanoates) (PHAs) are an emergent class of plastic materials available from renewable resources. Their properties are strictly correlated with the comonomeric composition and sequence, which may be determined by various mass spectrometry approaches. In this paper we compare fast-atom bombardment (FAB) and electrospray ionization (ESI) to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) of partially pyrolyzed samples. METHODS: We determined the compositions and sequences of the medium-chain-length PHAs (mcl-PHAs) prepared by bacterial fermentation of Pseudomonas aeruginosa ATCC 27853 cultured in media containing fatty acids with 8, 12, 14, 18, and 20 carbon atoms as carbon sources by means of MALDI-TOFMS of pyrolyzates, and compared the results with those obtained by FAB- and ESI-MS in previous studies. MALDI matrices used were 9-aminoacridine (9-AA) and indoleacrylic acid (IAA). RESULTS: MALDI-TOFMS was carried out in negative ion mode when using 9-AA as a matrix, giving a semi-quantitative estimation of the 3-hydroxyacids constituting the PHAs, and in positive mode when using IAA, allowing us, through statistical analysis of the relative intensity of the oligomers generated by pyrolysis, to establish that the polymers obtained are true random copolyesters and not a mixture of homopolymers or copolymers. CONCLUSIONS: MALDI-TOFMS in 9-AA and IAA of partial pyrolyzates of mcl-PHAs represents a powerful method for the structural analysis of these materials. In comparison with FAB and ESI, MALDI provided an extended mass range with better sensitivity at higher mass and a faster method of analysis.


Assuntos
Poli-Hidroxialcanoatos/análise , Poli-Hidroxialcanoatos/química , Pseudomonas aeruginosa/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
6.
Mater Sci Eng C Mater Biol Appl ; 46: 470-81, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25492012

RESUMO

Within a research directed to developing new polymeric materials, suitable for decorating the surface of colloidal drug carriers, PEG5000 polymers containing a free carboxyl or amine group at one end were conjugated to an α-lipoamino moiety (LAA). The conjugates were characterized by FT-IR, (1)H-NMR, and MALDI-TOF mass spectrometry. They showed the same profile of solubility as the parent PEGs in water and in some polar and apolar solvents of pharmaceutical use. Representative terms showed to be well tolerated when incubated with Caco-2 or L929 cell cultures. Dedicated differential scanning calorimetry (DSC) studies were performed to prove the interaction of increasing molar fractions of the PEG5000-LAA conjugates with dipalmitoylphosphatidylcholine (DPPC) bilayers, to gain information about their possible incorporation in drug nanocarriers. While the parent PEGs affected only the superficial structure of bilayers, the amphiphilic PEG-LAA conjugates induced a perturbing effect on the thermotropic behavior of DPPC liposomes, according to the structure of the linked LAA residue. A molar concentration of these PEG-LAA between 5 and 10% was individuated as the most suitable to produce stable vesicles.


Assuntos
Aminoácidos/química , Coloides , Portadores de Fármacos , Polietilenoglicóis/química , Materiais Biocompatíveis , Células CACO-2 , Varredura Diferencial de Calorimetria , Humanos , Espectroscopia de Prótons por Ressonância Magnética , Espectrofotometria Infravermelho , Análise Espectral , Propriedades de Superfície
7.
Carbohydr Res ; 346(18): 2905-12, 2011 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-22055818

RESUMO

Cepacian is an exopolysaccharide produced by the majority of the isolates belonging to the Burkholderia cepacia complex bacteria, a group of 17 species, some of which infect cystic fibrosis patients, sometime with fatal outcome. The repeating unit of cepacian consists of a backbone having a trisaccharidic repeating unit with three side chains, as reported in the formula below. The exopolysaccharide is also acetylated, carrying from one to three acetyl esters per repeating unit, depending on the strain examined. The consequences of O-acetyl substitution in a polysaccharide are important both for its biological functions and for industrial applications, including the preparation of conjugated vaccines, since O-acetyl groups are important immunogenic determinants. The location of acetyl groups was achieved by NMR spectroscopy and ESI mass spectrometry and revealed that these substituents are scattered in non-stoichiometric ratio on many sugar residues in different positions, a feature which adds to the already unique carbohydrate structure of the polysaccharide.


Assuntos
Burkholderia cepacia/química , Polissacarídeos Bacterianos/química , Burkholderia cepacia/crescimento & desenvolvimento , Burkholderia cepacia/metabolismo , Polissacarídeos Bacterianos/biossíntese , Polissacarídeos Bacterianos/isolamento & purificação
8.
Anal Chem ; 83(3): 654-60, 2011 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-21204561

RESUMO

A direct, rapid, and easy electrospray ionization mass spectrometry (ESI-MS) method to determine concentrations of sebacic acid (SA) and terephthalic acid (TA) residues in biodegradable copolymers was developed. Copolyester samples were synthesized from 1,4-butanediol and sebacic and terephthalic acids by melt polymerization. Extraction of monomers was performed in methanol. Their concentrations were determined by direct infusion ESI-MS, without chromatographic separation, using 1,12-dodecanedioic acid (DDA) as an internal standard. Calibration curves were obtained by plotting the ratio of the areas of the peaks relative to monomers and DDA standard as a function of their concentration ratio. We validated the method by determining the concentration of TA residue using both the ESI-MS protocol and high-performance liquid chromatography (HPLC) analysis with UV detection. The linearity range and the detection limit of this assay were 0.1-5.0 and 0.01 ppm for SA and 0.1-6.0 and 0.03 ppm for TA. This assay represents a useful alternative to conventional methods currently employed for acid quantification, resulting advantageous for its speed and high sensitivity.


Assuntos
Ácidos Decanoicos/análise , Ácidos Dicarboxílicos/análise , Ácidos Ftálicos/análise , Polímeros/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Calibragem , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Estrutura Molecular , Polímeros/metabolismo , Espectrometria de Massas por Ionização por Electrospray/instrumentação
9.
Int J Biol Macromol ; 48(1): 137-45, 2011 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-21035502

RESUMO

Pseudomonas aeruginosa produced medium chain length poly(3-hydroxyalkanoates) (mcl-PHAs) when grown on substrates containing very long chain fatty acids (VLCFA, C>20). Looking for low cost carbon sources, we tested Brassica carinata oil (erucic acid content 35-48%) as an intact triglyceride containing VLCFA. Oleic (C18:1), erucic (C22:1), and nervonic (C24:1) acids were also employed for mcl-PHA production as model substrates. The polymers obtained were analyzed by GC of methanolyzed samples, GPC, 1H and 13C NMR, ESI MS of partially pyrolyzed samples, and DSC. The repeating units of such polymers were saturated and unsaturated, with a higher content of the latter in the case of the PHA obtained from B. carinata oil. Statistical analysis of the ion intensity in the ESI mass spectra showed that the PHAs from pure fatty acids are random copolymers, while the PHA from B. carinata oil is either a pure polymer or a mixture of polymers. Weight-average molecular weight varied from ca. 56,000 g/mol for the PHA from B. carinata oil and oleic acid, to about 120,000 g/mol for those from erucic and nervonic acids. The PHAs from erucic and nervonic acids were partially crystalline, with rubbery characteristics and a melting point (Tm) of 50°C, while the PHAs from oleic acid and from B. carinata oil afforded totally amorphous materials, with glass transition temperatures (Tg) of -52°C and -47°C, respectively.


Assuntos
Brassica/química , Ácidos Erúcicos/metabolismo , Ácidos Graxos/metabolismo , Óleos de Plantas/química , Poli-Hidroxialcanoatos/biossíntese , Ácidos Erúcicos/química , Ácidos Graxos Monoinsaturados/química , Ácidos Graxos Monoinsaturados/metabolismo , Hidrólise , Espectroscopia de Ressonância Magnética , Poli-Hidroxialcanoatos/química , Poli-Hidroxialcanoatos/isolamento & purificação , Pseudomonas aeruginosa/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Temperatura
10.
Eur J Pharm Sci ; 37(3-4): 451-62, 2009 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-19504659

RESUMO

Poly(3-hydroxybutyrate-co-epsilon-caprolactone) copolymers and poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-epsilon-caprolactone) terpolymers were used by a solvent deposition technique to prepare either micro- or nanoparticles. In particular, the synthesis and analytical characterization of the terpolymers were described.On the basis of copolymer composition and properties, either micro- or nanoparticles were obtained; nanoparticle size was below 500 nm for the suspensions obtained from P(HB-co-CL) copolymers, and even smaller (200-300 nm) for those obtained using terpolymers. Particle size showed only a limited tendency to increase during storage, suggesting a good chemical and physical stability in the short-term storage at room temperature. Some copolymers produced hetero-dispersed microparticles under the same conditions, with a mean size between 10 and 30 microm. These systems showed a tendency to aggregate upon storage at room temperature.The nanoparticles showed a negative surface charge (around -20 mV for those prepared using an UltraTurrax and about -5 mV for those prepared by magnetic stirring). After storage at 4 degrees C the surface charge tend to decrease and these changes have been explained in terms of a partial hydrolysis of the polymeric matrix in aqueous suspension, which led to a change of chemical composition at the surface of the particles.The fluorescent probes calcein and Oil Red O were encapsulated in these systems as models of a hydrophilic and lipophilic drug molecule, respectively. Encapsulation efficiency and in vitro release profiles were studied to evaluate the effect of copolymer properties, such as molecular weight and composition, on their behaviour as potential materials to prepare controlled drug delivery carriers. Calcein was generally better encapsulated (up to 100%) than Oil Red O (10-30%); however, the zeta-potential measurement and in vitro release experiments suggested that a large amount of calcein was adsorbed onto the particle surface and was rapidly released within the first minutes of the test. Conversely, the lipophilic probe was dispersed within the polymeric matrix and its release profile from the nanoparticles was characterized by a considerable lag time (up to 8 h), followed by a slow and almost linear release.As a general trend, we observed that the composition and crystallinity of the tested polymers affected the type and size of obtained systems (micro- or nanoparticles), whereas the molecular weight mainly influenced the probes encapsulation and release.


Assuntos
Coloides/química , Sistemas de Liberação de Medicamentos , Poliésteres/química , Compostos Azo/química , Corantes/química , Preparações de Ação Retardada , Portadores de Fármacos , Excipientes , Fluoresceínas/química , Espectroscopia de Ressonância Magnética , Peso Molecular , Nanopartículas , Tamanho da Partícula , Solubilidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrofotometria Ultravioleta
11.
Carbohydr Res ; 343(1): 81-9, 2008 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-18028888

RESUMO

Cepacian is the exopolysaccharide produced by the majority of the so far investigated clinical strains of the Burkholderia cepacia complex. This is a group of nine closely related bacterial species that might cause serious lung infections in cystic fibrosis patients, in some cases leading to death. In this paper the aggregation ability and the conformational properties of cepacian chain were investigated to understand its role in biofilm formation. Viscosity and atomic force microscopy studies in water and in mixed (dimethylsulfoxide/water) solvent indicated the formation of double stranded molecular structures in aqueous solutions. Inter-residue short distances along cepacian chain were investigated by NOE NMR, which showed that two side chains of cepacian were not conformationally free due to strong interactions with the polymer backbone. These interactions were attributed to hydrogen bonding and contributed to structure rigidity.


Assuntos
Complexo Burkholderia cepacia/química , Polissacarídeos Bacterianos/química , Biofilmes , Configuração de Carboidratos , Dimetil Sulfóxido , Espectroscopia de Ressonância Magnética , Microscopia de Força Atômica , Viscosidade , Água
12.
Carbohydr Res ; 342(16): 2404-15, 2007 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-17719019

RESUMO

The major cause of morbidity and mortality in patients with cystic fibrosis, an autosomal recessive disorder, is chronic microbial colonisation of the major airways that leads to exacerbation of pulmonary infection. Several different microbes colonise cystic fibrosis lungs, and Pseudomonas aeruginosa is one of the most threatening, since the establishment of mucoid (alginate producing) strains is ultimately associated with the patient's death. Very recently a new bacterium, named Inquilinus limosus, was repeatedly found infecting the respiratory tract of cystic fibrosis patients. Its multi-resistance characteristic to antibiotics might result in the spreading of I. limosus infection among the cystic fibrosis community, as recently happened with strains of the Burkholderia cepacia complex. Since exopolysaccharides are recognised as important virulence factors in lung infections, the primary structure of the polysaccharide produced by I. limosus strain LMG 20952(T) was investigated as the first step in understanding its role in pathogenesis. The structure was determined by means of methylation analysis, acid degradations, mass spectrometry and NMR spectroscopy. The results showed that the bacterium produced a mixture constituted of the following polymers: [3)-[4,6-O-(1-carboxyethylidene)]-beta-D-Glcp(1-->]n; [2)-[4,6-O-(1-carboxyethylidene)]-alpha-D-Manp(1-->]n. Both polymers were completely substituted with pyruvyl ketal groups, a novel structural characteristic not previously found in bacterial polysaccharides. The absolute configuration of all pyruvyl groups was S. Inspection of possible local conformations assumed by the two polysaccharide chains showed features, which might provide interesting clues for understanding structure-function relationships.


Assuntos
Fibrose Cística , Polissacarídeos Bacterianos/química , Rhodospirillaceae/química , Rhodospirillaceae/patogenicidade , Cromatografia em Gel , Cromatografia por Troca Iônica , Glicosilação , Humanos , Espectroscopia de Ressonância Magnética , Metilação , Modelos Moleculares , Conformação Molecular , Monossacarídeos/química , Ácido Oxálico/química , Polissacarídeos Bacterianos/biossíntese , Polissacarídeos Bacterianos/toxicidade , Ácido Pirúvico/química , Rhodospirillaceae/metabolismo , Espectrometria de Massas por Ionização por Electrospray
13.
Biomacromolecules ; 8(3): 985-91, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17261066

RESUMO

Copolymers of 3-hydroxybutyrate (3HB) and 3-mercaptopropionate (3MP) or 3-mercaptobutyrate (3MB) units and minor amounts of 3-hydroxypropionate (3HP), 3-hydroxyvalerate (3HV), or 3-mercaptovalerate (3MV) were investigated regarding their microstructure by NMR, electrospray ionization mass spectrometry, and size exclusion chromatography NMR. These copolymers were produced by Ralstonia eutropha strain H16 when cells were cultivated in a mineral salts medium with gluconate as a carbon source for growth and 3MP or 3MB as precursor substrates for incorporation of 3-mercaptoalkanoates. Mass spectrometry analysis of partially methanolyzed or pyrolyzed samples proved the presence of true copolymers or terpolymers. (13)C NMR spectroscopy of intact polymer samples, with values of average block length and degree of randomness deviating from a random sequence model, suggested microblock structures; however, composition analysis by (1)H NMR of fractions obtained by size exclusion chromatography showed significant variations with molecular weight, revealing the presence of blends of poly(3HB-co-3MP-co-3HP) or poly(3HB-co-3MB) with poly(3HB). The experimental NMR carbonyl dyad signal intensities were satisfactorily matched by a random sequence model when the presence of poly(3HB) was taken into account.


Assuntos
Bioquímica/métodos , Cromatografia/métodos , Espectroscopia de Ressonância Magnética/métodos , Poliésteres/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Compostos de Sulfidrila/química , Ácido 3-Hidroxibutírico/química , Cupriavidus necator , Hidroxibutiratos/química , Espectrometria de Massas , Metano/química , Modelos Químicos , Conformação Molecular , Polímeros/química
14.
J Cyst Fibros ; 6(2): 145-52, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16860003

RESUMO

BACKGROUND: In the frame of a research line dedicated to better clarify the role of exopolysaccharides (EPS) in bacterial virulence, EPS produced by species of the Burkholderia cepacia complex (Bcc), namely Burkholderia multivorans, Burkholderia cenocepacia, and a Bcc member of undetermined genomovar, all isolated at the Cystic Fibrosis Regional Centre of Florence (Italy), were investigated for they structural properties. METHODS: Three strains of B. multivorans, three of B. cenocepacia and one of a Bcc member of undetermined genomovar were isolated from CF patients. The reference strains C1576 and J2315, for genomovar II and III, respectively, were included in the study. The bacteria were grown on solid media, the exopolysaccharides produced were purified, and their structures were determined. In addition, sugar analysis of sputum samples was accomplished to search for EPS produced in vivo. RESULTS: Six strains out of seven produced the exopolysaccharide cepacian, while one strain of B. multivorans produced a completely different polymer, previously known in the literature as PS1. Two strains synthesised very small amounts of EPS. No definitive evidence for the presence of cepacian in sputum samples was found. CONCLUSIONS: Most strains examined produced abundant amounts of polysaccharides. Cepacian was the most common EPS isolated and its production was not associated to a particular genomovar.


Assuntos
Infecções por Burkholderia/microbiologia , Complexo Burkholderia cepacia/metabolismo , Burkholderia cepacia/isolamento & purificação , Fibrose Cística/microbiologia , Polissacarídeos Bacterianos/metabolismo , Infecções por Burkholderia/complicações , Fibrose Cística/complicações , Ligação Genética , Humanos , Espectroscopia de Ressonância Magnética , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Escarro/microbiologia
15.
Biochem Biophys Res Commun ; 339(3): 821-6, 2006 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-16325774

RESUMO

Bacteria belonging to the Burkholderia cepacia complex (Bcc) are interesting for their involvement in pulmonary infections in patients affected by cystic fibrosis (CF) or chronic granulomatous disease. Many Bcc strains isolated from CF patients produce high amounts of exopolysaccharides (EPS). Although different strains sometimes biosynthesise different EPS, the majority of Bcc bacteria produce only one type of polysaccharide, which is called cepacian. The polymer has a unique heptasaccharidic repeating unit, containing three side chains, and up to three O-acetyl substituents.. We here report for the first time the isolation and characterisation of a lyase active towards cepacian produced by a Bacillus sp., which was isolated in our laboratory. The enzyme molecular mass, evaluated by size-exclusion chromatography, is 32,700+/-1500Da. The enzyme catalyses a beta-elimination reaction of the disaccharide side chain beta-d-Galp-(1-->2)-alpha-d-Rhap-(1--> from the C-4 of the glucuronic acid residue present in the polymer backbone. Although active on both native and de-acetylated cepacian, the enzyme showed higher activity on the latter polymer.


Assuntos
Complexo Burkholderia cepacia/metabolismo , Liases/química , Liases/metabolismo , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/metabolismo , Sítios de Ligação , Ativação Enzimática , Liases/isolamento & purificação , Peso Molecular , Polissacarídeos Bacterianos/isolamento & purificação , Ligação Proteica , Especificidade por Substrato
16.
Carbohydr Res ; 340(3): 439-47, 2005 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-15680599

RESUMO

The bacterial species Enterobacter amnigenus was isolated from sugar beets harvested in Finland. It produced an exopolysaccharide rich in l-fucose, which gave viscous water solutions. Its primary structure was determined mainly by NMR spectroscopy and ESIMS of oligosaccharides and a polysaccharide with decreased molecular weight, obtained by Smith degradation of the O-deacetylated native polymer [carbohydrate structure: see text]


Assuntos
Enterobacter/metabolismo , Polissacarídeos Bacterianos/química , Sequência de Carboidratos , Espectroscopia de Ressonância Magnética , Metilação , Dados de Sequência Molecular , Estrutura Molecular , Oligossacarídeos/química
17.
Biomacromolecules ; 5(6): 2469-78, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15530065

RESUMO

Pseudomonas aeruginosa ATCC 27853 accumulated poly(3-hydroxyalkanoates) (PHAs) after growth on saturated fatty acids with an odd number of carbon atoms. No nutrient limitation was required to induce PHA synthesis, although better yields were obtained when the medium was magnesium deprived. A comparative study was carried out between PHAs obtained from C-odd and those from C-even carbon sources. Repeating units identification was performed by gas chromatography (GC) and capillary liquid chromatography-electrospray mass spectrometry (LC-ESI MS) of methanolyzed samples. When C-odd n-alkanoic acids from nonanoic to pentadecanoic were used the lowest hydroxyalkanoate unit found was 3-hydroxyvalerate and the highest 3-hydroxypentadecanoate, whereas when C-even acids from octanoic to eicosanoic were used these were 3-hydroxycaproate and 3-hydroxyeicosanoate, respectively. Weight average molecular weights were in the range 187 000-596 000. DSC traces showed Tm and DeltaHm which varied from 43 to 58 degrees C and from 5.9 to 24.8 J/g, with the PHAs generated from C-odd carbon sources having lower values. ESI MS of partially pyrolyzed samples allowed the identification of oligomers up to heptamers, and statistical analysis of the ions intensity in the mass spectra showed that these PHAs are random copolyesters.


Assuntos
Ácidos Graxos/metabolismo , Poliésteres/química , Pseudomonas aeruginosa/metabolismo , Varredura Diferencial de Calorimetria , Cromatografia Gasosa , Ácidos Graxos/química , Hidroxiácidos/metabolismo , Magnésio/química , Espectroscopia de Ressonância Magnética , Modelos Químicos , Ácidos Pentanoicos/química , Poliésteres/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Temperatura , Termodinâmica
18.
J Cyst Fibros ; 3(3): 165-72, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15463903

RESUMO

Clinical and environmental strains of Burkholderia cenocepacia belonging to the recA lineages IIIA and IIIB were examined for exopolysaccharide (EPS) production. The exopolysaccharides structure was determined using mainly gas chromatography coupled to mass spectrometry and NMR spectroscopy. All the strains produced Cepacian, a highly branched polysaccharide constituted of a heptasaccharide repeating unit, composed of one rhamnose, one glucose, one glucuronic acid, one mannose and three galactose residues. This polymer is the most common exopolysaccharide produced by strains of the Burkholderia cepacia (Bcc) complex. One clinical strain produced also another polysaccharide constituted of three galactose units and one 3-deoxy-D-manno-2-octulosonic acid residues, a polymer that was previously isolated from two strains of B. cepacia genomovar I and B. cenocepacia IIIA.


Assuntos
Burkholderia cepacia/metabolismo , Polissacarídeos Bacterianos/biossíntese , Burkholderia cepacia/classificação , Humanos , Espectroscopia de Ressonância Magnética
19.
Biomacromolecules ; 5(2): 433-44, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15003003

RESUMO

High molar mass random poly(butylene succinate-co-butylene sebacate), P(BS-co-BSe), and poly(butylene succinate-co-butylene adipate), P(BS-co-BA), with different composition, were synthesized and subjected to enzymatic hydrolysis by Lipase from Mucor miehei or from Rhizopus arrhizus. The enzymatic hydrolysis of P(BS-co-BSe)s and P(BS-co-BA)s films produced a mixture of water-soluble monomers and co-oligomers that were separated and identified by on-line high performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS). Optimization of the HPLC analysis allowed the separation of isobar co-oligomers, differing only for the co-monomers sequence. Oligomers with the same monomer composition and molar mass but different sequence were identified by HPLC/ESI-MS-MS on-line analysis. The results obtained show a preferential hydrolytic cleavage induced by the lipases used. In particular, these enzymes prefer cleaving sebacic ester bonds in P(BS-co-BSe) copolymers, whereas succinic ester bonds appear to be hydrolyzed faster than adipic ester bonds in P(BS-co-BA) copolyesters. 1H NMR analysis further substantiates these findings. The primary products generated by lipase hydrolysis of polyester films underwent further degradation at longer reaction times. The HPLC/ESI-MS analysis of these mixtures at various times provided the first evidence that lipase catalysis is active also in water solution, a hydrophobic effect induced by the aliphatic units of these polyesters.


Assuntos
Lipase/química , Lipase/metabolismo , Poliésteres/química , Poliésteres/metabolismo , Catálise , Cromatografia Líquida de Alta Pressão , Hidrólise , Mucor/enzimologia , Rhizopus/enzimologia , Espectrometria de Massas por Ionização por Electrospray
20.
Rapid Commun Mass Spectrom ; 18(4): 392-8, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-14966845

RESUMO

The spectra recorded by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS/MS) of complex carbohydrates from human milk are presented. Besides ions originating from glycosidic cleavages and from sugar ring fragmentations, these spectra show intense peaks that may be assigned to ions produced by three new fragmentation pathways involving a six-atom rearrangement. These ions, together with the A fragments from sugar ring fragmentations, open the possibility of obtaining a complete mapping of the linkage positions present in the carbohydrates investigated by MALDI-TOF/TOF.


Assuntos
Carboidratos/análise , Carboidratos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Algoritmos , Hexoses/química , Estrutura Molecular
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