Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 9.594
Filtrar
1.
Microbiol Res ; 227: 126303, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31421717

RESUMO

The inhibitory action that a Brevibacillus laterosporus strain isolated from the honeybee body causes against the American Foulbrood (AFB) etiological agent Paenibacillus larvae was studied by in-vitro experiments. A protein fraction isolated from B. laterosporus culture supernatant was involved in the observed inhibition of P. larvae vegetative growth and spore germination. As a result of LC-MS/MS proteomic analyses, the bacteriocin laterosporulin was found to be the major component of this fraction, followed by other antimicrobial proteins and substances including lectins, chaperonins, various enzymes and a number of putative uncharacterized proteins. The results obtained in this study highlight the potential of B. laterosporus as a biological control agent for preserving and improving honeybee health.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/farmacologia , Abelhas/microbiologia , Brevibacillus/metabolismo , Paenibacillus larvae/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Bacteriocinas/isolamento & purificação , Bacteriocinas/farmacologia , Brevibacillus/isolamento & purificação , Cromatografia Líquida , Testes de Sensibilidade Microbiana , Proteômica , Espectrometria de Massas em Tandem
2.
Sheng Wu Gong Cheng Xue Bao ; 35(5): 847-856, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31223003

RESUMO

Pectobacterium carotovorum subsp. carotovorum is one of the world's top ten plant pathogens, mainly infecting cruciferous economic crops and ornamental flowers. In this study, an antibacterial gene cpxP (Gene ID: 29704421) was cloned from the genome of Pectobacterium carotovorum subsp. carotovorum, and constructed on the prokaryotic expression plasmid pET-15b, and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3), then stability and bacteriostatic experiments of the purified CpxP protein were performed. The final concentration of IPTG was 1 mmol/L, obtaining high-efficiency exogenous expression of the CpxP protein. There was no other protein after purification, and the destined protein exhibited good thermal stability and pH stability. The antibacterial test results showed that the inhibition rate of the CpxP protein on carrot slice was 44.89% while the inhibition rate on potato slice was 59.41%. To further explain its antibacterial mechanism, studying the spatial structure of this protein can provide new ideas for the control of soft rot and new protein pesticide targets.


Assuntos
Bactérias , Proteínas de Bactérias , Proteínas de Membrana , Pectobacterium carotovorum , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/farmacologia , Escherichia coli/genética , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/farmacologia , Pectobacterium carotovorum/genética , Pectobacterium carotovorum/metabolismo , Plasmídeos/genética
3.
Nat Commun ; 10(1): 2649, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201333

RESUMO

In human and other mammalian cells, transport of L-lactate across plasma membranes is mainly catalyzed by monocarboxylate transporters (MCTs) of the SLC16 solute carrier family. MCTs play an important role in cancer metabolism and are promising targets for tumor treatment. Here, we report the crystal structures of an SLC16 family homologue with two different bound ligands at 2.54 and 2.69 Å resolution. The structures show the transporter in the pharmacologically relevant outward-open conformation. Structural information together with a detailed structure-based analysis of the transport function provide important insights into the molecular working mechanisms of ligand binding and L-lactate transport.


Assuntos
Proteínas de Bactérias/química , Ácido Láctico/metabolismo , Transportadores de Ácidos Monocarboxílicos/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Cristalografia por Raios X , Transporte de Íons/fisiologia , Ligantes , Transportadores de Ácidos Monocarboxílicos/isolamento & purificação , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/química , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Simportadores/química
4.
Nat Commun ; 10(1): 2032, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31048734

RESUMO

The SLC26 family of transporters maintains anion equilibria in all kingdoms of life. The family shares a 7 + 7 transmembrane segments inverted repeat architecture with the SLC4 and SLC23 families, but holds a regulatory STAS domain in addition. While the only experimental SLC26 structure is monomeric, SLC26 proteins form structural and functional dimers in the lipid membrane. Here we resolve the structure of an SLC26 dimer embedded in a lipid membrane and characterize its functional relevance by combining PELDOR/DEER distance measurements and biochemical studies with MD simulations and spin-label ensemble refinement. Our structural model reveals a unique interface different from the SLC4 and SLC23 families. The functionally relevant STAS domain is no prerequisite for dimerization. Characterization of heterodimers indicates that protomers in the dimer functionally interact. The combined structural and functional data define the framework for a mechanistic understanding of functional cooperativity in SLC26 dimers.


Assuntos
Proteínas de Bactérias/metabolismo , Simulação de Dinâmica Molecular , Multimerização Proteica , Estrutura Quaternária de Proteína , Transportadores de Sulfato/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Deinococcus , Espectroscopia de Ressonância de Spin Eletrônica , Mutagênese Sítio-Dirigida , Transportadores de Ânions Orgânicos Dependentes de Sódio/química , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas SLC4A/química , Proteínas SLC4A/metabolismo , Transportadores de Sulfato/química , Transportadores de Sulfato/genética , Transportadores de Sulfato/isolamento & purificação
5.
Nat Commun ; 10(1): 2074, 2019 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-31061390

RESUMO

Hydride transfers play a crucial role in a multitude of biological redox reactions and are mediated by flavin, deazaflavin or nicotinamide adenine dinucleotide cofactors at standard redox potentials ranging from 0 to -340 mV. 2-Naphthoyl-CoA reductase, a key enzyme of oxygen-independent bacterial naphthalene degradation, uses a low-potential one-electron donor for the two-electron dearomatization of its substrate below the redox limit of known biological hydride transfer processes at E°' = -493 mV. Here we demonstrate by X-ray structural analyses, QM/MM computational studies, and multiple spectroscopy/activity based titrations that highly cooperative electron transfer (n = 3) from a low-potential one-electron (FAD) to a two-electron (FMN) transferring flavin cofactor is the key to overcome the resonance stabilized aromatic system by hydride transfer in a highly hydrophobic pocket. The results evidence how the protein environment inversely functionalizes two flavins to switch from low-potential one-electron to hydride transfer at the thermodynamic limit of flavin redox chemistry.


Assuntos
Proteínas de Bactérias/química , Coenzimas/química , Flavinas/química , Modelos Moleculares , Oxirredutases/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Coenzimas/metabolismo , Simulação por Computador , Cristalografia por Raios X , Transporte de Elétrons , Flavinas/metabolismo , Naftalenos/química , Naftalenos/metabolismo , Oxirredutases/isolamento & purificação , Oxirredutases/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise Espectral
6.
Nat Commun ; 10(1): 1609, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30962433

RESUMO

Bacterial degradation of organosulfonates plays an important role in sulfur recycling, and has been extensively studied. However, this process in anaerobic bacteria especially gut bacteria is little known despite of its potential significant impact on human health with the production of toxic H2S. Here, we describe the structural and biochemical characterization of an oxygen-sensitive enzyme that catalyzes the radical-mediated C-S bond cleavage of isethionate to form sulfite and acetaldehyde. We demonstrate its involvement in pathways that enables C2 sulfonates to be used as terminal electron acceptors for anaerobic respiration in sulfate- and sulfite-reducing bacteria. Furthermore, it plays a key role in converting bile salt-derived taurine into H2S in the disease-associated gut bacterium Bilophila wadsworthia. The enzymes and transporters in these anaerobic pathways expand our understanding of microbial sulfur metabolism, and help deciphering the complex web of microbial pathways involved in the transformation of sulfur compounds in the gut.


Assuntos
Acetiltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Desulfovibrio/metabolismo , Sulfeto de Hidrogênio/toxicidade , Taurina/metabolismo , Acetiltransferases/genética , Acetiltransferases/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Ácidos e Sais Biliares/metabolismo , Bilophila/metabolismo , Ensaios Enzimáticos , Microbioma Gastrointestinal/fisiologia , Sulfeto de Hidrogênio/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Mercaptoetanol/análogos & derivados , Mercaptoetanol/metabolismo , Redes e Vias Metabólicas/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Enxofre/metabolismo
7.
Prep Biochem Biotechnol ; 49(5): 521-528, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31017522

RESUMO

Staphylococcus aureus, among other staphylococcal species, developed multidrug resistance and causes serious health risks that require complex treatments. Therefore, the development of novel and effective strategies to combat these bacteria has been gaining importance. Since Staphylococcus simulans lysostaphin is a peptidoglycan hydrolase effective against staphylococcal species, the enzyme has a significant potential for biotechnological applications. Despite promising results of lysostaphin as a bacteriocin capable of killing staphylococcal pathogens, it is still not widely used in healthcare settings due to its high production cost. In this study, medium engineering techniques were applied to improve the expression yield of recombinant lysostaphin in E. coli. A new effective inducible araBAD promoter system and different mediums were used to enhance lysostaphin production. Our results showed that the composition of autoinduction media enhanced the amount of lysostaphin production 5-fold with the highest level of active lysostaphin at 30 °C. The production cost of 1000 U of lysostaphin was determined as 4-fold lower than the previously proposed technologies. Therefore, the currently developed bench scale study has a great potential as a large-scale fermentation procedure to produce lysostaphin efficiently.


Assuntos
Proteínas de Bactérias/biossíntese , Meios de Cultura/metabolismo , Lisostafina/biossíntese , Engenharia Metabólica/métodos , Proteínas Recombinantes/biossíntese , Arabinose/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Meios de Cultura/química , Indução Enzimática/efeitos dos fármacos , Estabilidade Enzimática , Escherichia coli/genética , Fermentação , Lisostafina/isolamento & purificação , Engenharia Metabólica/economia , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Staphylococcus/química , Staphylococcus/metabolismo , Temperatura Ambiente , Fatores de Tempo
8.
Dalton Trans ; 48(16): 5408-5416, 2019 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-30946408

RESUMO

HutZ from Vibrio cholerae is a dimeric enzyme that catalyzes oxygen-dependent degradation of heme via a similar catalytic mechanism to mammalian heme oxygenase. However, HutZ oxidizes the ß- or δ-meso position of heme at a ∼1 : 1 ratio distinct from heme oxygenase, which initiates the degradation of heme solely at the α-meso position. His63 is a residue that potentially forms hydrogen bond with the heme 7-propionate group. To establish the role of His63 in regioselectivity of heme degradation by HutZ and heme binding, we constructed mutants of His63. Interestingly, the H63L mutant retained a comparable level of ß- or δ-regioselectivity as wild-type HutZ. Ascorbic acid-assisted heme degradation by HutZ is pH-dependent, showing activity at pH 6.0 but not above pH 8.0. Compared to the wild-type protein, the H63L mutant was inactive, even at pH 6.0, and affinity for heme was significantly decreased in contrast with a comparable heme binding affinity at pH 8.0, as observed for the mutant of Asp132 to Val, which is located within hydrogen bonding distance of the heme axial ligand His170, but in a different protomer. In addition, the distance between heme and Trp109 increased from 16-18 Å for wild-type HutZ to 24-28 Å for the H63L mutant, indicating that protomer orientation is altered by the mutation, since Trp109 is in another subunit of the heme axial ligand. Our results collectively suggest that His63 positioned near heme does not contribute to regioselectivity of heme degradation but plays a key role in maintaining the orientation of subunits for HutZ to function of heme degradation.


Assuntos
Proteínas de Bactérias/metabolismo , Heme/metabolismo , Histonas/metabolismo , Vibrio cholerae/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Sítios de Ligação , Heme/química , Histonas/química , Concentração de Íons de Hidrogênio , Modelos Moleculares , Análise Espectral Raman
9.
Nat Chem ; 11(5): 463-469, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31011175

RESUMO

Staphylococci secrete autoinducing peptides (AIPs) as signalling molecules to regulate population-wide behaviour. AIPs from non-Staphylococcus aureus staphylococci have received attention as potential antivirulence agents to inhibit quorum sensing and virulence gene expression in the human pathogen Staphylococcus aureus. However, only a limited number of AIP structures from non-S. aureus staphylococci have been identified to date, as the minute amounts secreted in complex media render it difficult. Here, we report a method for the identification of AIPs by exploiting their thiolactone functionality for chemoselective trapping and enrichment of the compounds from the bacterial supernatant. Standard liquid chromatography mass spectrometry analysis, guided by genome sequencing data, then readily provides the AIP identities. Using this approach, we confirm the identity of five known AIPs and identify the AIPs of eleven non-S. aureus species, and we expect that the method should be extendable to AIP-expressing Gram-positive bacteria beyond the Staphylococcus genus.


Assuntos
Proteínas de Bactérias/análise , Depsipeptídeos/análise , Staphylococcus/química , Sequência de Aminoácidos , Proteínas de Bactérias/síntese química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/farmacologia , Cisteína/química , Depsipeptídeos/síntese química , Depsipeptídeos/isolamento & purificação , Depsipeptídeos/farmacologia , Limite de Detecção , Listeria monocytogenes/química , Estrutura Molecular , Percepção de Quorum/efeitos dos fármacos , Staphylococcus/metabolismo
10.
Mar Drugs ; 17(4)2019 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-30934874

RESUMO

Seaweeds are of significant interest in the food, pharmaceutical, and agricultural industries as they contain several commercially relevant bioactive compounds. Current extraction methods for macroalgal-derived metabolites are, however, problematic due to the complexity of the algal cell wall which hinders extraction efficiencies. The use of advanced extraction methods, such as enzyme-assisted extraction (EAE), which involve the application of commercial algal cell wall degrading enzymes to hydrolyze the cell wall carbohydrate network, are becoming more popular. Ascophyllum nodosum samples were collected from the Irish coast and incubated in artificial seawater for six weeks at three different temperatures (18 °C, 25 °C, and 30 °C) to induce decay. Microbial communities associated with the intact and decaying macroalga were examined using Illumina sequencing and culture-dependent approaches, including the novel ichip device. The bacterial populations associated with the seaweed were observed to change markedly upon decay. Over 800 bacterial isolates cultured from the macroalga were screened for the production of algal cell wall polysaccharidases and a range of species which displayed multiple hydrolytic enzyme activities were identified. Extracts from these enzyme-active bacterial isolates were then used in EAE of phenolics from Fucus vesiculosus and were shown to be more efficient than commercial enzyme preparations in their extraction efficiencies.


Assuntos
Ascophyllum/microbiologia , Bactérias/crescimento & desenvolvimento , Bactérias/metabolismo , Fracionamento Químico/métodos , Polissacarídeo-Liase/biossíntese , Polissacarídeo-Liase/química , Bactérias/enzimologia , Bactérias/isolamento & purificação , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Fucus/química , Hidrólise , Microbiota , Fenóis/isolamento & purificação , Polissacarídeo-Liase/isolamento & purificação , Proteólise , Alga Marinha/microbiologia
11.
Mar Drugs ; 17(4)2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30974812

RESUMO

Chitosanase has attracted great attention due to its potential applications in medicine, agriculture, and nutraceuticals. In this study, P. mucilaginosus TKU032, a bacterial strain isolated from Taiwanese soil, exhibited the highest chitosanase activity (0.53 U/mL) on medium containing shrimp heads as the sole carbon and nitrogen (C/N) source. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, a chitosanase isolated from P. mucilaginosus TKU032 cultured on shrimp head medium was determined at approximately 59 kDa. The characterized chitosanase showed interesting properties with optimal temperature and thermal stability up to 70 °C. Three chitosan oligosaccharide (COS) fractions were isolated from hydrolyzed colloidal chitosan that was catalyzed by TKU032 chitosanase. Of these, fraction I showed the highest α-glucosidase inhibitor (aGI) activity (65.86% at 20 mg/mL); its inhibitory mechanism followed the mixed noncompetitive inhibition model. Fractions II and III exhibited strong 2,2-diphenyl1-picrylhydrazyl (DPPH) radical scavenging activity (79.00% at 12 mg/mL and 73.29% at 16 mg/mL, respectively). In summary, the COS fractions obtained by hydrolyzing colloidal chitosan with TKU032 chitosanase may have potential use in medical or nutraceutical fields due to their aGI and antioxidant activities.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Fatores Biológicos/biossíntese , Glicosídeo Hidrolases/isolamento & purificação , Oligossacarídeos/biossíntese , Paenibacillus/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Biocatálise , Fatores Biológicos/farmacologia , Quitosana/metabolismo , Crustáceos/química , Ensaios Enzimáticos/métodos , Depuradores de Radicais Livres/metabolismo , Depuradores de Radicais Livres/farmacologia , Proteínas Fúngicas/metabolismo , Inibidores de Glicosídeo Hidrolases/metabolismo , Inibidores de Glicosídeo Hidrolases/farmacologia , Glicosídeo Hidrolases/metabolismo , Temperatura Alta , Oligossacarídeos/farmacologia , Paenibacillus/isolamento & purificação , Estabilidade Proteica , Microbiologia do Solo , Especificidade por Substrato , alfa-Glucosidases/metabolismo
12.
Mar Drugs ; 17(4)2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30987346

RESUMO

Toxin-antitoxin (TA) systems are ubiquitous and abundant genetic elements in bacteria and archaea. Most previous TA studies have focused on commensal and pathogenic bacteria, but have rarely focused on marine bacteria, especially those isolated from the deep sea. Here, we identified and characterized three putative TA pairs in the deep-sea-derived Streptomyces sp. strain SCSIO 02999. Our results showed that Orf5461/Orf5462 and Orf2769/Orf2770 are bona fide TA pairs. We provide several lines of evidence to demonstrate that Orf5461 and Orf5462 constitute a type-II TA pair that are homologous to the YoeB/YefM TA pair from Escherichia coli. Although YoeB from SCSIO 02999 was toxic to an E. coli host, the homologous YefM antitoxin from SCSIO 02999 did not neutralize the toxic effect of YoeB from E. coli. For the Orf2769/Orf2770 TA pair, Orf2769 overexpression caused significant cell elongation and could lead to cell death in E. coli, and the neighboring Orf2770 could neutralize the toxic effect of Orf2769. However, no homologous toxin or antitoxin was found for this pair, and no direct interaction was found between Orf2769 and Orf2770. These results suggest that Orf2769 and Orf2770 may constitute a novel TA pair. Thus, deep-sea bacteria harbor typical and novel TA pairs. The biochemical and physiological functions of different TAs in deep-sea bacteria warrant further investigation.


Assuntos
Organismos Aquáticos/fisiologia , Proteínas de Bactérias/genética , Streptomyces/fisiologia , Sistemas Toxina-Antitoxina/genética , Proteínas de Bactérias/isolamento & purificação , Toxinas Bacterianas , Escherichia coli/fisiologia , Proteínas de Escherichia coli/fisiologia , Loci Gênicos/fisiologia , Sedimentos Geológicos/microbiologia , Interações Microbianas/fisiologia , Oceanos e Mares , Homologia de Sequência do Ácido Nucleico
13.
Yakugaku Zasshi ; 139(4): 617-627, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-30930397

RESUMO

Salmonella is a Gram-negative [Gram(-)] bacteria, distributed widely in such natural environments as soil, dust, or river water, causing food poisoning as well as oral infections such as Typhi or Paratyphi. Salmonella is highly tissue invasive, easily spreading throughout the whole body after initial growth in the phagocytic vesicles of macrophages as an intra-cellular parasite. Because there remain many unknown elements in the Salmonella-macrophage interaction, I started my study by focusing on the molecules and mechanisms underlying the interaction; for example, how Salmonella escapes natural biodefense systems armed by macrophages, and how macrophages surround and inactivate Salmonella. In addition, I developed insight into Salmonella survival in the face of both environmental stresses and immunological stresses, including attacks from macrophages, based on the idea that "pathogenicity" is not limited simply to an attack, but to both the attack and defense against hazards. In this study, I found a novel pathogenicity-related protein of Salmonella, SEp22, an iron-chelating protein of MW 18.7 kDa, to cope with reactive-oxygen intermediates (ROIs) generated by activated macrophages pre-treated with lipopolysaccharides (LPS), one of the major components of Salmonella outer membrane. We also showed that Salmonella attacks macrophages by a novel mechanism through the induction of apoptosis with large amounts of LPS and protein synthesis inhibition, in addition to the well-known mechanisms of type-three secretion system (TTSS)-induced cell damage, including InvA, an attacking, virulent factor of Salmonella. We showed that macrophages could escape from this type of cell death by LPS-induced macrophage activation and LPS-tolerance.


Assuntos
Proteínas de Bactérias , Macrófagos/imunologia , Espécies Reativas de Oxigênio , Salmonella/genética , Salmonella/patogenicidade , Animais , Apoptose , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Células Cultivadas , Humanos , Imunidade Inata , Macrófagos/metabolismo , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos Endogâmicos BALB C , Fagocitose/imunologia , Salmonella/crescimento & desenvolvimento , Virulência
14.
Ultrason Sonochem ; 54: 48-60, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30827903

RESUMO

A green and innovative method, manothermosonication (MTS), for proteins extraction from dry Arthrospira platensis cyanobacteria assisted by ultrasound was designed in this work. Manothermosonication (probe, 20 kHz) was compared to a conventional process performed in the same conditions without ultrasounds. The extraction was carried out with a continuous flow rate at 15 mL/hour. Extraction parameters were optimized using a central composite design. Moreover, mathematic modelling and microscopic investigations were realized to allow a better understanding of ultrasound physical and structural effects on spirulina filaments and mass transfer phenomena over time. Crude extract and sections stained with toluidine blue were analyzed with optical and scanning electron microscopies. According to experimental results, MTS promoted mass transfer (high effective diffusivity, De) and enabled to get 229% more proteins (28.42 ±â€¯1.15 g/100 g DW) than conventional process without ultrasound (8.63 ±â€¯1.15 g/100 g DW). With 28.42 g of proteins per 100 g of dry spirulina biomass in the extract, a protein recovery rate of 50% was achieved in 6 effective minutes with a continuous MTS process. Microscopic observations showed that acoustic cavitation impacted spirulina filaments by different mechanisms such as fragmentation, sonoporation, detexturation. These various phenomena make extraction, release and solubilization of spirulina bioactive compounds easier.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Fracionamento Químico/métodos , Indústrias , Modelos Teóricos , Spirulina/química , Ondas Ultrassônicas , Aminoácidos/análise , Proteínas de Bactérias/química , Fracionamento Químico/instrumentação , Estudos de Viabilidade , Química Verde , Cinética , Temperatura Ambiente
15.
Photosynth Res ; 140(1): 103-113, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30826949

RESUMO

The remarkable drought-resistance of the terrestrial cyanobacterium Nostoc flagelliforme (N. flagelliforme) has attracted attention for many years. In this study, we purified a group of red proteins that accumulate in dried field samples of N. flagelliforme. These red proteins contain canthaxanthin as the bound chromophore. Native-PAGE analysis revealed that the purified red proteins resolved into six visible red bands and were composed of four helical carotenoid proteins (HCPs), HCP1, HCP2, HCP3, and HCP6 (homologs to the N-terminal domain of the orange carotenoid protein (OCP)). Seven genes encode homologs of the OCP in the genome of N. flagelliforme: two full-length ocp genes (ocpx1 and ocpx2), four N-terminal domain hcp genes (hcp1, hcp2, hcp3, and hcp6), and one C-terminal domain ccp gene. The expression levels of hcp1, hcp2, and hcp6 were highly dependent on the water status of field N. flagelliforme samples, being downregulated during rehydration and upregulated during subsequent dehydration. Transcripts of ocpx2 were dominant in the dried field samples, which we confirmed by detecting the presence of OCPx2-derived peptides in the purified red proteins. The results shed light on the relationship between carotenoid-binding proteins and the desiccation resistance of terrestrial cyanobacteria, and the physiological functions of carotenoid-binding protein complexes in relation to desiccation are discussed.


Assuntos
Adaptação Fisiológica , Proteínas de Bactérias/metabolismo , Carotenoides/metabolismo , Nostoc/fisiologia , Peptídeos/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Cantaxantina/genética , Cantaxantina/metabolismo , Carotenoides/genética , Carotenoides/isolamento & purificação , Dessecação , Nostoc/genética , Peptídeos/genética , Filogenia , Alinhamento de Sequência
16.
Microb Pathog ; 130: 146-155, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30826430

RESUMO

Application of antibiotics to combat bacterial diseases in fish has been criticized due to likely emergence of drug resistance. Therefore, investigation of new bioactive compounds from natural sources has been taken into account. This study was designed to purify and characterize the bioactive compound in the cell free supernatant (CFSs) of autochthonous gut bacteria (Bacillus methylotrophicus KU556164, B. amyloliquefaciens KU556165, Pseudomonas fluorescens KU556166 and B. licheniformis KU556167) isolated from rohu, Labeo rohita. CFSs were antagonistic to fish pathogenic Aeromonas spp., moderately thermo-tolerant and active in wide range of pH (5-11). Antibacterial activity of the CFSs was reduced by the action of proteases (e.g., Proteinase K and Trypsin), indicating proteinaceous nature of the bioactive compound like the bacteriocins. Three-step purification procedure resulted in recovery of 16.97%, 18.04%, 33.33% and 6.38% activity of the antimicrobial protein produced by B. methylotrophicus, B. amyloliquefaciens, P. fluorescens and B. licheniformis, respectively. Purification at each step revealed decrease in protein content with gradual increase in the specific activity of the antimicrobial protein. The purified antibacterial compound ranged between 18.2 and 25.6 kDa. Identification through MALDI-TOF MS/MS and database search through Mascot search engine predicted that the bactericidal compound belonged to either alkaline proteases, or, transcriptional regulator and some hypothetical proteins. Apart from potential technological application of the antibacterial compound, the present study might show promise for application of gut-associated bacteriocinogenic bacteria to control diseases in fish caused by pathogenic bacteria.


Assuntos
Antibacterianos/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Produtos Biológicos/metabolismo , Cyprinidae/microbiologia , Microbioma Gastrointestinal , Aeromonas/efeitos dos fármacos , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Antibiose , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/farmacologia , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Concentração de Íons de Hidrogênio , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
17.
Dalton Trans ; 48(12): 3973-3983, 2019 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-30834412

RESUMO

HutZ, a dimeric protein, from Vibrio cholerae is a protein that catalyzes the oxygen-dependent degradation of heme. Interestingly, the ascorbic acid-supported heme-degradation activity of HutZ depends on pH: less than 10% of heme is degraded by HutZ at pH 8.0, but nearly 90% of heme is degraded at pH 6.0. We examined here pH-dependent conformational changes in HutZ using fluorescence spectroscopy. Trp109 is estimated to be located approximately 21 Å from heme and is present in a different subunit containing a heme axial ligand. Thus, we postulated that the distance between heme and Trp109 reflects subunit-subunit orientational changes. On the basis of resonance energy transfer from Trp109 to heme, we estimated the distance between heme and Trp109 to be approximately 17 Å at pH 8.0, while the distance increased by less than 2 Å at pH 6.0. We presumed that such changes led to a decrease in electron donation from the proximal histidine, resulting in enhancement of the heme-degradation activity. To confirm this scenario, we mutated Ala31, located at the dimer interface, to valine to alter the distance through the subunit-subunit interaction. The distance between heme and Trp109 for the A31V mutant was elongated to 24-27 Å. Although resonance Raman spectra and reduction rate of heme suggested that this mutation resulted in diminished electron donation from the heme axial ligand, ascorbic acid-supported heme-degradation activity was not observed. Based on our findings, it can be proposed that the relative positioning of two protomers is important in determining the heme degradation rate by HutZ.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Heme/química , Heme/metabolismo , Vibrio cholerae/química , Proteínas de Bactérias/isolamento & purificação , Concentração de Íons de Hidrogênio , Modelos Moleculares , Estrutura Molecular , Oxirredução , Espectrometria de Fluorescência , Vibrio cholerae/metabolismo
18.
Methods Mol Biol ; 1969: 105-112, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30877672

RESUMO

Mass spectrometry (MS) proteomics allows systematic identification, characterization, and relative quantification of the full suite of proteins in a biological sample, and is a powerful analytical approach for investigation of many aspects of the biology of Neisseria meningitidis. Here, we describe methods for robust and efficient sample preparation of the proteome of N. meningitidis suitable for diverse MS proteomics workflows.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Neisseria meningitidis/metabolismo , Proteoma/análise , Proteômica/métodos , Manejo de Espécimes/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
19.
J Infect Chemother ; 25(6): 444-451, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30824303

RESUMO

Global emergence and dissemination of carbapenemases are clinically threatening, notably in countries with endemic blaNDM. To analyze the extent of carbapenemases in Bangladesh, 71 isolates were collected from 7 different clinical sources: wound swab (n = 38), pus (n = 13), urine (n = 9), blood (n = 4), tracheal aspirate (n = 3), pleural fluid (n = 1) and vaginal swab (n = 3) from Dhaka Medical College Hospital, Bangladesh. Among the isolates, 25 were resistant to at least one of the three carbapenems (imipenem, meropenem and doripenem), including 15 being resistant to all. These resistant isolates were identified as Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, P. hibiscicola, Proteus mirabilis, Providencia stuartii and Citrobacter sedlakii. Carbapenemase detection among these 25 isolates varied in individual phenotypic assays (83% in Modified Hodge Test, 50% in Combined Disk Test for Metallo-ß-lactamase prediction) as compared with the genotypes observed (96% prevalence of various carbapenemases including blaOXA-1,48, blaNDM-1,5, blaVIM-2,5). blaOXA-48 was the most prevalent gene (84%) followed by blaNDM (72%). Coexistence of multiple gene combination such as blaNDM+blaOXA-48+blaOXA-1 was prevalent (48%). Harborage of blaVIM-5 (n = 1) was characterized for the first time, while blaNDM-5 (n = 5) was reported contemporarily with a recent study in Bangladesh. Presence of plasmids (64%) and integron class 1 (100%) signifies the transferable potential of resistant traits. The emergence of such new variants along with the presence of the mobile genetic elements demands strict surveillance and combating strategies.


Assuntos
Antibacterianos/farmacologia , Bactérias/genética , Infecções Bacterianas/tratamento farmacológico , Proteínas de Bactérias/genética , Carbapenêmicos/farmacologia , beta-Lactamases/genética , Antibacterianos/uso terapêutico , Bactérias/efeitos dos fármacos , Bactérias/isolamento & purificação , Infecções Bacterianas/microbiologia , Proteínas de Bactérias/isolamento & purificação , Bangladesh , Carbapenêmicos/uso terapêutico , Farmacorresistência Bacteriana Múltipla/genética , Humanos , Integrons , Testes de Sensibilidade Microbiana , Plasmídeos , Resistência beta-Lactâmica/genética , beta-Lactamases/isolamento & purificação
20.
Mar Drugs ; 17(3)2019 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-30871149

RESUMO

Fibrinolytic enzymes have received more attention due to their medicinal potential for thrombolytic diseases. The aim of this study is to characterize the in vitro fibrinolytic nature of purified protease producing Streptomyces radiopugnans VITSD8 from marine brown tube sponges Agelas conifera. Three varieties of sponge were collected from the Rameshwaram Sea coast, Tamil Nadu, India. The fibrinolytic activity of Streptomyces sp. was screened and determined by casein plasminogen plate and fibrin plate methods respectively. The crude caseinolytic protease was purified using ammonium sulfate fractionation, affinity and ion-exchange chromatography. Based on the morphological, biochemical, and molecular characterization, the isolate VITSD8 was confirmed as Streptomyces radiopugnans. Maltose and peptone were found to be the best carbon and nitrogen sources for the production of fibrinolytic protease. The carbon and nitrogen source peptone showed (781 U/mL) enzyme activity. The optimum pH and temperature for fibrinolytic protease production was found to be 7.0 and 33 °C respectively. The purified enzyme showed a maximum specific activity of 3891 U. The blood clot lysis activity was compared with the standard, and it was concluded that a minimum of 0.18 U (10 µL) of purified protease was required to dissolve the blood clot. This is the first report which exploits the fibrinolytic protease activity of Streptomyces radiopugnans VITSD8 extracted from a marine sponge. Hence the investigation suggests a potential benefit of purified fibrinolytic protease which will serve as an excellent clot buster alternative.


Assuntos
Proteínas de Bactérias/química , Endopeptidases/biossíntese , Fibrinolíticos/química , Peptídeo Hidrolases/química , Poríferos/microbiologia , Streptomyces/química , Streptomyces/enzimologia , Animais , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/farmacologia , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Fibrinolíticos/isolamento & purificação , Fibrinolíticos/farmacologia , Peptídeo Hidrolases/isolamento & purificação , Peptídeo Hidrolases/farmacologia , Filogenia , Espectroscopia de Infravermelho com Transformada de Fourier , Trombose/tratamento farmacológico
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA