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1.
Int J Syst Evol Microbiol ; 70(3): 1639-1643, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32125262

RESUMO

A Gram-stain-negative, aerobic, rod-shaped bacterium with peritrichous flagella, designated strain HB161719T, was isolated from coastal sand collected from Tanmen Port in Hainan, PR China. The isolate was found to grow with 2-11 % (w/v) NaCl, at 15-45 °C and pH 6.0-10.0, with an optima of 2-3 % NaCl, 37 °C and pH 7.0, respectively. Chemotaxonomic analysis showed that Q-8 was detected as the sole respiratory quinone and that iso-C15 : 0 and summed features 3, 8 and 9 were the major cellular fatty acids. The G+C content of the genomic DNA was 58.2 mol%. Analysis of the 16S rRNA gene sequence of the strain showed an affiliation with the genus Microbulbifer, sharing 98.7, 98.4, 97.8 and 97.8 % sequence similarities to the closest relatives of Microbulbifer okinawensis ABABA23T, Microbulbifer pacificus SPO729T, Microbulbifer taiwanensis CC-LN1-12T and Microbulbifer gwangyangensis GY2T, respectively. Low DNA-DNA hybridization values showed that it formed a distinct genomic species. The combined phenotypic and molecular features supported that strain HB161719T represents a novel species of the genus Microbulbifer, for which the name Microbulbifer harenosus sp. nov. is proposed. The type strain is HB161719T (=CGMCC 1.13584T=JCM 32688T).


Assuntos
Alteromonadaceae/classificação , Filogenia , Areia/microbiologia , Alginatos/metabolismo , Alteromonadaceae/isolamento & purificação , Técnicas de Tipagem Bacteriana , Composição de Bases , China , DNA Bacteriano/genética , Ácidos Graxos/química , Hibridização de Ácido Nucleico , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Ubiquinona/química
2.
Appl Microbiol Biotechnol ; 104(5): 2179-2191, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31900562

RESUMO

O-acetylation of alginate produced by the opportunistic human pathogen Pseudomonas aeruginosa significantly contributes to its pathogenesis. Three proteins, AlgI, AlgJ and AlgF have been implicated to form a complex and act together with AlgX for O-acetylation of alginate. AlgI was proposed to transfer the acetyl group across the cytoplasmic membrane, while periplasmic AlgJ was hypothesised to transfer the acetyl group to AlgX that acetylates alginate. To elucidate the proposed O-acetylation multiprotein complex, isogenic knockout mutants of algI, algJ and algF genes were generated in the constitutively alginate overproducing P. aeruginosa PDO300 to enable mutual stability studies. All knockout mutants were O-acetylation negative and complementation with the respective genes in cis or trans restored O-acetylation of alginate. Interestingly, only the AlgF deletion impaired alginate production suggesting a link to the alginate polymerisation/secretion multiprotein complex. Mutual stability experiments indicated that AlgI and AlgF interact independent of AlgJ as well as impact on stability of the alginate polymerisation/secretion multiprotein complex. Deletion of AlgJ did not destabilise AlgX and vice versa. When the alginate polymerase, Alg8, was absent, then AlgI and AlgF stability was strongly impaired supporting a link of the O-acetylation machinery with alginate polymerisation. Pull-down experiments suggested that AlgI interacts with AlgJ, while AlgF interacts with AlgJ and AlgI. Overall, these results suggested that AlgI-AlgJ-AlgF form a multiprotein complex linked via Alg8 to the envelope-spanning alginate polymerisation/secretion multiprotein complex to mediate O-acetylation of nascent alginate. Here, we provide the first insight on how the O-acetylation machinery is associated with alginate production.


Assuntos
Alginatos/metabolismo , Proteínas de Bactérias/metabolismo , Complexos Multiproteicos/metabolismo , Pseudomonas aeruginosa/metabolismo , Acetilação , Alginatos/química , Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Técnicas de Inativação de Genes , Teste de Complementação Genética , Complexos Multiproteicos/genética , Periplasma/metabolismo , Polimerização , Ligação Proteica , Estabilidade Proteica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade
3.
Chemosphere ; 238: 124652, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31473524

RESUMO

This research aimed to find the best phenotype of the brown algae Fucus vesiculosus (kelp) which has the greater potential to become a sorption byproduct for Zn removal from contaminated waters. Thus, the Zn uptake capacity and sorption mechanisms of the kelp collected from the Baltic Sea shore was, for the first time, investigated under various conditions, and compared to the phenotype habiting on the Irish Sea shore. Sorption studies were performed investigating the effect of algal dosage, Zn sources as well as algal harvesting time of the year on Zn uptake capacity. The results suggested that the Baltic algae is a better bio-sorbent for Zn uptake. Sorption mechanisms were studied by employing various indirect and direct approaches, more importantly, including high resolution synchrotron X-Ray Fluorescence and X-Ray Absorption Spectroscopy (XAS) and molecular modelling (MM). The results revealed that alginate and cellulose are among the main polysaccharide bonding Zn at algal surface, via coordination with O atoms from carboxyl and hydroxyl groups. XAS results giving direct measurements of Zn bonding environment on algal surface are supported by MM outputs and suggested that Zn is surrounded by ca. 5 O atoms at interatomic distances varying from 1.94 to 2.02 Å. The results contribute to understanding sorption mechanisms which can further lead to finding the best eluent for Zn desorption from the used biomass, bio sorbent reconditioning and reuse in multiple sorption desorption cycles as well as process optimization before industrial scaling up.


Assuntos
Biodegradação Ambiental , Fucus/metabolismo , Zinco/isolamento & purificação , Absorção Fisico-Química , Alginatos/metabolismo , Biomassa , Celulose/metabolismo , Fucus/química , Reciclagem/métodos , Zinco/farmacocinética
4.
mBio ; 10(6)2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31848278

RESUMO

It is well appreciated that oxygen- and other nutrient-limiting gradients characterize microenvironments within chronic infections that foster bacterial tolerance to treatment and the immune response. However, determining how bacteria respond to these microenvironments has been limited by a lack of tools to study bacterial functions at the relevant spatial scales in situ Here, we report the application of the hybridization chain reaction (HCR) v3.0 to provide analog mRNA relative quantitation of Pseudomonas aeruginosa single cells as a step toward this end. To assess the potential for this method to be applied to bacterial populations, we visualized the expression of genes needed for the production of alginate (algD) and the dissimilatory nitrate reductase (narG) at single-cell resolution within laboratory-grown aggregates. After validating new HCR probes, we quantified algD and narG expression across microenvironmental gradients within both single aggregates and aggregate populations using the agar block biofilm assay (ABBA). For mucoid and nonmucoid ABBA populations, narG was expressed in hypoxic and anoxic regions, while alginate expression was restricted to the hypoxic zone (∼40 to 200 µM O2). Within individual aggregates, surface-adjacent cells expressed alginate genes at higher levels than interior cells, revealing that alginate expression is not constitutive in mucoid P. aeruginosa but instead varies with oxygen availability. These results establish HCR v3.0 as a versatile and robust tool to resolve subtle differences in gene expression at spatial scales relevant to microbial assemblages. This advance has the potential to enable quantitative studies of microbial gene expression in diverse contexts, including pathogen activities during infections.IMPORTANCE A goal for microbial ecophysiological research is to reveal microbial activities in natural environments, including sediments, soils, or infected human tissues. Here, we report the application of the hybridization chain reaction (HCR) v3.0 to quantitatively measure microbial gene expression in situ at single-cell resolution in bacterial aggregates. Using quantitative image analysis of thousands of Pseudomonas aeruginosa cells, we validated new P. aeruginosa HCR probes. Within in vitro P. aeruginosa aggregates, we found that bacteria just below the aggregate surface are the primary cells expressing genes that protect the population against antibiotics and the immune system. This observation suggests that therapies targeting bacteria growing with small amounts of oxygen may be most effective against these hard-to-treat infections. More generally, this proof-of-concept study demonstrates that HCR v3.0 has the potential to identify microbial activities in situ at small spatial scales in diverse contexts.


Assuntos
Alginatos/metabolismo , Expressão Gênica , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/fisiologia , Biofilmes , Biomarcadores , Humanos , Hipóxia/genética , Hipóxia/metabolismo , Mucosa Intestinal/fisiologia , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/patologia
5.
Mar Drugs ; 17(10)2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31547564

RESUMO

Alginase lyase is an important enzyme for the preparation of alginate oligosaccharides (AOS), that possess special biological activities and is widely used in various fields, such as medicine, food, and chemical industry. In this study, a novel bifunctional alginate lyase (AlgH) belonging to the PL7 family was screened and characterized. The AlgH exhibited the highest activity at 45 °C and pH 10.0, and was an alkaline enzyme that was stable at pH 6.0-10.0. The enzyme showed no significant dependence on metal ions, and exhibited unchanged activity at high concentration of NaCl. To determine the function of non-catalytic domains in the multi-domain enzyme, the recombinant AlgH-I containing only the catalysis domain and AlgH-II containing the catalysis domain and the carbohydrate binding module (CBM) domain were constructed and characterized. The results showed that the activity and thermostability of the reconstructed enzymes were significantly improved by deletion of the F5/8 type C domain. On the other hand, the substrate specificity and the mode of action of the reconstructed enzymes showed no change. Alginate could be completely degraded by the full-length and modified enzymes, and the main end-products were alginate disaccharide, trisaccharide, and tetrasaccharide. Due to the thermo and pH-stability, salt-tolerance, and bifunctionality, the modified alginate lyase was a robust enzyme which could be applied in industrial production of AOS.


Assuntos
Alginatos/metabolismo , Gammaproteobacteria/metabolismo , Oligossacarídeos/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Concentração de Íons de Hidrogênio , Especificidade por Substrato
6.
Biotechnol Lett ; 41(10): 1187-1200, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31418101

RESUMO

OBJECTIVES: Bifunctional alginate lyase can efficiently saccharify alginate biomass and prepare functional oligosaccharides of alginate. RESULTS: A new BP-2 strain that produces alginate lyase was screened and identified from rotted Sargassum. A new alginate lyase, Alg17B, belonging to the polysaccharide lyase family 17, was isolated and purified from BP-2 fermentation broth by freeze-drying, dialysis, and ion exchange chromatography. The enzymatic properties of the purified lyase were investigated. The molecular weight of Alg17B was approximately 77 kDa, its optimum reaction temperature was 40-45 °C, and its optimum reaction pH was 7.5-8.0. The enzyme was relatively stable at pH 7.0-8.0, with a temperature range of 25-35 °C, and the specific activity of the purified enzyme reached 4036 U/mg. A low Na+ concentration stimulated Alg17B enzyme activity, but Ca2+, Zn2+, and other metal ions inhibited it. Substrate specificity analysis, thin-layer chromatography, and mass spectrometry showed that Alg17B is an alginate lyase that catalyses the hydrolysis of sodium alginate, polymannuronic acid (polyM) and polyguluronic acid to produce monosaccharides and low molecular weight oligosaccharides. Alg17B is also bifunctional, exhibiting both endolytic and exolytic activities toward alginate, and has a wide substrate utilization range with a preference for polyM. CONCLUSIONS: Alg17B can be used to saccharify the main carbohydrate, alginate, in the ethanolic production of brown algae fuel as well as in preparing and researching oligosaccharides.


Assuntos
Organismos Aquáticos/enzimologia , Gammaproteobacteria/enzimologia , Polissacarídeo-Liase/isolamento & purificação , Polissacarídeo-Liase/metabolismo , Sargassum/microbiologia , Alginatos/metabolismo , Ácido Algínico/metabolismo , Organismos Aquáticos/classificação , Organismos Aquáticos/genética , Organismos Aquáticos/isolamento & purificação , Ativadores de Enzimas/análise , Inibidores Enzimáticos/análise , Estabilidade Enzimática , Gammaproteobacteria/classificação , Gammaproteobacteria/genética , Gammaproteobacteria/isolamento & purificação , Concentração de Íons de Hidrogênio , Hidrólise , Peso Molecular , Monossacarídeos/metabolismo , Oligossacarídeos/metabolismo , Polissacarídeo-Liase/química , Polissacarídeo-Liase/genética , Polissacarídeos Bacterianos/metabolismo , Especificidade por Substrato , Temperatura
7.
Biofabrication ; 11(4): 045020, 2019 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-31387086

RESUMO

Bioink is of paramount importance in the process of three-dimensional extrusive bioprinting technology. Alginate is extensively used in cell-laden extrusive bioprinters with the advantage of biocompatibility, gelling and crosslinking features; however, the bioinert properties of alginate made it hard to degrade in vivo, and restrict cellular adhesion, extension and migration. In this study, we incorporated two concentrations of alginate lyase (0.5 mU ml-1 and 5 mU ml-1) into alginate/gelatin bioink to improve its degradation properties and effects on cellular behavior. The enzymatically degradable bioink demonstrated lower stiffness and higher porosity. Cellular proliferation, adhesion and extension were facilitated in the degradable bioink without sacrifice of cell viability. Additionally, the property of degradation still worked in vivo, with cellular infiltration and retention being observed in the grafted bioprinted constructs. The results suggest that alginate lyase could be incorporated into alginate/gelatin bioink. Degradation properties and cellular behavior could be promoted both in vitro and in vivo, providing a new avenue for the upgrade and modification of alginate-based bioink for further applications.


Assuntos
Alginatos/metabolismo , Fibroblastos/citologia , Gelatina/metabolismo , Tinta , Polissacarídeo-Liase/metabolismo , Animais , Bioimpressão , Adesão Celular , Proliferação de Células , Sobrevivência Celular , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Impressão Tridimensional , Ratos , Tecidos Suporte/química
8.
Biofouling ; 35(5): 541-550, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31269803

RESUMO

Biofilm formation is an important virulence factor which is controlled by complex regulatory circuits in Pseudomonas aeruginosa. In this work, a biofilm hyper-producing strain, P2-7, was selected from a collection of transposon insertion mutants in which the PA2121 gene was disrupted. PA2121 was predicted as a putative LysR-type regulator. Analyses showed that it was involved in early biofilm formation, mature biofilm development, and colony morphology. Quantitative measurements revealed that PA2121 repressed biosynthesis of extracellular polysaccharides (alginate, psl and pel). Furthermore, it was observed that PA2121 was self-regulated, highly expressed in the early phase of biofilm development, and subject to the negative regulation by a biofilm synthesis regulator SrpA that binds directly to the PA2121 gene promoter. Collectively, this study proposes that PA2121 is a novel biofilm synthesis repressor (BsrA) in P. aeruginosa.


Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes , Pseudomonas aeruginosa/fisiologia , Fatores de Transcrição/metabolismo , Alginatos/metabolismo , Regulação Bacteriana da Expressão Gênica , Polissacarídeos Bacterianos/biossíntese
9.
Int J Biol Macromol ; 138: 831-836, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31351956

RESUMO

To reduce animal testing, there is a need to develop novel in-vitro models for evaluating the retention of bioactive compounds in food and pharmaceutical products. Here, a mucus-mimetic platform was developed through a one-step approach based on encapsulating mucin within alginate gel beads. We found that mucins form micron sized aggregates distributed across the surface of the calcium-alginate bead, as shown by environmental scanning electron microscopy (ESEM). Retention of bioactive compounds on the mucin-functionalised surface was tested using a commercial orange drink formulation. To aid flavour retention, different mucoadhesive polymers with varying charge, including anionic, neutral and strongly cationic, were tested for their ability to interact with mucin and aid retaining flavour compounds within the mucin-alginate bead. The alginate-mucin mucus mimic was validated using an ex-vivo bovine tongue, with the flavour retention results showing qualitative agreement. The developed method proved to be a convenient, efficient tool for providing information on the effectiveness of mucoadhesive polymers without variability, safety and sustainability issues associated with an ex-vivo or in-vivo system. We propose that by encapsulating other relevant oral proteins, alongside mucins, current gaps between in-vitro and the ex-vivo systems may be narrowed.


Assuntos
Alginatos/química , Mucinas/química , Adesividade , Alginatos/metabolismo , Animais , Fenômenos Químicos , Cromatografia Gasosa-Espectrometria de Massas , Mucinas/metabolismo , Muco/metabolismo , Suínos
10.
Food Chem ; 299: 125142, 2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31325715

RESUMO

Alginate lyases can be used for alginate oligosaccharide production and for structural characterization or modification of alginates. For these applications it is important to obtain detailed information on mode of action and substrate specificities of alginate lyases. In this study, five alginate lyase genes were cloned from Cellulophaga algicola DSM 14237 genomic DNA, heterologously expressed, and characterized by using HPSEC-RI and HPAEC-PAD/MS. It was demonstrated that these analytical approaches can provide detailed information on preferred substrates, extent of hydrolysis, and the liberated products. The recombinant enzymes cleaved alginates endolytically (CaAly1, CaAly2, CaAly3) or exolytically (CaAly4, CaAly5). The three endolytic alginate lyases predominantly hydrolyzed guluronic acid-rich alginates, only CaAly1 also showed activity on mannuronic acid-rich alginates. The oligosaccharide profiles further demonstrated that the endolytic enzymes have rather narrow but slightly different substrate specificities and that the two exolytic alginate lyases mainly cleaved unsaturated guluronic acid oligosaccharides to monomers.


Assuntos
Alginatos/metabolismo , Cromatografia em Gel/métodos , Cromatografia por Troca Iônica/métodos , Flavobacteriaceae/enzimologia , Polissacarídeo-Liase/metabolismo , Alginatos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Flavobacteriaceae/genética , Ácidos Hexurônicos/metabolismo , Hidrólise , Polissacarídeo-Liase/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
11.
Nat Commun ; 10(1): 2486, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31171782

RESUMO

Although brown macroalgae holds potential as an alternative feedstock, its utilization by conventional microbial platforms has been limited due to the inability to metabolize one of the principal sugars, alginate. Here, we isolate Vibrio sp. dhg, a fast-growing bacterium that can efficiently assimilate alginate. Based on systematic characterization of the genomic information of Vibrio sp. dhg, we establish a genetic toolbox for its engineering. We also demonstrate its ability to rapidly produce ethanol, 2,3-butanediol, and lycopene from brown macroalgae sugar mixture with high productivities and yields. Collectively, Vibrio sp. dhg can be used as a platform for the efficient conversion of brown macroalgae sugars into diverse value-added biochemicals.


Assuntos
Feófitas/metabolismo , Alga Marinha/metabolismo , Vibrio/metabolismo , Alginatos/metabolismo , Butileno Glicóis/metabolismo , Etanol/metabolismo , Licopeno/metabolismo , Manitol/metabolismo
12.
Mar Drugs ; 17(6)2019 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-31159265

RESUMO

Alginate lyases have been widely used to prepare alginate oligosaccharides in food, agricultural, and medical industries. Therefore, discovering and characterizing novel alginate lyases with excellent properties has drawn increasing attention. Herein, a novel alginate lyase FsAlyPL6 of Polysaccharide Lyase (PL) 6 family is identified and biochemically characterized from Flammeovirga sp. NJ-04. It shows highest activity at 45 °C and could retain 50% of activity after being incubated at 45 °C for 1 h. The Thin-Layer Chromatography (TLC) and Electrospray Ionization Mass Spectrometry (ESI-MS) analysis indicates that FsAlyPL6 endolytically degrades alginate polysaccharide into oligosaccharides ranging from monosaccharides to pentasaccharides. In addition, the action pattern of the enzyme is also elucidated and the result suggests that FsAlyPL6 could recognize tetrasaccharide as the minimal substrate and cleave the glycosidic bonds between the subsites of -1 and +3. The research provides extended insights into the substrate recognition and degradation pattern of PL6 alginate lyases, which may further expand the application of alginate lyases.


Assuntos
Alginatos/metabolismo , Organismos Aquáticos/enzimologia , Bacteroidetes/química , Bacteroidetes/enzimologia , Polissacarídeo-Liase/metabolismo , Cromatografia em Camada Delgada , Microbiologia Industrial , Polissacarídeo-Liase/química , Espectrometria de Massas por Ionização por Electrospray
13.
J Colloid Interface Sci ; 553: 494-502, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31229868

RESUMO

We prepared ionic liquids (ILs) modified magnetic alginate nanoparticles and used these as supports for lipase immobilization. The novel supports were characterized using Fourier transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (1HNMR), vibrating sample magnetometry (VSM), thermogravimetry (TG), transmission electron microscopy (TEM) and water contact angle (WCA) measurements. The immobilized lipase (PPL-IL-MSA) exhibited high activity, 2.1-fold higher than that compared to free lipase and 1.59-fold higher compared to immobilized lipase without IL (PPL-MSA). In addition, the pH and temperature application range of PPL-IL-MSA were both found to be broader than that of free lipase and PPL-MSA. The thermal stability, denaturation stability, and reusing stability of PPL-IL-MSA were also higher than those of other samples. After 10 times of reuse, the residual activity of PPL-IL-MSA was 89.7% higher than that of PPL-MSA (84.4%). Furthermore, the kinetic constant Km of PPL-IL-MSA was 13.7 mg/mL lower than that of free lipase (21.2 mg/mL) and PPL-MSA (18.4 mg/mL). Circular dichroism (CD) was used to study the secondary structure of enzymes in order to explain the mechanism of the performance improvement of PPL-IL-MSA. This work involving the development of a new supports for enzyme immobilization may serve as a reference for further studies in this field.


Assuntos
Alginatos/metabolismo , Líquidos Iônicos/metabolismo , Lipase/metabolismo , Nanopartículas de Magnetita/química , Alginatos/química , Biocatálise , Líquidos Iônicos/química , Lipase/química , Modelos Moleculares , Estrutura Molecular , Tamanho da Partícula , Propriedades de Superfície
14.
Mar Drugs ; 17(5)2019 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-31137680

RESUMO

Pseudomonas aeruginosa biofilms are typically associated with the chronic lung infection of cystic fibrosis (CF) patients and represent a major challenge for treatment. This opportunistic bacterial pathogen secretes alginate, a polysaccharide that is one of the main components of its biofilm. Targeting this major biofilm component has emerged as a tempting therapeutic strategy for tackling biofilm-associated bacterial infections. The enormous potential in genetic diversity of the marine microbial community make it a valuable resource for mining activities responsible for a broad range of metabolic processes, including the alginolytic activity responsible for degrading alginate. A collection of 36 bacterial isolates were purified from marine water based on their alginolytic activity. These isolates were identified based on their 16S rRNA gene sequences. Pseudoalteromonas sp. 1400 showed the highest alginolytic activity and was further confirmed to produce the enzyme alginate lyase. The purified alginate lyase (AlyP1400) produced by Pseudoalteromonas sp. 1400 showed a band of 23 KDa on a protein electrophoresis gel and exhibited a bifunctional lyase activity for both poly-mannuronic acid and poly-glucuronic acid degradation. A tryptic digestion of this gel band analyzed by liquid chromatography-tandem mass spectrometry confirmed high similarity to the alginate lyases in polysaccharide lyase family 18. The purified alginate lyase showed a maximum relative activity at 30 °C at a slightly acidic condition. It decreased the sodium alginate viscosity by over 90% and reduced the P. aeruginosa (strain PA14) biofilms by 69% after 24 h of incubation. The combined activity of AlyP1400 with carbenicillin or ciprofloxacin reduced the P. aeruginosa biofilm thickness, biovolume and surface area in a flow cell system. The present data revealed that AlyP1400 combined with conventional antibiotics helped to disrupt the biofilms produced by P. aeruginosa and can be used as a promising combinational therapeutic strategy.


Assuntos
Biofilmes/efeitos dos fármacos , Polissacarídeo-Liase/farmacologia , Pseudoalteromonas/enzimologia , Pseudomonas aeruginosa/efeitos dos fármacos , Alginatos/metabolismo , Antibacterianos/farmacologia , Organismos Aquáticos/enzimologia , Organismos Aquáticos/genética , Carbenicilina/farmacologia , Ciprofloxacino/farmacologia , Polissacarídeo-Liase/genética , Polissacarídeo-Liase/metabolismo , Pseudoalteromonas/genética , Pseudomonas aeruginosa/fisiologia , RNA Ribossômico 16S/genética
15.
J Colloid Interface Sci ; 552: 186-195, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31125829

RESUMO

Advances in pharmaceutical technology have promoted the development of colon-targeted delivery system for oral administration of bioactive peptides or proteins to enhance their bioavailability. In this study, a multi-unit nanofiber mat was fabricated by coaxial electrospinning and its feasibility as the colon-targeted delivery system for a bioactive peptide, salmon calcitonin (sCT), was investigated. Sodium alginate and sCT-loaded liposome coated with pectin served as the shell layer and core layer, respectively. An in vitro study demonstrated that the encapsulated sCT was released in a sustained and colon-targeted way. Analysis using different mathematical models showed that release followed a complex mechanism. In addition, greater amounts of sCT were released from the core-shell nanofiber mat into simulated colon fluid (SCF) than was released from a uniaxial nanofiber mat (65.2% vs. 47.8%). The use of a core-shell nanofiber mat further alleviated the burst release of sCT into simulated gastric and intestinal fluid (SGF and SIF), demonstrating the superiority of a multi-unit vehicle for colon-targeted delivery of sCT. Furthermore, 88% of the bioactivity of encapsulated sCT was retained. This multi-unit vehicle offers a better-designed vehicle for the colon-targeted sustained release of bioactive peptides or proteins and, thus, should improve oral bioavailability.


Assuntos
Calcitonina/metabolismo , Colo/metabolismo , Nanofibras/química , Pectinas/metabolismo , Administração Oral , Alginatos/administração & dosagem , Alginatos/química , Alginatos/metabolismo , Disponibilidade Biológica , Calcitonina/administração & dosagem , Calcitonina/química , Colo/química , Sistemas de Liberação de Medicamentos , Lipossomos/administração & dosagem , Lipossomos/química , Lipossomos/metabolismo , Nanofibras/administração & dosagem , Tamanho da Partícula , Pectinas/administração & dosagem , Pectinas/química , Propriedades de Superfície
16.
Sci Total Environ ; 681: 312-319, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31103668

RESUMO

Plants are naturally colonized by bacteria that can exert beneficial effects on growth and stress tolerance. These bacteria can be used as inoculants to boost crop productivity and plants resilience, and can be especially interesting if they are able to survive to abiotic stresses, such as drought. Herein we report the mechanisms that soil bacteria resort to tolerate drought and we also explore the influence of each mechanism to the level of drought tolerance exhibited, in order to test the hypothesis that different levels of tolerance displayed by bacteria are linked to differential efficiency of osmotolerance mechanisms. For this, the biochemical and physiological responses of bacterial strains of different genera and displaying different levels of tolerance to osmotic stress (sensitive, moderately tolerant and tolerant) induced by polyethylene glycol-6000 (PEG) were studied. Betaine, trehalose and alginate content increased in the majority of the strains exposed to PEG. Betaine was the osmolyte with higher increases, evidencing the important role of this compound in the tolerance of bacteria to drought. However, betaine and trehalose levels were not significantly different among bacteria with different osmotolerance levels. Several biochemical endpoints (protein content, superoxide dismutase, catalase, glutathione-S-transferases) related to oxidative stress were assessed, since oxidative damage has been reported in drought conditions, but little information is available. The oxidative stress parameters were not sufficient to explain differences in the osmotolerance observed for the tested strains. In contrast, alginate showed significant differences among the three levels of osmotolerance, linking the level of osmotolerance with the ability of soil bacteria to synthesize and accumulate alginate intracellularly for the first time. Moreover, our results show that this ability is present in different bacteria genera. Thus, evaluating the ability to synthesize alginate might be an important cue when considering bacterial inoculants for osmotically stressful conditions.


Assuntos
Adaptação Fisiológica , Alginatos/metabolismo , Bactérias/metabolismo , Fabaceae/microbiologia , Catalase/metabolismo , Secas , Portugal , Microbiologia do Solo , Superóxido Dismutase/metabolismo
17.
Drug Des Devel Ther ; 13: 925-940, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30962675

RESUMO

Background: Association of long hydrocarbon chain with alginate molecule imparts surface active properties and increases chain flexibility. Purpose: This work studied the efficacy of synthesized hexyl alginate derivative as a film-forming material with unique amphiphilic and mechanical properties for the preparation of rapidly disintegrating repaglinide oral films with higher drug release rate and improved bioavailability. Methods: Alginate hexyl amide derivative was prepared and used in the formulation of oral films by solvent casting technique. Using Box-Behnken experimental design, formulations were optimized at different polymer, plasticizer, and disintegrant levels as independent variables for maximum drug release rate, higher tensile strength, and shortest disintegration time as responses. Optimized film formulae were fully evaluated and subjected to further in vivo bioavailability studies in rabbits. Results: Higher dependency of response results on the selected variables was observed. Optimized formula showed satisfactory tensile strength (145.862 g/cm2), rapid disintegration (22.2 seconds), and higher drug release rate (97.799% within 30 minutes). The drug bioavailability was significantly improved in comparison with plain drug and conventional alginate oral films, where the AUC and Cmax values reached 296.072 µg.h/mL and 116.932 µg/mL in comparison with 164.917 µg.h/mL and 56.568 µg/mL for alginate film and 95.368 µg.h/mL and 31.925 µg/mL for plain drug, respectively. Tmax also showed significant reduction to be only 30 minutes in comparison with 60 minutes for other forms. Conclusion: This led to the final conclusion that the synthesized alginate derivative is an innovative promising film-forming material with unique mechanical and drug release properties for application in buccal drug delivery especially of Biopharmaceutics Classification System (BCS) class II drugs to increase solubility and improve bioavailability.


Assuntos
Alginatos/química , Carbamatos/metabolismo , Piperidinas/metabolismo , Tensoativos/química , Administração Oral , Alginatos/administração & dosagem , Alginatos/metabolismo , Disponibilidade Biológica , Carbamatos/administração & dosagem , Carbamatos/química , Liberação Controlada de Fármacos , Piperidinas/administração & dosagem , Piperidinas/química , Tensoativos/administração & dosagem , Tensoativos/metabolismo , Resistência à Tração
18.
Sci Total Environ ; 673: 750-755, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31003102

RESUMO

A process to produce both biodiesel and alginate in an integrated manner from a brown seaweed, Laminaria japonica, was established. Mannitol, a major carbon constituent in L. japonica, served to produce neutral lipids via the heterotrophic cultivation of an oleaginous yeast, Cryptococcus sp.; and simultaneously alginate, a high value product, was extracted to enhance the economic feasibility of the entire process. Only autoclave pretreatment, without need of any chemical agents, was enough to recover all the essential nutrients for the yeast cultivation. Specifically, it could recover 6.4 g L-1 of mannitol to a degree comparable to 6.6 g L-1 obtained by acid-aided pretreatment using 1.5% (v/v) of H2SO4. Maximum fatty acids methyl esters (FAME) content was 30.37% with FAME productivity of 0.56 g L-1 d-1, and the produced FAME could meet the biodiesel quality standards. Na2CO3-based method showed the best efficiency of alginate recovery, yielding 21.06% (w/w). This study supports that L. japonica can indeed be a promising low-cost feedstock for biodiesel production, and it is more so when a high-value product alginate is co-produced.


Assuntos
Alginatos/metabolismo , Biocombustíveis , Laminaria/fisiologia , Cryptococcus/fisiologia , Alga Marinha/fisiologia
19.
J Ethnopharmacol ; 239: 111909, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31026553

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: The Shuanghuanglian formula (SF) is a famous antimicrobial and antiviral traditional Chinese medicine that is made of Lonicera japonica Thunb., Scutellaria baicalensis Georgi, and Forsythia suspensa (Thunb.) Vahl. According to the Chinese Pharmacopoeia, the SF is commonly administered in the forms of oral liquid, tablets, and injection. It has long been used to treat acute respiratory tract infections, especially lung infection. AIM OF THE STUDY: In the light of the increasing incidence of multidrug resistance to conventional antibiotics, the aim of this study was to screen potential anti-virulence agents against Pseudomonas aeruginosa from the extract of the SF. MATERIALS AND METHODS: The SF was used for effective compounds screening via the combination of the molecule docking approach and ultra-high-performance liquid chromatography-quadrupole/time of flight mass spectrometry. Fifty-one anti-virulence-related proteins were docked, 26 identified compounds were from SF. Subsequently, the top-scoring screened compound was assessed via bioactive-related assays, including the quantification of alginate biosynthesis, anti-biofilm assays, and the A549 human lung cells infection. RESULT: A flavonoid Lonicerin was found to be bonded with the active site of the alginate secretion protein (AlgE) with the highest score in molecule docking. Furthermore, we validated that Lonicerin could significantly reduce alginate secretion (25 µg/mL) and biofilm formation (12.5 µg/mL) at a sub-MIC concentration without inhibiting the proliferation of P. aeruginosa or influencing the expression of AlgE, which suggested that Lonicerin may directly inhibit AlgE. In addition, Lonicerin was proven to inhibit the infection of P. aeruginosa in the A549 cells. CONCLUSION: This work reported on the first potential AlgE antagonist that was derived from herbal resources. Lonicerin was proven to be an effective inhibitor in-vitro of P. aeruginosa infection.


Assuntos
Antibacterianos/farmacologia , Medicamentos de Ervas Chinesas/química , Luteolina/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Células A549 , Alginatos/metabolismo , Humanos , Simulação de Acoplamento Molecular , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Virulência/efeitos dos fármacos
20.
J Biosci Bioeng ; 128(2): 203-208, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30852124

RESUMO

A novel alginolytic bacterium Hydrogenophaga sp. strain UMI-18 that produces poly(3-hydroyxybutylate) (PHB) in the alginate-mineral salt (AMS) medium containing 1% (w/v) sodium alginate as a sole carbon source was isolated from a decayed brown seaweed litter. The yield of PHB produced by strain UMI-18 was 1.1 ± 0.15 g/L of AMS and the PHB content in dried cell pellet was 58 ± 4% (w/w). Glucose, fructose, galactose, mannose, mannitol, sucrose and lactose were also available for the production of PHB by strain UMI-18. The yield of PHB in 1% (w/v) carbohydrate media reached 2.03-2.24 g/L for glucose and fructose, 0.75-1.64 g/L for alginate, galactose, mannitol and sucrose, and ∼0.15 g/L for lactose. The PHB produced by strain UMI-18 showed a glass-transition temperature (Tg) at 4°C, a melting temperature at 175°C, and an average molecular mass of 860 kDa. Draft genome analysis of the strain UMI-18 revealed that an alginate-assimilating gene cluster is located in contig 8 comprising 453,520 bp and the PHB-synthesis gene cluster is in contig 15 comprising 653,793 bp.


Assuntos
Alginatos/metabolismo , Carbono/metabolismo , Comamonadaceae/metabolismo , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Hidrólise
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