RESUMO
Ferredoxin/flavodoxin-NADPH reductases (FPRs) catalyze the reversible electron transfer between NADPH and ferredoxin/flavodoxin. The Acinetobacter sp. Ver3 isolated from high-altitude Andean lakes contains two isoenzymes, FPR1ver3 and FPR2ver3. Absorption spectra of these FPRs revealed typical features of flavoproteins, consistent with the use of FAD as a prosthetic group. Spectral differences indicate distinct electronic arrangements for the flavin in each enzyme. Steady-state kinetic measurements show that the enzymes display catalytic efficiencies in the order of 1-6 µm-1·s-1, although FPR1ver3 exhibited higher kcat values compared to FPR2ver3. When flavodoxinver3 was used as a substrate, both reductases exhibited dissimilar behavior. Moreover, only FPR1ver3 is induced by oxidative stimuli, indicating that the polyextremophile Ver3 has evolved diverse strategies to cope with oxidative environments.
Assuntos
Ferredoxinas , Flavodoxina , Flavodoxina/metabolismo , NADP/metabolismo , Ferredoxinas/metabolismo , Ferredoxina-NADP Redutase/química , Ferredoxina-NADP Redutase/metabolismo , Isoformas de Proteínas , CinéticaRESUMO
Cadmium (Cd) is a toxic heavy metal that causes serious health problems and is present in agriculturally important soils in Colombia, such as the ones used for cocoa farming. Recently, the use of ureolytic bacteria by the Microbiologically Induced Carbonate Precipitation (MICP) activity has been proposed as an alternative to mitigate the availability of Cd in contaminated soils. In this study, 12 urease-positive bacteria able to grow in the presence of Cd(II) were isolated and identified. Three were selected based on urease activity, precipitates formation and growth, with two belonging to the genus Serratia (codes 4.1a and 5b) and one to Acinetobacter (code 6a). These isolates exhibited low urease activity levels (3.09, 1.34 and 0.31 µmol mL-1 h-1, respectively), but could raise the pH to values close to 9.0 and to produce carbonate precipitates. It was shown that the presence of Cd affects the growth of the selected isolates. However, urease activity was not negatively influenced. In addition, the three isolates were observed to efficiently remove Cd from solution. The two Serratia isolates presented maximum removals of 99.70% and 99.62%, with initial 0.05 mM Cd(II) in the culture medium (supplemented with urea and Ca(II)) at 30 °C and 144 h of incubation. For the Acinetobacter isolate, the maximum removal was 91.23% at the same conditions. Thus, this study evidences the potential use of these bacteria for bioremediation treatments in samples contaminated with Cd, and it is one of the few reports that shows the high cadmium removal capacity of bacteria from the genus Serratia. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03495-1.
RESUMO
BACKGROUND: The genus Acinetobacter sp. comprises more than 50 species, and four are closely related and difficult to be distinguished by either phenotypic or genotypic methods: the Acinetobacter calcoaceticus-baumannii complex (ABC). The correct identification at species level is necessary mainly due to the epidemiological aspects. METHODS: We evaluated a multiplex PCR for gyrB gene to identify the species of the ABC using the sequencing of the ITS 16S-23S fragment as a gold standard. Isolates identified as Acinetobacter calcoaceticus-baumannii from three hospitals at southern Brazil in 2011 were included in this study. RESULTS: A total of 117 isolates were obtained and 106 (90.6%) were confirmed as A. baumannii, 6 (5.1%) as A. nosocomialis and 4 (3.4%) as A. pittii by PCR for gyrB gene. Only one isolate did not present a product of the PCR for the gyrB gene; this isolate was identified as Acinetobacter genospecie 10 by sequencing of ITS. We also noted that the non-A. baumannii isolates were recovered from respiratory tract (8/72.7%), blood (2/18.2%) and urine (1/9.1%), suggesting that these species can cause serious infection. CONCLUSION: These findings evidenced that the multiplex PCR of the gyrB is a feasible and simple method to identify isolates of the ABC at the species level.
Assuntos
Infecções por Acinetobacter , Acinetobacter baumannii/genética , DNA Girase/genética , DNA Bacteriano/análise , Reação em Cadeia da Polimerase Multiplex/métodos , Infecções por Acinetobacter/diagnóstico , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/classificação , DNA Bacteriano/química , DNA Bacteriano/genética , HumanosRESUMO
ABSTRACT The purpose of this study was to isolate, purify and optimize the production conditions of an organic solvent tolerant and thermostable lipase from Acinetobacter sp. AU07 isolated from distillery waste. The lipase production was optimized by response surface methodology, and a maximum production of 14.5 U/mL was observed at 30 ºC and pH 7, using a 0.5% (v/v) inoculum, 2% (v/v) castor oil (inducer), and agitation 150 rpm. The optimized conditions from the shake flask experiments were validated in a 3 L lab scale bioreactor, and the lipase production increased to 48 U/mL. The enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography and the overall yield was 36%. SDS-PAGE indicated a molecular weight of 45 kDa for the purified protein, and Matrix assisted laser desorption/ionization time of flight analysis of the purified lipase showed sequence similarity with GDSL family of lipases. The optimum temperature and pH for activity of the enzyme was found to be 50 ºC and 8.0, respectively. The lipase was completely inhibited by phenylmethylsulfonyl fluoride but minimal inhibition was observed when incubated with ethylenediaminetetraacetic acid and dithiothreitol. The enzyme was stable in the presence of non-polar hydrophobic solvents. Detergents like SDS inhibited enzyme activity; however, there was minimal loss of enzyme activity when incubated with hydrogen peroxide, Tween 80 and Triton X-100. The kinetic constants (Km and Vmax) revealed that the hydrolytic activity of the lipase was specific to moderate chain fatty acid esters. The Vmax, Km and Vmax/Km ratio of the enzyme were 16.98 U/mg, 0.51 mM, and 33.29, respectively when 4-nitrophenyl palmitate was used as a substrate.
Assuntos
Compostos Orgânicos , Solventes , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/biossíntese , Acinetobacter/enzimologia , Lipase/isolamento & purificação , Lipase/biossíntese , Compostos Orgânicos/química , Solventes/química , Especificidade por Substrato , Temperatura , Proteínas de Bactérias/química , Estabilidade Enzimática , Cinética , Cromatografia por Troca Iônica , Ativação Enzimática , Espaço Extracelular/enzimologia , Concentração de Íons de Hidrogênio , Íons , Lipase/química , Lipólise , Metais , Peso MolecularRESUMO
The purpose of this study was to isolate, purify and optimize the production conditions of an organic solvent tolerant and thermostable lipase from Acinetobacter sp. AU07 isolated from distillery waste. The lipase production was optimized by response surface methodology, and a maximum production of 14.5 U/mL was observed at 30 ºC and pH 7, using a 0.5% (v/v) inoculum, 2% (v/v) castor oil (inducer), and agitation 150 rpm. The optimized conditions from the shake flask experiments were validated in a 3 L lab scale bioreactor, and the lipase production increased to 48 U/mL. The enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography and the overall yield was 36%. SDS-PAGE indicated a molecular weight of 45 kDa for the purified protein, and Matrix assisted laser desorption/ionization time of flight analysis of the purified lipase showed sequence similarity with GDSL family of lipases. The optimum temperature and pH for activity of the enzyme was found to be 50 ºC and 8.0, respectively. The lipase was completely inhibited by phenylmethylsulfonyl fluoride but minimal inhibition was observed when incubated with ethylenediaminetetraacetic acid and dithiothreitol. The enzyme was stable in the presence of non-polar hydrophobic solvents. Detergents like SDS inhibited enzyme activity; however, there was minimal loss of enzyme activity when incubated with hydrogen peroxide, Tween 80 and Triton X-100. The kinetic constants (Km and Vmax) revealed that the hydrolytic activity of the lipase was specific to moderate chain fatty acid esters. The Vmax, Km and Vmax/Km ratio of the enzyme were 16.98 U/mg, 0.51 mM, and 33.29, respectively when 4-nitrophenyl palmitate was used as a substrate.(AU)
Assuntos
Lipase/análise , Lipase/isolamento & purificação , Solventes/isolamento & purificação , Acinetobacter/imunologia , Acinetobacter/fisiologiaRESUMO
The purpose of this study was to isolate, purify and optimize the production conditions of an organic solvent tolerant and thermostable lipase from Acinetobacter sp. AU07 isolated from distillery waste. The lipase production was optimized by response surface methodology, and a maximum production of 14.5U/mL was observed at 30°C and pH 7, using a 0.5% (v/v) inoculum, 2% (v/v) castor oil (inducer), and agitation 150rpm. The optimized conditions from the shake flask experiments were validated in a 3L lab scale bioreactor, and the lipase production increased to 48U/mL. The enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography and the overall yield was 36%. SDS-PAGE indicated a molecular weight of 45kDa for the purified protein, and Matrix assisted laser desorption/ionization time of flight analysis of the purified lipase showed sequence similarity with GDSL family of lipases. The optimum temperature and pH for activity of the enzyme was found to be 50°C and 8.0, respectively. The lipase was completely inhibited by phenylmethylsulfonyl fluoride but minimal inhibition was observed when incubated with ethylenediaminetetraacetic acid and dithiothreitol. The enzyme was stable in the presence of non-polar hydrophobic solvents. Detergents like SDS inhibited enzyme activity; however, there was minimal loss of enzyme activity when incubated with hydrogen peroxide, Tween 80 and Triton X-100. The kinetic constants (Km and Vmax) revealed that the hydrolytic activity of the lipase was specific to moderate chain fatty acid esters. The Vmax, Km and Vmax/Km ratio of the enzyme were 16.98U/mg, 0.51mM, and 33.29, respectively when 4-nitrophenyl palmitate was used as a substrate.
Assuntos
Acinetobacter/enzimologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/isolamento & purificação , Lipase/biossíntese , Lipase/isolamento & purificação , Compostos Orgânicos , Solventes , Proteínas de Bactérias/química , Cromatografia por Troca Iônica , Ativação Enzimática , Estabilidade Enzimática , Espaço Extracelular/enzimologia , Concentração de Íons de Hidrogênio , Íons , Cinética , Lipase/química , Lipólise , Metais , Peso Molecular , Compostos Orgânicos/química , Solventes/química , Especificidade por Substrato , TemperaturaRESUMO
INTRODUCTION: Hospitals around the world have presented multiresistant Acinetobacter sp. outbreaks. The spread of these isolates that harbor an increasing variety of resistance genes makes the treatment of these infections and their control within the hospital environment more difficult. This study aimed to evaluate the occurrence and dissemination of Acinetobacter sp. multiresistant isolates and to identify acquired resistance genes. METHODS: We analyzed 274 clinical isolates of Acinetobacter sp. from five hospitals in Porto Alegre, RS, Brazil. We evaluated the susceptibility to antimicrobial, acquired resistance genes from Ambler's classes B and D, and performed molecular typing of the isolates using enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) technique. RESULTS: A high (68 percent) percentage of multiresistant isolates of Acinetobacter sp. was observed, and 69 percent were resistant to carbapenems. We identified 84 percent of isolates belonging to species A. baumannii because they presented the gene blaOXA-51. The gene blaOXA-23 was detected in 62 percent of the isolates, and among these, 98 percent were resistant to carbapenems. Using the ERIC-PCR technique, we identified clones of Acinetobacter sp. spread among the four hospitals analyzed during the sampling period. CONCLUSIONS: The data indicate the dissemination of Acinetobacter sp. isolates among hospitals and their permanence in the hospital after one year.
INTRODUÇÃO: Hospitais no mundo todo têm apresentado surtos de Acinetobacter sp. multirresistentes. A disseminação destes isolados com uma variedade cada vez maior de genes de resistência torna difícil o tratamento destas infecções e seu controle dentro do ambiente hospitalar. Este trabalho teve como objetivo avaliar a ocorrência e disseminação de isolados de Acinetobacter sp. multirresistentes e identificar genes de resistência adquirida. MÉTODOS: Foram avaliados 274 isolados clínicos de Acinetobacter sp. obtidos de cinco hospitais da Cidade de Porto Alegre, RS, Brasil. Avaliamos o perfil de suscetibilidade a antimicrobianos, genes de resistência adquirida das classes B e D de Ambler e realizamos a tipificação molecular dos isolados utilizando a técnica de enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). RESULTADOS: Encontramos uma alta (68 por cento) porcentagem de isolados de Acinetobacter sp. multirresistentes e 69 por cento dos isolados apresentaram resistência aos carbapenêmicos. Foram identificados 84 por cento de isolados pertencentes a espécie A. baumannii, pois apresentaram o gene blaOXA-51. Em 62 por cento dos isolados, foi detectado o gene blaOXA-23, sendo que 98 por cento destes isolados foram resistentes aos carbapenêmicos. Através da tipificação molecular pela técnica de ERIC-PCR identificamos clones de Acinetobacter sp. disseminados entre quatro dos hospitais analisados e nos anos de 2006 e 2007. CONCLUSÕES: Os dados obtidos indicam a disseminação de isolados de Acinetobacter sp. entre hospitais assim como sua permanência no ambiente hospitalar após um ano.
Assuntos
Humanos , Acinetobacter/efeitos dos fármacos , Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Genes Bacterianos/genética , Acinetobacter/genética , Acinetobacter/isolamento & purificação , Técnicas de Tipagem Bacteriana , Brasil , Infecção Hospitalar/microbiologia , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , DNA Bacteriano/análise , Fenótipo , Reação em Cadeia da Polimerase/métodosRESUMO
Este projeto demonstra a importância no contexto hospitalar da adequação da rede de aspiração de secreções pela implantação do vácuo clínico para prevenção de infecções. As Unidades de Internação do hospital em estudo apresentam o sistema de sucção para fluidos e secreções corporais de pacientes pelo uso de válvulas de venturi conectadas em rede de oxigênio medicinal. Entretanto, por este sistema, ocorre a dispersão de micro-gotículas contaminantes no ambiente. A execução deste projeto busca reduzir a contaminação ambiental por microorganismos colonizantes, especialmente o Acinetobacter sp, envolvido em surtos hospitalares. Pretende-se intervir sobre o risco de colonização microbiana nos profissionais, entre pacientes e nos visitantes pela exposição durante procedimento de aspiração. A confiança da sociedade sobre a atenção terciária no SUS passa por projetos de intervenção que visem maior segurança assistencial prestada. Esta adequação física atende a legislações ministeriais sobre regulamentos para projetos em estabelecimentos assistenciais de saúde. Na perspectiva de custos, pretende-se reduzir gastos correlatos a médio e longo prazo.