RESUMEN
Green synthesis of bimetallic nanoparticles of noble metals is highly desirable in nanomedicine because of their potential use as anticoagulant, thrombolytic and anticancer agents. In this study, it was discovered that the filamentous fungus Aspergillus niger proved effective in producing bimetallic Ag-Au nanoparticles. A. niger culture supernatant was able to produce Ag-AuNPs by reducing the solution of chloroauric acid/silver nitrate (1.0:1.0 mM) within 2 min at 100 °C and pH 8. Experimental Ag-AuNP detection was performed by visually observing the color change to reddish brown. The produced nanoparticles displayed maximal absorbance at 530 nm in UV-vis spectroscopy. According to transmission electron microscopy, most of the nanoparticles were spherical, with a mean diameter of 8-10 nm. The biosynthesis of Ag-AuNPs by A. niger was confirmed by Fourier transform infrared spectroscopy, X-ray diffraction and energy dispersive X-ray analytical techniques. Its zeta potential was discovered to be -34.01 mV. The biosynthesized Ag-AuNPs exhibited effective thrombolytic and antiplatelet aggregation actions by totally preventing and dissolving the blood clot which was verified by microscopic examination, amelioration of blood coagulation assays, and carrageenan-induced tail thrombosis model. The findings verified the effectiveness of biosynthesized Ag-AuNPs as a powerful antitumor agent against HepG2 and A549 cell lines with IC50 values of 15.57 and 27.07 µg/mL, respectively. Crystal violet assay validated the cytopathic effects of Ag-AuNPs on A549 and HepG2 cell lines. Therefore, the produced Ag-AuNPs from A. niger are a promising candidate in the management of thrombosis.
RESUMEN
Cochliobolus hawaiiensis Alcorn Assiut University Mycological Centre 8606 was chosen from the screened 20 fungal species as the potent producer of fibrinolytic enzyme on skimmed-milk agar plates. The greatest enzyme yield was attained when the submerged fermentation (SmF) conditions were optimized, and it was around (39.7 U/mg protein). Moreover, upon optimization of fibrinolytic enzyme production under solid-state fermentation (SSF), the maximum productivity of fibrinolytic enzyme was greatly increased recorded a bout (405 U/mg protein) on sugarcane bagasse, incubation period of 5 days, moisture level of 100%, initial pH of salt basal medium 7.8, incubation temperature at 35°C, and supplementation of the salt basal medium with corn steep liquor (80ï¼ , v/v). The yield of fibrinolytic enzyme by C. hawaiiensis under SSF was higher than that of SmF with about 10.20-fold. The purification procedures of fibrinolytic enzyme by ammonium sulfate (70%), gel filtration, and ion-exchange columns chromatography caused a great increase in its specific activity to 2581.6 U/mg protein with an overall yield of 55.89%, 6.37 purification fold and molecular weight of 35 kDa. Maximal activity was recorded at pH 7 and 37°C. Significant pH stability was recorded at pH 6.6-7.2, and thermal stability was recorded at 33-41°C. The enzyme showed the highest affinity toward fibrin, with Vmax of 240 U/mL and an apparent Km value of 47.61 mmol. Mg2+ and Ca2+ moderately induced fibrinolytic activity, whereas Cu2+ and Zn2+ greatly suppressed the enzyme activity. The produced enzyme is categorized as serine protease and non-metalloprotease. The purified fibrinolytic enzyme showed efficient thrombolytic and antiplatelet aggregation activities by completely prevention and dissolution of the blood clot which confirmed by microscopic examination and amelioration of blood coagulation assays. These findings suggested that the produced fibrinolytic enzyme is a promising agent in management of blood coagulation disorders.
Asunto(s)
Celulosa , Saccharum , Humanos , Celulosa/metabolismo , Fermentación , Concentración de Iones de Hidrógeno , Saccharum/metabolismo , Fibrinolíticos/farmacología , Fibrinolíticos/química , Fibrinolíticos/metabolismo , Temperatura , Peso MolecularRESUMEN
The most important tools in killing and overcoming on the microbes and pathogens that cause diseases in medicine and/or in agriculture are the antibiotics. The discovery and synthesis of the microbial natural products or antibiotics has greatly developed genetically and biotechnologically quickly in the last decades. It is necessary to access this great genetic diversity by finding ways to increase the level of expression of these biosynthetic pathways. In this study, we carried out an improvement in the antibiotic production of weak Streptomyces graminofaciens strain NBR9 that has high lipid content; using Ultra-Violet irradiation mutagenesis. This strain was isolated from the Northern Region in the kingdom of Saudi Arabia and identified biochemically and confirmed genetically by sequencing of the 16S rRNA gene as Streptomyces graminofaciens NBR9; Accession No. (MN640578). The resultant mutant strain showed increasing in their antimicrobial activities. The methods and techniques used for the antibiotic extraction, purification, characterization and identification proved that the obtained antibiotic is same with antibiotic Carbomycin.
Asunto(s)
Vías Biosintéticas/genética , Microbiología Industrial , Leucomicinas/biosíntesis , Lípidos/genética , Mutación/genética , Streptomyces/genética , Antibacterianos/biosíntesis , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Leucomicinas/aislamiento & purificación , Leucomicinas/farmacología , Mutagénesis , ARN Ribosómico 16S/genéticaRESUMEN
Aspergillus terreus was chosen for production of alkaline protease using solid-state fermentation (SSF). The maximum enzyme yield reached about 34.87 U/mg protein after optimization of fermentation parameters. The produced alkaline protease was purified by precipitation with iso-propanol and then purified through gel filtration and ion exchange column chromatography with a yield of 53.58% and 5.09- fold purification. The enzyme has shown to have a molecular weight of 35 kDa. Optimal pH and temperature for the enzyme activity were 9.5 and 50°C respectively. The highest activity was reported towards casein, with an apparent Km value of 6.66 mg/mL and Vmax was 30 U/mL. The enzyme activity was greatly repressed by phenylmethylsulfonyl fluoride (PMSF). Sodium dodecyl sulfate (SDS) caused activation in enzyme activity. The enzyme retained about 83.8, 70.6, 74.5, 76.4 and 66.4% of its original activity after incubation with Aerial, Leader, Oxi, Persil and Tide, respectively for 8 h at 60°C. Adding of the enzyme in detergents improved the cleansing performance to the blood stains and suggested to be used as a detergent additive. Our outcomes showed that protease could be used as environment green-approach in dehairing process.
Asunto(s)
Aspergillus/metabolismo , Detergentes , Fermentación , Serina Endopeptidasas , Activación Enzimática/efectos de los fármacos , Concentración de Iones de Hidrógeno , Fluoruro de Fenilmetilsulfonilo/farmacología , Serina Endopeptidasas/aislamiento & purificación , Serina Endopeptidasas/metabolismo , Dodecil Sulfato de Sodio/farmacología , TemperaturaRESUMEN
The aim of this work was to purify L-glutaminase from Aspergillus flavus. The enzyme was purified 12.47-fold from a cell-free extract with a final specific activity of 613.3 U/mg and the yield was 51.11%. The molecular weight of the enzyme, as estimated by SDS-PAGE, was found to be 69 kDa. The maximal activity of L-glutaminase was recorded at pH 8 and 40°C. The highest activity was reported towards L-glutamine as substrate, with an apparent Km value of 4.5 mmol and Vmax was 20 Uml-1. The enzyme was activated by Na+ and Co2+, while it was greatly suppressed by iodoacetate, NEM, Zn2+ and Hg2+ at 10 mM. L-glutaminase activity increased with a gradual increase of sodium chloride concentration up to 15%. In vivo, the median lethal dose (LD50) was approximately 39.4 mg/kg body weight after intraperitoneal injection in Sprague Dawley rats. Also, L-glutaminase showed no observed changes in liver and kidney functions and hematological parameters on rates. Purified A. flavus L-glutaminase had neither a cognizable effect on human platelet aggregation nor hemolytic activity. In addition, MTT assay showed that the purified L-glutaminase has a high toxic impact on Hela and Hep G2 cell lines with an IC50 value 18 and 12 µg/ml, respectively, and a moderate cytotoxic effect on HCT-116 and MCF7 cells, with an IC50 value 44 and 58 µg/ml, respectively.
Asunto(s)
Antineoplásicos/farmacología , Aspergillus flavus/enzimología , Glutaminasa/farmacología , Animales , Antineoplásicos/aislamiento & purificación , Plaquetas/efectos de los fármacos , Estabilidad de Enzimas , Glutaminasa/aislamiento & purificación , Células HeLa , Células Hep G2 , Humanos , Concentración de Iones de Hidrógeno , Concentración 50 Inhibidora , Cinética , Dosificación Letal Mediana , Peso Molecular , Ratas , Ratas Sprague-Dawley , Especificidad por SustratoRESUMEN
Trichoderma viride AUMC 13021 isolated from Mangrove soil of Ras Mohammed protected area at Sharm El-Sheikh, Egypt, was optimized to promote chitinase activity under submerged fermentation. The maximum enzyme yield (38.33 U/mg protein) was obtained at 1.4% of colloidal chitin, 96 h of incubation, 35°C, pH 6.5 and 125, rpm and using maltose (1%) and yeast extract (1%) as supplementation of salt basal medium. The enzyme has been purified with an overall yield of 73.1% and 5.48 purification fold, and a specific activity of 210.16 U/mg protein. The molecular mass of the purified chitinase was 62 kDa. Maximal activity of chitinase was recorded at pH 6.5 and 40°C. The highest activity was recorded in the case of colloidal chitin, with an apparent Km value of 6.66 mg/ml and Vmax of 90.8 U/ml. The purified chitinase was activated by Ca2+ and Mn2+ while the activity was inhibited by Hg2+, Zn2+, Cu2+, Co2+, dodecyl sulphate and EDTA. In vivo, the median lethal dose (LD50) was approximately 18.43 mg/kg body weight of Sprague Dawley rats. MTT assay showed that the purified chitinase has a toxic effect to MCF7 with an IC50 value 20 µg/ml, and HCT-116 cell lines with an IC50 value 44 µg/ml. Moreover, the purified enzyme showed significant antifungal activity against Fusarium oxysporum f. sp. lycopersici race 3 the causal agent of tomato wilt.
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Antifúngicos/farmacología , Antineoplásicos/farmacología , Quitinasas/biosíntesis , Quitinasas/farmacología , Fermentación , Trichoderma/enzimología , Animales , Supervivencia Celular/efectos de los fármacos , Fusarium/efectos de los fármacos , Células HCT116 , Células Hep G2 , Humanos , Cinética , Dosificación Letal Mediana , Células MCF-7 , Ratas , Ratas Sprague-Dawley , Microbiología del SueloRESUMEN
Twenty four fungal species were screened for their ability to produce alkaline L-methioninase on methionine-glucose liquid medium. Aspergillus ustus AUMC 10151 displayed the highest yield of enzyme (10.8 U/mg protein), followed by A. ochraceus and Fusarium proliferatum. Upon optimization of the submerged fermentation (SmF)conditions, the maximum enzyme yield (18.23 U/mg protein) was obtained on a medium containing L-methionine (0.5%), sucrose (0.95%), KH2PO4 (0.1%) and 175 rpm. Seven agro-industrial by-products were screened as substrates for L-methioninase production under solid-state fermentation (SSF). Wheat bran resulted 38.1 U/mg protein, followed by rice bran (27.6 U/mg protein) and soya bean meal (26.6 U/mg protein). Maximum alkaline L-methioninase (99.56U/mg protein) was achieved at initial moisture content of 71.5%, inoculum size of 2.0 mL of spore suspension, initial pH 8.5, incubation period eight days at 30°C and supplementation of the salt basal medium with pyridoxine(100 μg/mL) and beet molasses (20% v/v). The productivity of L-methioninase by A. ustus under SSF was higher than that of SmF about 5.45 fold under optimum conditions.
RESUMEN
A thermohalophilic fungus, Aspergillus terreus AUMC 10138, isolated from the Wadi El-Natrun soda lakes in northern Egypt was exposed successively to gamma and UV-radiation (physical mutagens) and ethyl methan-sulfonate (EMS; chemical mutagen) to enhance alkaline cellulase production under solid state fermentation (SSF) conditions. The effects of different carbon sources, initial moisture, incubation temperature, initial pH, incubation period, inoculum levels and different concentrations of NaCl on production of alkaline filter paper activity (FPase), carboxymethyl cellulase (CMCase) and ß-glucosidase by the wild-type and mutant strains of A. terreus were evaluated under SSF. The optimum conditions for maximum production of FPase, CMCase and ß-glucosidase were found to be the corn stover: moisture ratio of 1:3(w/v), temperature 45 °C, pH range, 9.0-11.0, and fermentation for 4, 4 and 7 day, respectively. Inoculum levels of 30% for ß-glucosidase and 40% for FPase, CMCase gave the higher cellulase production by the wild-type and mutant strains, respectively. Higher production of all three enzymes was obtained at a 5% NaCl. Under the optimized conditions, the mutant strain A. terreus M-17 produced FPase (729 U/g), CMCase (1,783 U/g), and ß-glucosidase (342 U/g), which is, 1.85, 1.97 and 2.31-fold higher than the wild-type strain. Our results confirmed that mutant strain M-17 could be a promising alkaline cellulase enzyme producer employing lignocellulosics especially corn stover.
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Aspergillus/enzimología , Aspergillus/metabolismo , Celulasas/metabolismo , Mutagénesis , Zea mays/metabolismo , Aspergillus/efectos de los fármacos , Aspergillus/efectos de la radiación , Medios de Cultivo/química , Egipto , Metanosulfonato de Etilo , Concentración de Iones de Hidrógeno , Lagos/microbiología , Técnicas Microbiológicas , Cloruro de Sodio/metabolismo , Temperatura , Rayos UltravioletaRESUMEN
Abstract A thermohalophilic fungus, Aspergillus terreus AUMC 10138, isolated from the Wadi El-Natrun soda lakes in northern Egypt was exposed successively to gamma and UV-radiation (physical mutagens) and ethyl methan-sulfonate (EMS; chemical mutagen) to enhance alkaline cellulase production under solid state fermentation (SSF) conditions. The effects of different carbon sources, initial moisture, incubation temperature, initial pH, incubation period, inoculum levels and different concentrations of NaCl on production of alkaline filter paper activity (FPase), carboxymethyl cellulase (CMCase) and β-glucosidase by the wild-type and mutant strains of A. terreus were evaluated under SSF. The optimum conditions for maximum production of FPase, CMCase and β-glucosidase were found to be the corn stover: moisture ratio of 1:3(w/v), temperature 45 °C, pH range, 9.0–11.0, and fermentation for 4, 4 and 7 day, respectively. Inoculum levels of 30% for β-glucosidase and 40% for FPase, CMCase gave the higher cellulase production by the wild-type and mutant strains, respectively. Higher production of all three enzymes was obtained at a 5% NaCl. Under the optimized conditions, the mutant strain A. terreus M-17 produced FPase (729 U/g), CMCase (1,783 U/g), and β-glucosidase (342 U/g), which is, 1.85, 1.97 and 2.31-fold higher than the wild-type strain. Our results confirmed that mutant strain M-17 could be a promising alkaline cellulase enzyme producer employing lignocellulosics especially corn stover.