RESUMO
An actinobacteria strain was isolated from an olive waste mill and tested for protease production on skimmed milk media. The strain identification was achieved through both 16 S rDNA sequencing and phenotypic characterization. The enzyme was purified using the ammonium sulfate/t-butanol three-phase partitioning (TPP) method, followed by characterization to investigate the effect of pH, temperature, and various chemical agents. Subsequently, the enzyme was assessed for its milk coagulation activity. The strain belonging to the Streptomyces genera, exhibits significant phylogenetic and phenotypic differences from the aligned species, suggesting its novelty as a new strain. The enzyme was best separated in the TPP aqueous phase with a 5.35 fold and 56.25% yield. Optimal activity was observed at pH 9.0 and 60 °C, with more than half of the activity retained within the pH range of 7-10 over one hour. The protease demonstrated complete stability between 30 and 60 °C. While metallic ions enhanced enzyme activity, EDTA acted as an inhibitor. The enzyme displayed resistance to H2O2, SDS, Tween 80, and Triton X-100. Notably, it was activated in organic solvents (ethyl acetate, petroleum ether, and xylene), maintaining > 75% of its original activity in butanol, ethanol, and methanol. Additionally, the enzyme yielded high milk coagulant activity of 11,478 SU/mL. The new Streptomyces sp. protease revealed high activity and stability under a wide range of biochemical conditions. Its use in the dairy industry appears particularly promising. Further industrial process investigations will be valuable in determining potential uses for this enzyme.
Assuntos
Estabilidade Enzimática , Leite , Peptídeo Hidrolases , Filogenia , Streptomyces , Temperatura , Streptomyces/isolamento & purificação , Streptomyces/enzimologia , Streptomyces/genética , Streptomyces/classificação , Leite/microbiologia , Animais , Concentração de Íons de Hidrogênio , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/isolamento & purificação , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , RNA Ribossômico 16S/genéticaRESUMO
Two new actinobacteria, designated strains IBSBF 2807T and IBSBF 2953T, isolated from scab lesions on potato tubers grown in the southern Brazilian states of Rio Grande do Sul and Santa Catarina, respectively, were characterized and identified through a polyphasic approach. Phylogenetic analyses of 16S rRNA sequences revealed that these two strains belong to the genus Streptomyces. Multilocus sequence analysis using five concatenated genes, atpD, gyrB, recA, rpoB and trpB, allocated strains IBSBF 2807T and IBSBF 2953T in distinct branches of Streptomyces phytopathogenic strains. PCR-RFLP analysis of the atpD gene also confirmed that these strains differ from the type strains of Streptomyces associated with potato scab. The morphological, physiological and biochemical characterization, along with the overall genome-related index properties, indicated that these two strains could be distinguished from their closest phylogenetic relatives and each other. According to the data, IBSBF 2807T and IBSBF 2953T represent two new Streptomyces species related to potato scab. The proposed names for these strains are Streptomyces hilarionis sp. nov. (IBSBF 2807T=CBMAI 2674T=ICMP 24297T=MUM 22.66T) and Streptomyces hayashii sp. nov (IBSBF 2953T=CBMAI 2675T=ICMP 24301T=MUM 22.68T).
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Solanum tuberosum , Streptomyces , Ácidos Graxos/química , Análise de Sequência de DNA , Solanum tuberosum/microbiologia , Brasil , Filogenia , RNA Ribossômico 16S/genética , DNA Bacteriano/genética , Técnicas de Tipagem Bacteriana , Composição de BasesRESUMO
Streptomyces sp. BRA-346 is an Actinobacteria isolated from the Brazilian endemic tunicate Euherdmania sp. We have reported that this strain produces epoxyketone peptides, as dihydroeponemycin (DHE) and structurally related analogs. This cocktail of epoxyketone peptides inhibits the proteasome chymotrypsin-like activity and shows high cytotoxicity to glioma cells. However, low yields and poor reproducibility of epoxyketone peptides production by BRA-346 under laboratory cultivation have limited the isolation of epoxyketone peptides for additional studies. Here, we evaluated several cultivation methods using different culture media and chemical elicitors to increase the repertoire of peptide epoxyketone production by this bacterium. Furthermore, BRA-346 genome was sequenced, revealing its broad genetic potential, which is mostly hidden under laboratory conditions. By using specific growth conditions, we were able to evidence different classes of secondary metabolites produced by BRA-346. In addition, by combining genome mining with untargeted metabolomics, we could link the metabolites produced by BRA-346 to its genetic capacity and potential regulators. A single biosynthetic gene cluster (BGC) was related to the production of the target epoxyketone peptides by BRA-346. The candidate BGC displays conserved biosynthetic enzymes with the reported eponemycin (EPN) and TMC-86A (TMC) BGCs. The core of the putative epoxyketone peptide BGC (ORFs A-L), in which ORF A is a LuxR-like transcription factor, was cloned into a heterologous host. The recombinant organism was capable to produce TMC and EPN natural products, along with the biosynthetic intermediates DH-TMC and DHE, and additional congeners. A phylogenetic analysis of the epn/tmc BGC revealed related BGCs in public databases. Most of them carry a proteasome beta-subunit, however, lacking an assigned specialized metabolite. The retrieved BGCs also display a diversity of regulatory genes and TTA codons, indicating tight regulation of this BGC at the transcription and translational levels. These results demonstrate the plasticity of the epn/tmc BGC of BRA-346 in producing epoxyketone peptides and the feasibility of their production in a heterologous host. This work also highlights the capacity of BRA-346 to tightly regulate its secondary metabolism and shed light on how to awake silent gene clusters of Streptomyces sp. BRA-346 to allow the production of pharmacologically important biosynthetic products.
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BACKGROUND: The native potatoes (Solanum tuberosum subsp. tuberosum L.) grown in Chile (Chiloé) represent a new, unexplored source of endophytes to find potential biological control agents for the prevention of bacterial diseases, like blackleg and soft rot, in potato crops. RESULT: The objective of this study was the selection of endophytic actinobacteria from native potatoes for antagonistic activity against Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum, and their potential to suppress tissue maceration symptoms in potato tubers. This potential was determined through the quorum quenching activity using a Chromobacterium violaceaum ATCC 12472 Wild type (WT) bioassay and its colonization behavior of the potato plant root system (S. tuberosum) by means of the Double labeling of oligonucleotide probes for fluorescence in situ hybridization (DOPE-FISH) targeting technique. The results showed that although Streptomyces sp. TP199 and Streptomyces sp. A2R31 were able to inhibit the growth of the pathogens, only the Streptomyces sp. TP199 isolate inhibited Pectobacterium sp. growth and diminished tissue maceration in tubers (p ≤ 0.05). Streptomyces sp. TP199 had metal-dependent acyl homoserine lactones (AHL) quorum quenching activity in vitro and was able to colonize the root endosphere 10 days after inoculation. CONCLUSIONS: We concluded that native potatoes from southern Chile possess endophyte actinobacteria that are potential agents for the disease management of soft rot and blackleg.
Assuntos
Actinobacteria/fisiologia , Antibiose/fisiologia , Endófitos/fisiologia , Solanum tuberosum/microbiologia , Actinobacteria/classificação , Actinobacteria/genética , Actinobacteria/isolamento & purificação , Agentes de Controle Biológico/isolamento & purificação , Chile , Endófitos/classificação , Endófitos/genética , Endófitos/isolamento & purificação , Pectobacterium/fisiologia , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Tubérculos/microbiologia , Percepção de Quorum , Streptomyces/classificação , Streptomyces/genética , Streptomyces/isolamento & purificação , Streptomyces/fisiologiaRESUMO
The extracellular polymeric substances (EPS) have shown free radical scavenging and antitumor activity against both breast and colon cell lines. In this regard, actinobacteria have become an increasingly popular sources of EPS. Therefore, in this study four Streptomyces strains isolated from contaminated soil (M7, A5, A14 and MC1) were evaluated for determining its biofilm-forming capacity including under pesticide stress. In addition, chemical composition of EPS and its cytotoxic effects over 4T1 breast cancer cell and Caco-2 human tumor colon cells were evaluated. The results demonstrated that Streptomyces sp. A5 had the highest capability to develop biofilm more than other strains tested, even under pesticide stress. Moreover, this strain produced EPS with a total protein/total polysaccharide rate of 1.59 ± 0.05. On the other hand, cytotoxicity assays of EPS showed that Streptomyces sp. A5 display a higher toxic effect against 4T1 Breast cancer cells (96.2 ± 13.5 %), Caco-2 (73.9 ± 6.4 %) and low toxicity (29.9 % ± 9.1 %) against non-transformed intestinal cells (IEC-18). Data do not show cytotoxic effect relationship with biofilm-forming capabilities of strains, nor the chemical composition of EPS matrix. The gene that codes for polysaccharide deacetylase, parB-like and transRDD proteins, were identified. These results contribute to the knowledge about the variability of chemical composition and potential cytotoxic properties of EPS produced by Streptomyces biofilms. It proposes interesting future challenges for linking Streptomyces-based pesticide remediation technology with the development of new antitumor drugs.
Assuntos
Matriz Extracelular de Substâncias Poliméricas , Streptomyces , Antineoplásicos/química , Antineoplásicos/farmacologia , Neoplasias da Mama , Células CACO-2 , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Matriz Extracelular de Substâncias Poliméricas/química , Matriz Extracelular de Substâncias Poliméricas/parasitologia , Humanos , Streptomyces/químicaRESUMO
ABSTRACT Objective: To investigate the secondary metabolites from the cultures of Streptomyces sp CSDX076. Methods: The compounds were isolated using column chromatography and RP-18 medium-pressure liquid chromatography. Their structures were elucidated by one-dimensional and two-dimensional nuclear magnetic resonance spectroscopic methods in combination with mass spectrometry experiments. Results: Four compounds were isolated from the cultures of Streptomyces sp CSDX076 and identified as aurantiamide benzoate, deoxytryptoquivaline, 2-acetyl-3,5-dihydroxyl-benzene acetic acid, and 2-acetyl-3,5-dihydroxyl-benzene ester. Conclusion: It was the first time that the four isolated compounds were obtained from the Streptomyces genus.
RESUMEN Objetivo: Investigar los metabolitos secundarios de los cultivos de Streptomyces sp CSDX076. Métodos: Los compuestos fueron aislados usando la cromatografía de columna y cromatografía líquida RP-18 de presión media. Sus estructuras fueron dilucidadas mediante métodos espectroscópicos de resonancia magnética nuclear unidimensional y bidimensional, combinados con experimentos de espectrometría de masa. Resultados: Cuatro compuestos de culturas de Streptomyces sp CSDX076 fueron aislados e identificados como benzoato de aurantiamida, deoxitriptoquivalina, ácido acético 2-acetil-3,5-dihidroxil-benzeno, y éster 2-acetil-3,5-dihidroxil-benzeno. Conclusión: Fue la primera vez que los cuatro compuestos aislados se obtuvieron del género Streptomyces.
Assuntos
Streptomyces/química , Benzoatos/isolamento & purificação , Espectrometria de Massas , Espectroscopia de Ressonância Magnética , Cromatografia LíquidaRESUMO
The most practical approach to reduce morbidity and mortality of coronary heart disease (CHD) is to delay the process of thrombus by usage of clot-dissolving agents. The necessities of such safer compounds are to be critically examined for thrombolytic activity especially, from marine sources. Thrombolytic agents have been investigated as a possible treatment for thrombus. The aim of this study was to investigate the in vitro thrombolytic potential of Streptomyces sp.VITJS4 (NCIM No. 5574); (ACC No: JQ234978.1) active compounds. The fibrin degradation revealed a clear transparent zone of clearance with 500µg/mL concentration showing 24mm hydrolysis. The thrombolytic effect of Streptomyces sp.VITJS4 compounds was also demonstrated in vitro clot lysis assay where The percent of thrombolysis by the crude extract showed 90±1.7% at the concentration of 1000µg/mL, whereas percent of thrombolysis by streptokinase was found 100± 00%%. The bioactive compounds were further studied for spectrophotometric analysis. The UV-VIS profile showed different peaks ranging from 400-700 nm with different absorption respectively. The data confirmed the presence of both analogues with absorption maxima at 210 and 310 nm. A sensitive method using LC-MS technique was optimized for the separation and identification of bioactive metabolites which was indicated by the fingerprints. The results of the LC-MS analysis provided different peaks determining the presence of compounds with different therapeutic activities. The current study refers the bioactive compound as impressive thrombolytic agent for further laboratory study. Further studies should be conducted to ensure the efficacy and safety of different concentration of bioactive compounds for drug development. Hence the results reported perhaps useful for the discovery of novel thrombolytic drugs from marine origin.
A abordagem mais prática para reduzir a morbidade e a mortalidade da doença arterial coronariana (CHD, do inglês coronary heart disease) consiste em retardar o processo de trombo através da utilização de agentes de dissolução de coágulos. As necessidades de tais compostos mais seguros devem ser criticamente examinadas para a atividade trombolítica, especialmente de fontes marinhas. Agentes trombolíticos tem sido estudados como um possível tratamento para o trombo. O objetivo deste estudo foi investigar o potencial trombolítico in vitro dos compostos ativos do Streptomyces sp.VITJS4 (NCIM No. 5574); (ACC No: JQ234978.1). A degradação da fibrina revelou um clara zona livre transparente com concentração de 500µg/mL mostrando uma hidrólise de 24mm. O efeito trombolítico dos compostos de Streptomyces sp.VITJS4 também foi demonstrado no ensaio in vitro de lise dos coágulos em que a percentagem de trombólise pelo extrato bruto mostrou 90±1.7% a uma concentração de 1000µg/mL, enquanto que a percentagem de trombólise pela estreptoquinase foi de 100± 00%. Os compostos bioativos foram estudados posteriormente através da análise espectrofotométrica. O perfil ultra violeta visível (UV-VIS profile, em inglês) mostrou diferentes picos variando entre 400-700 nm com diferentes absorções respectivamente. Os dados confirmaram a presença de ambos os análogos com absorção máxima em 210 e 300 nm. Um método sensível usando a técnica LC-MS (Liquid chromatographymass spectrometry) foi otimizado para a separação e identificação metabólitos bioativos que foram indicados pelas impressões digitais (?). Os resultados da análise LC-MS forneceram diferentes picos determinando a presença de compostos com diferentes atividades terapêuticas. O estudo atual refere-se ao composto bioativo como um agente trombolítico impressionante para futuros estudos em laboratório. Estudos futuros devem ser conduzidos para assegurar a eficácia e segurança de diferentes concentrações dos compostos bioativos para o desenvolvimento de drogas. Assim, os resultados reportados talvez sejam úteis para a descoberta de novas drogas trombolíticas de origem marinha.
Assuntos
Streptomyces , Trombose , Técnicas In Vitro , Actinobacteria , FibrinolíticosRESUMO
The organochlorine insecticide γ-hexachlorocyclohexane (γ-HCH, lindane) and its non-insecticidal α- and ß-isomers continue to pose serious environmental and health concerns, although their use has been restricted or completely banned for decades. In this study we report the first evidence of the growth ability of a Streptomyces strain in a mineral salt medium containing high doses of α- and ß-HCH (16.6 mg l(-1)) as a carbon source. Degradation of HCH isomers by Streptomyces sp. M7 was investigated after 1, 4, and 7 days of incubation, determining chloride ion release, and residues in the supernatants by GC with µECD detection. The results show that both the α- and ß-HCH isomers were effectively metabolized by Streptomyces sp. M7, with 80 and 78 % degradation respectively, after 7 days of incubation. Moreover, pentachlorocyclohexenes and tetrachlorocyclohexenes were detected as metabolites. In addition, the formation of possible persistent compounds such as chlorobenzenes and chlorophenols were studied by GC-MS, while no phenolic compounds were detected. In conclusion, we have demonstrated for the first time that Streptomyces sp. M7 can degrade α- and ß-isomers individually or combined with γ-HCH and could be considered as a potential agent for bioremediation of environments contaminated by organochlorine isomers.
Assuntos
Hexaclorocicloexano/química , Hexaclorocicloexano/metabolismo , Poluentes do Solo/química , Poluentes do Solo/metabolismo , Streptomyces/metabolismo , Anaerobiose , Biodegradação Ambiental , Inseticidas/química , Inseticidas/metabolismo , IsomerismoRESUMO
ABSTRACT This study evaluated the influence of glucose and stirring in the fermentation process in order to produce anti-Candida metabolites produced by Streptomyces sp. MPO4 isolated from Amazon soil. The anti-Candida metabolites production was registered after 24 h of fermentation in stirred ISP2 medium, having antifungal inhibition halos between 12.3 mm and 25.3 mm, yielding higher production of anti-Candida agents after 96 h. Stirring was a determining factor for the production of anti-Candida secondary metabolites, since the absence of glucose reflected in the late production of the antifungal starting from Streptomyces sp.
RESUMO Este estudo avaliou a influência da glicose e agitação no processo de fermentação para a produção de metabólitos anti-Candida produzidos por Streptomyces sp. MPO4 isolado do solo da Amazônia. A produção dos metabólitos anti-Candida foi registrada a partir de 24 h de fermentação sob agitação em meio ISP2, apresentando halos de inibição entre 12,3 mm e 25,3 mm, obtendo-se maior produção do antifúngico em 96 h. A agitação foi um fator determinante para a produção de metabólitos secundários anti-Candida e a ausência de glicose refletiu na produção tardia do antifúngico a partir do Streptomyces sp.
Assuntos
Streptomyces/química , Candida albicans/química , Fermentação/efeitos dos fármacos , Glucose/análise , Antifúngicos/farmacologiaRESUMO
Atrazine is still one of the most used agricultural pesticides worldwide and it has been recognized as a major contaminant of surface and ground water. The aims of this research were to isolate an endophytic microorganism from leaves of sugarcane, evaluate its ability to degrade atrazine, and investigate the formation of metabolites. By sequencing of the 16S rRNA gene, the endophytic isolate atz2 was identified as Streptomyces sp. The reduction in atrazine concentration by Streptomyces sp. atz2 was 98 % and UHPLC-MS/MS analyses showed the appearance of an unknown metabolite observed as m/z 311. Ecotoxicity tests with an aquatic organism, Daphnia similis, confirmed that this metabolite was nontoxic. This mechanism of detoxification of atrazine is different from the ones of other free-living microorganisms that inhabit the soil or rhizosphere. The results show new aspects of atrazine detoxification, highlighting a new role of endophytic bacteria in plants.
Assuntos
Atrazina/metabolismo , Streptomyces/metabolismo , Agricultura , Animais , Biodegradação Ambiental , Daphnia/efeitos dos fármacos , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , Saccharum/metabolismo , Saccharum/microbiologia , Solo/química , Microbiologia do Solo , Streptomyces/isolamento & purificação , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Actinomycetes are gram positive bacteria with high G + C content in their DNA and are capable of producing variety of secondary metabolites. Many of these metabolites possess different biological activities and have the potential to be developed as therapeutic agents. The aim of the present study was to screen actinomycetes inhabiting halophilic environment such as Khewra salt mines present in Pakistan for cytotoxic and antitumor compounds. RESULTS: An actiomycetes strain designated as Streptomyces sp. KML-2 was isolated from a saline soil of Khewra salt mines, Pakistan. The strain Streptomyces sp. KML-2 showed 84 % cytotoxic activity against larvae of Artemiasalina. In the screening phase, the strain exhibited significant antitumor activity with IC50 values of 12, 48 and 56 µg/ml against Hela, MDBK and Vero cell lines, respectively. After that extract from 20 l fermentation was used to purify secondary metabolites by several chromatographic techniques. Structure elucidation of isolated compounds revealed that it is highly stable producer of Chromomycin SA (1) and 1-(1H-indol-3-yl)-propane-1,2,3-triol (2). Both of the isolated compounds showed significant antitumor activity against Hela and MCF-7 cancer cell lines (IC50 values 8.9 and 7.8 µg/ml against Hela; 12.6 and 0.97 µg/ml against MCF-7, respectively). The 16S rRNA gene sequence (1437 bp) of the strain confirm its identity (99 %) with Streptomyces griseus. CONCLUSIONS: From this research work we were successful in isolating two potent antitumor compounds, Chromomycin SA and 1-(1H-indol-3-yl)-propane-1,2,3-triol from Streptomyces KML-2 strain, isolated from Khewra salt mine. As such this is the second report which confirms that S. griseus can produce Chromomycin SA without introducing any mutagenesis in its biosynthesizing gene cluster and isolated indole derivative is being reported first time from any member of actinomycetes group with having novel antitumor activity against Hela and MCF-7 cells Nucleotide sequences: Nucleotide sequence data reported are available in the GenBank database under the accession number: GenBank KJ009562.
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Humanos , Animais , Bovinos , Microbiologia do Solo , Streptomyces/química , Antineoplásicos/farmacologia , Paquistão , Filogenia , Artemia/classificação , Artemia/efeitos dos fármacos , Sais , Solo/química , Streptomyces/isolamento & purificação , Streptomyces/ultraestrutura , Streptomyces griseus/classificação , Sais de Tetrazólio , Células Vero , RNA Ribossômico 16S/genética , Cromomicinas/classificação , Cromomicinas/farmacologia , Células HeLa , Microscopia Eletrônica de Varredura , Linhagem Celular , Chlorocebus aethiops , Cromatografia/métodos , Análise de Sequência de RNA , Concentração Inibidora 50 , Células MCF-7 , Formazans , Glicerol/análogos & derivados , Glicerol/farmacologia , Larva/efeitos dos fármacos , Mineração , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Antineoplásicos/isolamento & purificaçãoRESUMO
In this study, attempts were made to isolate Streptomyces sp. from soil samples of two different regions of Bangladesh and evaluate their antagonistic activity against fish and human pathogenic bacteria. A total of 10 isolates were identified as Streptomyces sp. based on several morphological, physiological and biochemical tests. Cross streak method was used to observe the antagonistic activity of the Streptomyces sp. isolates against different fish pathogens belonging to the genus Aeromonas, Pseudomonas and Edwardsiella and human clinical isolates belonging to the genus Klebsiella, Salmonella and Streptococcus. Seven Streptomyces sp. isolates showed antagonism against both fish and human pathogenic bacteria. Four isolates viz., N24, N26, N28 and N47 showed broad spectrum of antagonistic activity (80-100%) against all genera of fish and human pathogenic bacteria. The isolate N49 exhibited highest spectrum of antagonism against all fish pathogens (90-100%) but comparatively lower degree of antagonism against human pathogens (50-60%). Rest of the two isolates (N21 and N23) showed variability in their antagonism. Results showed that broad spectrum antibiotic(s) could be developed from the isolates N24, N26, N28 and N47against several human and fish pathogens. The isolate N49 could be a potential source of antibiotic, especially for fish pathogenic bacteria.
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The fluorinase is an enzyme that catalyses the combination of S-adenosyl-L-methionine (SAM) and a fluoride ion to generate 5'-fluorodeoxy adenosine (FDA) and L-methionine through a nucleophilic substitution reaction with a fluoride ion as the nucleophile. It is the only native fluorination enzyme that has been characterised. The fluorinase was isolated in 2002 from Streptomyces cattleya, and, to date, this has been the only source of the fluorinase enzyme. Herein, we report three new fluorinase isolates that have been identified by genome mining. The novel fluorinases from Streptomyces sp. MA37, Nocardia brasiliensis, and an Actinoplanes sp. have high homology (80-87 % identity) to the original S. cattleya enzyme. They all possess a characteristic 21-residue loop. The three newly identified genes were overexpressed in E. coli and shown to be fluorination enzymes. An X-ray crystallographic study of the Streptomyces sp. MA37 enzyme demonstrated that it is almost identical in structure to the original fluorinase. Culturing of the Streptomyces sp. MA37 strain demonstrated that it not only also elaborates the fluorometabolites, fluoroacetate and 4-fluorothreonine, similar to S. cattleya, but this strain also produces a range of unidentified fluorometabolites. These are the first new fluorinases to be reported since the first isolate, over a decade ago, and their identification extends the range of fluorination genes available for fluorination biotechnology.
Assuntos
Proteínas de Bactérias/metabolismo , Genoma Bacteriano , Micromonosporaceae/genética , Nocardia/genética , Oxirredutases/metabolismo , Streptomyces/genética , Proteínas de Bactérias/genética , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/metabolismo , Fluoretação , Fluoretos/química , Fluoretos/metabolismo , Cinética , Micromonosporaceae/enzimologia , Família Multigênica , Nocardia/enzimologia , Oxirredutases/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Streptomyces/enzimologiaRESUMO
Approximately 1.5 trillion tons are the estimated yearly biomass production, making it an essentially unlimited source of raw material for environmentally friendly and biocompatible products transformed by microorganism, specially fungi and actinomycetes. Several lignocellulosic residues, such as sisal waste and sugarcane bagasse contain starch in their structures which could become important sources for the production of amylases. This study evaluated the production of amylolytic enzymes using Streptomyces sp. SLBA-08 strain, isolated from a semi-arid soil, according to their ability to grow on soluble starch as the sole carbon source. The effect of the carbon source (sisal waste and sugarcane bagasse) on α-amylase production was studied using submerged cultivations at 30 ºC. The highest level of α-amylase activity corresponded to 10.1 U. mL-1 and was obtained using sisal waste (2.7%) and urea (0.8%) in submerged fermentation after 3 days of cultivation. The partial characterization showed the best α-amylase activity at 50ºC and pH 7.0. These results are of great importance for the use of sisal waste as a substrate for biotechnological proposes.
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A new Streptomyces sp. IF 5 was isolated from the feather dumped soil and found to have a tremendous keratinase activity. The strain enabled the degradation of the chicken feathers very effectively in 60 h. The 16S rRNA sequence of 1474 bp long was submitted to the National centre for Biotechnological information. The keratinolytic activity in the culture medium was 1181 U/ml. The release and analyses of sulphydryl groups in the culture medium evident the degradation activity by the Streptomyces sp. IF 5. The idea of the present study was to use the degraded chicken feathers as the substrate for the growth and cultivation of microorganisms. We have designed a very economical culture medium that includes the usage of some basal salts alone and degraded chicken feathers (10 g/l). The results of the specific growth rate of the tested microbes confirm the usage of the new designed medium for microbial culturing.
Assuntos
Animais , Sequência de Bases , Ensaios Enzimáticos Clínicos , Microbiologia Ambiental , Análise de Alimentos , Microbiologia de Alimentos , Genética Microbiana , Meios de Cultura/isolamento & purificação , Estabilidade de RNA , Streptomyces/isolamento & purificação , Galinhas , Ativação Enzimática , Alimentos , Amostras de Alimentos , Métodos , MétodosRESUMO
The aim of this study was to isolate novel enzyme-producing bacteria from vegetation samples from East Antarctica and also to characterize them genetically and biochemically in order to establish their phylogeny. The ability to grow at low temperature and to produce amylases and proteases cold-active was also tested. The results of the 16S rRNA gene sequence analysis showed that the 4 Alga rRNA was 100 percent identical to the sequences of Streptomyces sp. rRNA from Norway and from the Solomon Islands. The Streptomyces grew well in submerged system at 20ºC, cells multiplication up to stationary phase being drastically increased after 120 h of submerged cultivation. The beta-amylase production reached a maximum peak after seven days, while alpha-amylase and proteases were performing biosynthesis after nine days of submerged cultivation at 20ºC. Newly Streptomyces were able to produce amylase and proteases in a cold environment. The ability to adapt to low temperature of these enzymes could make them valuable ingredients for detergents, the food industry and bioremediation processes which require low temperatures.
Assuntos
Amilases , Microbiologia Ambiental , RNA Bacteriano/análise , Streptomyces/crescimento & desenvolvimento , Streptomyces/isolamento & purificação , beta-Amilase/análise , Métodos , Filogenia , MétodosRESUMO
The response of two marine actinomycetes such as Streptomyces sp. MADO2 and Nocardiopsis sp. MADO3 to osmotic stress in minimal medium M63 and in glycerol-asparagine medium (ISP5) was studied. The two strains were moderately halophilic and the behavior of the strain Streptomyces sp. MADO2 and Nocardiopsis sp. MADO3 towards the salt stress was varied depends on the media composition and the salinity concentration. The strain Streptomyces sp. was more sensitive to salt stress than Nocardiopsis sp. The growth of both Streptomyces sp. and Nocardiopsis sp. were inhibited at 1 M NaCl irrespective of the medium used. The Nocardiopsis sp. acquired osmoadaptation on ISP5 medium whereas the Streptomyces sp. showed poor growth on M63 medium. Glycine betaine (GB), proline and trehalose played a critical role in osmotic adaptation at high osmolarity whereas at low osmolarity they showed an inhibitory effect on the bacterial growth. The present findings confirmed that GB was the powerful osmoprotectant for Streptomyces sp. and Nocardiopsis sp. grown at 1 M NaCl both in M63 and ISP5 media.
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An indigenous Streptomyces isolate CTF9, exhibiting promising antifungal activity against Mucor miehei and Candida albicans in pre-screening studies, was investigated by cultivation in a 50-L fermenter and by subsequent isolation, purification, and structure elucidation of the active metabolites. Based on the morphological, biochemical, and physiological characterization, as well as the 16S rRNA gene sequence, the isolate CTF9 was identified as Streptomyces malachitofuscus. Using a series of chromatographic techniques, two pure compounds were isolated from the obtained extracts after the fermentation of the isolate CTF9. The isolated compounds were identified as phenylacetic acid and indolyl-3-lactic acid by mass spectrometry (MS) and NMR analysis. The culture optimization studies revealed that the isolate CTF9 can use a variety of low-cost carbon and nitrogen sources to generate the maximum quantity of industrially important metabolites at an elevated temperature of 35°C and at a pH 7.8.
RESUMO
Clavulanic acid is a β-lactam antibiotic which has a potent β-lactamase inhibiting activity. In order to optimize its production by the new isolate Streptomyces DAUFPE 3060, the influence of two independent variables, temperature and soybean flour concentration, on clavulanic acid and biomass concentrations was investigated in 250 mL-Erlenmeyers according to a 2² central composite design. To this purpose, temperature and soybean flour (SF) concentration were varied in the ranges 26-34°C and 10-50 g/L, respectively, and the results evaluated utilizing the Response Surface Methodology. The experimental maximum production of clavulanic acid (629 mg/L) was obtained at 32°C and 40 g/L SF after 48 h, while the maximum biomass concentration (3.9 g/L) at 30°C and 50 g/L soybean flour, respectively. These values are satisfactorily close to those (640 mg/L and 3.75 g/L, respectively) predicted by the model, thereby demonstrating the validity of the mathematical approach adopted in this study.
RESUMO
The response of two marine actinomycetes such as Streptomyces sp. MADO2 and Nocardiopsis sp. MADO3 to osmotic stress in minimal medium M63 and in glycerol-asparagine medium (ISP5) was studied. The two strains were moderately halophilic and the behavior of the strain Streptomyces sp. MADO2 and Nocardiopsis sp. MADO3 towards the salt stress was varied depends on the media composition and the salinity concentration. The strain Streptomyces sp. was more sensitive to salt stress than Nocardiopsis sp. The growth of both Streptomyces sp. and Nocardiopsis sp. were inhibited at 1 M NaCl irrespective of the medium used. The Nocardiopsis sp. acquired osmoadaptation on ISP5 medium whereas the Streptomyces sp. showed poor growth on M63 medium. Glycine betaine (GB), proline and trehalose played a critical role in osmotic adaptation at high osmolarity whereas at low osmolarity they showed an inhibitory effect on the bacterial growth. The present findings confirmed that GB was the powerful osmoprotectant for Streptomyces sp. and Nocardiopsis sp. grown at 1 M NaCl both in M63 and ISP5 media.