RESUMO
Among the several threats to humanity by anthropogenic activities, contamination of the environment by heavy metals is of great concern. Upon entry into the food chain, these metals cause serious hazards to plants and other organisms including humans. Use of microbes for bioremediation of the soil and stress mitigation in plants are among the preferred strategies to provide an efficient, cost-effective, eco-friendly solution of the problem. The current investigation is an attempt in this direction where fungal strain PH1 was isolated from the rhizosphere of Parthenium hysterophorus which was identified as Aspergillus niger by sequence homology of the ITS 1 and ITS 4 regions of the rRNA. The strain was tested for its effect on growth and biochemical parameters as reflection of its potential to mitigate Pb stress in Zea mays exposed to 100, 200 and 500 µg of Pb/g of soil. In the initial screening, it was revealed that the strain has the ability to tolerate lead stress, solubilize insoluble phosphate and produce plant growth promoting hormones (IAA and SA) and other metabolites like phenolics, flavonoids, sugar, protein and lipids. Under 500 µg of Pb/g of soil, Z. mays exhibited significant growth retardation with a reduction of 31% in root length, 30.5% in shoot length, 57.5% in fresh weight and 45.2% in dry weight as compared to control plants. Inoculation of A. niger to Pb treated plants not only restored root and shoot length, rather promoted it to a level significantly higher than the control plants. Association of the strain modulated the physio-hormonal attributes of maize plants that resulted in their better growth which indicated a state of low stress. Additionally, the strain boosted the antioxidant defence system of the maize there by causing a significant reduction in the ascorbic acid peroxidase (1.5%), catalase (19%) and 1,1-diphenyl-2 picrylhydrazyl (DPPH) radical scavenging activity (33.3%), indicating a lower stress condition as compared to their non-inoculated stressed plants. Based on current evidence, this strain can potentially be used as a biofertilizer for Pb-contaminated sites where it will improve overall plant health with the hope of achieving better biological and agricultural yields.
Assuntos
Antioxidantes , Aspergillus niger , Chumbo , Fosfatos , Fotossíntese , Zea mays , Zea mays/crescimento & desenvolvimento , Zea mays/microbiologia , Zea mays/efeitos dos fármacos , Zea mays/metabolismo , Aspergillus niger/metabolismo , Chumbo/metabolismo , Antioxidantes/metabolismo , Fotossíntese/efeitos dos fármacos , Fosfatos/metabolismo , Poluentes do Solo/metabolismo , Estresse Fisiológico , Biodegradação AmbientalRESUMO
Inactivation of flbA in Aspergillus niger results in thinner cell walls, increased cell lysis, abolished sporulation, and an increased secretome complexity. A total of 36 transcription factor (TF) genes are differentially expressed in ΔflbA. Here, seven of these genes (abaA, aslA, aslB, azf1, htfA, nosA, and srbA) were inactivated. Inactivation of each of these genes affected sporulation and, with the exception of abaA, cell wall integrity and protein secretion. The impact on secretion was strongest in the case of ΔaslA and ΔaslB that showed increased pepsin, cellulase, and amylase activity. Biomass was reduced of agar cultures of ΔabaA, ΔaslA, ΔnosA, and ΔsrbA, while biomass was higher in liquid shaken cultures of ΔaslA and ΔaslB. The ΔaslA and ΔhtfA strains showed increased resistance to H2O2, while ΔaslB was more sensitive to this reactive oxygen species. Together, inactivation of the seven TF genes impacted biomass formation, sporulation, protein secretion, and stress resistance, and thereby these genes explain at least part of the pleiotropic phenotype of ΔflbA of A. niger.
Assuntos
Aspergillus niger , Parede Celular , Proteínas Fúngicas , Regulação Fúngica da Expressão Gênica , Fenótipo , Esporos Fúngicos , Fatores de Transcrição , Aspergillus niger/genética , Aspergillus niger/metabolismo , Aspergillus niger/crescimento & desenvolvimento , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Parede Celular/metabolismo , Parede Celular/genética , Peróxido de Hidrogênio/farmacologia , Pleiotropia GenéticaRESUMO
Hydrogen polysulfides (H2Sn) have emerged as critical physiological mediators that are closely associated with hydrogen sulfide (H2S) signaling. H2Sn exhibit greater nucleophilicity than H2S while also having electrophilic characteristics, enabling unique activities such as protein S-persulfidation. Despite their physiological importance, mechanisms and reactivities of H2Sn remain inadequately explored due to their inherent instability in aqueous environments. Consequently, there is a need to develop biocompatible methods for controlled H2Sn generation to elucidate their behaviors in biological contexts. Herein, we present a dual enzyme system (containing glucose oxidase (GOx) and chloroperoxidase (CPO)) with thioglucose as the substrate to facilitate the controlled release of H2Sn. Fluorescence measurements with SSP4 and the trapping studies allowed us to confirm the production of H2Sn. Such a method may be useful in elucidating the reactivity of hydrogen polysulfides in biological systems as well as provide a potential delivery of H2Sn to target sites for biological applications.
Assuntos
Cloreto Peroxidase , Glucose Oxidase , Sulfetos , Glucose Oxidase/metabolismo , Glucose Oxidase/química , Cloreto Peroxidase/metabolismo , Cloreto Peroxidase/química , Sulfetos/química , Sulfetos/metabolismo , Sulfeto de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/química , Aspergillus niger/enzimologiaRESUMO
Currently, the L-malic acid titer achieved through Aspergillus niger fermentation reaches 201 g/L, meeting industrial demands satisfactorily. However, the co-presence of structurally similar fumaric acid and succinic acid in fermentation products suggests a theoretical potential for further improvement in L-malic acid production. In the tricarboxylic acid cycle, fumarate reductase mediates the conversion of succinic acid to fumaric acid. Subsequently, fumarase catalyzes the conversion of fumaric acid to L-malic acid. Notably, both enzymatic reactions are reversible. Our investigation revealed that A. niger contains only one mitochondria-located fumarase FumA. Employing CRISPR-Cas9 technology, we performed a replacement of the fumA promoter with a doxycycline-induced promoter Tet. Under non-inducing condition, the conditional strain exhibited increased levels of fumaric acid and succinic acid. It strongly suggests that FumA mainly promotes the flow of fumaric acid to L-malic acid. Furthermore, a promoter PmfsA that is exclusively activated in a fermentation medium by calcium carbonate was identified through RNA-sequencing screening. Utilizing PmfsA to regulate fumA expression led to a 9.0% increase in L-malic acid titer, an 8.75% increase in yield (glucose to L-malic acid), and an 8.86% enhancement in productivity. This research serves as a significant step toward expediting the industrialization of L-malic acid synthesis via biological fermentation. Additionally, it offers valuable insights for the biosynthesis of other organic acids.IMPORTANCEThis study focuses on enhancing L-malic acid synthesis by modifying the tricarboxylic acid cycle within the mitochondria of Aspergillus niger. We emphasize the significant role of fumarase in converting fumaric acid into L-malic acid, enhancing our understanding of metabolic pathways in A. niger. The precise regulation of fumA is highlighted as a key factor in enhancing L-malic acid production. Furthermore, this research introduces a stringent conditional promoter (PmfsA), exclusively activated by CaCO3. The utilization of PmfsA for fumA expression resulted in heightened L-malic acid titers. The progress in metabolic engineering and bioprocess optimization holds promise for expediting industrial L-malic acid synthesis via biological fermentation. Moreover, it carries implications for the biosynthesis of various other organic acids.
Assuntos
Aspergillus niger , Fumarato Hidratase , Fumaratos , Aspergillus niger/genética , Aspergillus niger/metabolismo , Fumarato Hidratase/genética , Fumarato Hidratase/metabolismo , Malatos/metabolismo , Ácido SuccínicoRESUMO
Nano-biotechnology is quickly developing as an important field of modern research, generating the most promising applications in medicine and agriculture. Biosynthesis of silver nanoparticles using biogenic or green approach provide ecofriendly, clean and effective way out for the synthesis of nanoparticles. The main aim of the study was to synthesize silver nanoparticles (AgNPs) from Aspergillus niger, Aspergillus flavus and Pencillium chrysogenum using a green approach and to test the antifungal activity of these synthesized AgNPs against a variety of pathogenic fungi. The characterization of samples was done by using UV-visible spectroscopy, SEM (scanning electron microscopy), FTIR (Fourier transmission infrared spectroscopy), and XRD (X-ray diffractometry). The investigation confirmed the creation of AgNPs by the fungi Aspergillus niger, Aspergillus flavus and Pencillium chrysogenum, as evidenced by prominent plasmon absorbance bands at 420 and 450 nm.The biosynthesized AgNPs were 80-100 nm in size, asymmetrical in shape and became spherical to sub-spherical when aggregated. Agar well diffusion method was performed to evaluate the antifungal activity of AgNPs against various plant pathogenic fungi. An efficient and strong antifungal activity was shown by these biosynthesized nanoparticles against serious plant pathogenic fungi, viz. Aspergillus terreus, Fusarium oxysporum, Penicillium citrinum, Rhizopus stolonifer and Mucor mucedo. The biosynthesized AgNPs at various concentrations caused significant zone of inhibition in the test fungal pathogens. Silver nanoparticles (AgNPs) biosynthesized from Aspergillus niger at highest concentrations showed maximum zone of inhibition against Penicillium citrinum (19.33 ± 0.57 mm) followed by Rhizopus stolonifer (17.66 ± 0.57), Aspergillus terreus (16.33 ± 1.54 mm), Fusarium oxysporum (14.00 ± 1.00 mm) and Mucor mucedo (13.33 ± 1.15 mm) respectively. Therefore, the findings clearly indicate that silver nanoparticles could play a significant role in managing diverse plant diseases caused by fungi.
Assuntos
Antifúngicos , Aspergillus flavus , Aspergillus niger , Fusarium , Nanopartículas Metálicas , Testes de Sensibilidade Microbiana , Prata , Prata/farmacologia , Prata/química , Prata/metabolismo , Antifúngicos/farmacologia , Antifúngicos/química , Antifúngicos/síntese química , Nanopartículas Metálicas/química , Fusarium/efeitos dos fármacos , Espectroscopia de Infravermelho com Transformada de Fourier , Aspergillus flavus/efeitos dos fármacos , Aspergillus flavus/metabolismo , Aspergillus niger/efeitos dos fármacos , Aspergillus/efeitos dos fármacos , Aspergillus/metabolismo , Fungos/efeitos dos fármacos , Difração de Raios X , Microscopia Eletrônica de Varredura , Química Verde , Doenças das Plantas/microbiologiaRESUMO
In this comprehensive study, we delved into the capabilities of five fungal strains: Aspergillus flavus, Aspergillus niger, Penicillium chrysogenum, Penicillium glabrum, and Penicillium rubens (the latter isolated from heavy crude oil [HCO]) in metabolizing HCO as a carbon source. Employing a meticulously designed experimental approach, conducted at room temperature (25 °C), we systematically explored various culture media and incubation periods. The results unveiled the exceptional resilience of all these fungi to HCO, with A. flavus standing out as the top performer. Notably, A. flavus exhibited robust growth, achieving a remarkable 59.1% expansion across the medium's surface, accompanied by distinctive macroscopic traits, including a cottony appearance and vibrant coloration. In an effort to further scrutinize its biotransformation prowess, we conducted experiments in a liquid medium, quantifying CO2 production through gas chromatography, which reached its zenith at day 30, signifying substantial bioconversion with a 38% increase in CO2 production. Additionally, we monitored changes in surface tension using the Du Noüy ring method, revealing a reduction in aqueous phase tension from 72.3 to 47 mN/m. This compelling evidence confirms that A. flavus adeptly metabolizes HCO to fuel its growth, while concurrently generating valuable biosurfactants. These findings underscore the immense biotechnological potential of A. flavus in addressing challenges related to HCO, thereby offering promising prospects for bioremediation and crude oil bioupgrading endeavors.
Assuntos
Aspergillus flavus , Dióxido de Carbono , Biodegradação Ambiental , Aspergillus niger , BiotecnologiaRESUMO
Acid protease is widely used in industries such as food processing and feed additives. In the study, low frequency magnetic field (LF-MF) as an aid enhances acid protease production by Aspergillus niger (A. niger). The study assessed mycelial biomass, the enzymic activity of the acidic protease and underlying mechanism. At low intensities, alternating magnetic field (AMF) is more effective than static magnetic fields (SMF). Under optimal magnetic field conditions, acid protease activity and biomass increased by 91.44% and 16.31%, as compared with the control, respectively. Maximum 19.87% increase in enzyme activity after magnetic field treatment of crude enzyme solution in control group. Transcriptomics analyses showed that low frequency alternating magnetic field (LF-AMF) treatment significantly upregulated genes related to hydrolases and cell growth. Our results showed that low-frequency magnetic fields can enhance the acid protease production ability of A. niger, and the effect of AMF is better at low intensities. The results revealed that the effect of magnetic field on the metabolic mechanism of A. niger and provided a reference for magnetic field-assisted fermentation of A. niger.
Assuntos
Aspergillus niger , Campos Magnéticos , Peptídeo Hidrolases , Aspergillus niger/enzimologia , Aspergillus niger/genética , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/genética , Fermentação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Biomassa , Micélio/enzimologia , Micélio/crescimento & desenvolvimento , Micélio/genéticaRESUMO
Xylanase plays the most important role in catalyzing xylan to xylose moieties. GH11 xylanases have been widely used in many fields, but most GH11 xylanases are mesophilic enzymes. To improve the catalytic activity and thermostability of Aspergillus niger xylanase (Xyn-WT), we predicted potential key mutation sites of Xyn-WT through multiple computer-aided enzyme engineering strategies. We introduce a simple and economical Ni affinity chromatography purification method to obtain high-purity xylanase and its mutants. Ten mutants (Xyn-A, Xyn-B, Xyn-C, E45T, Q93R, E45T/Q93R, A161P, Xyn-D, Xyn-E, Xyn-F) were identified. Among the ten mutants, four (Xyn-A, Xyn-C, A161P, Xyn-F) presented improved thermal stability and activity, with Xyn-F(A161P/E45T/Q93R) being the most thermally stable and active. Compared with Xyn-WT, after heat treatment at 55 °C and 60 °C for 10 min, the remaining enzyme activity of Xyn-F was 12 and 6 times greater than that of Xyn-WT, respectively, and Xyn-F was approximately 1.5 times greater than Xyn-WT when not heat treated. The pH adaptation of Xyn-F was also significantly enhanced. In summary, an improved catalytic activity and thermostability of the design variant Xyn-F has been reported.
Assuntos
Aspergillus niger , Endo-1,4-beta-Xilanases , Estabilidade Enzimática , Aspergillus niger/enzimologia , Aspergillus niger/genética , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/metabolismo , Endo-1,4-beta-Xilanases/isolamento & purificação , Engenharia de Proteínas/métodos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/isolamento & purificação , Temperatura Alta , Desenho Assistido por ComputadorRESUMO
BACKGROUND: Aspergillus niger ATCC 20611 is an industrially important fructooligosaccharides (FOS) producer since it produces the ß-fructofuranosidase with superior transglycosylation activity, which is responsible for the conversion of sucrose to FOS accompanied by the by-product (glucose) generation. This study aims to consume glucose to enhance the content of FOS by heterologously expressing glucose oxidase and peroxidase in engineered A. niger. RESULTS: Glucose oxidase was successfully expressed and co-localized with ß-fructofuranosidase in mycelia. These mycelia were applied to synthesis of FOS, which possessed an increased purity of 60.63% from 52.07%. Furthermore, peroxidase was expressed in A. niger and reached 7.70 U/g, which could remove the potential inhibitor of glucose oxidase to facilitate the FOS synthesis. Finally, the glucose oxidase-expressing strain and the peroxidase-expressing strain were jointly used to synthesize FOS, which content achieved 71.00%. CONCLUSIONS: This strategy allows for obtaining high-content FOS by the multiple enzymes expressed in the industrial fungus, avoiding additional purification processes used in the production of oligosaccharides. This study not only facilitated the high-purity FOS synthesis, but also demonstrated the potential of A. niger ATCC 20611 as an enzyme-producing cell factory.
Assuntos
Aspergillus niger , Aspergillus , beta-Frutofuranosidase , Aspergillus niger/genética , Glucose Oxidase/genética , Oligossacarídeos , Peroxidases , GlucoseRESUMO
The biosynthetic potency of Taxol by fungi raises their prospective to be a platform for commercial production of Taxol, nevertheless, the attenuation of its productivity with the fungal storage, is the challenge. Thus, screening for a novel fungal isolate inhabiting ethnopharmacological plants, with a plausible metabolic stability for Taxol production could be one of the most affordable approaches. Aspergillus niger OR414905.1, an endophyte of Encephalartos whitelockii, had the highest Taxol productivity (173.9 µg/L). The chemical identity of the purified Taxol was confirmed by HPLC, FTIR, and LC-MS/MS analyses, exhibiting the same molecular mass (854.5 m/z) and molecular fragmentation pattern of the authentic Taxol. The purified Taxol exhibited a potent antiproliferative activity against HepG-2, MCF-7 and Caco-2, with IC50 values 0.011, 0.016, and 0.067 µM, respectively, in addition to a significant activity against A. flavus, as a model of human fungal pathogen. The purified Taxol displayed a significant effect against the cellular migration of HepG-2 and MCF-7 cells, by ~ 52-59% after 72 h, compared to the control, confirming its interference with the cellular matrix formation. Furthermore, the purified Taxol exhibited a significant ability to prompt apoptosis in MCF-7 cells, by about 11-fold compared to control cells, suppressing their division at G2/M phase. Taxol productivity by A. niger has been optimized by the response surface methodology with Plackett-Burman Design and Central Composite Design, resulting in a remarkable ~ 1.6-fold increase (279.8 µg/L), over the control. The biological half-life time of Taxol productivity by A. niger was ~ 6 months of preservation at 4 â, however, the Taxol yield by A. niger was partially restored in response to ethyl acetate extracts of E. whitelockii, ensuring the presence of plant-derived signals that triggers the cryptic Taxol encoding genes.
Assuntos
Aspergillus , Paclitaxel , Zamiaceae , Humanos , Aspergillus niger , Endófitos/metabolismo , Células CACO-2 , Cromatografia Líquida , Estudos Prospectivos , Espectrometria de Massas em Tandem , Ciclo CelularRESUMO
Epothilones are one of the common prescribed anticancer drugs for solid tumors, for their exceptional binding affinity with ß-tubulin microtubule, stabilizing their disassembly, causing an ultimate arrest to the cellular growth. Epothilones were initially isolated from Sornagium cellulosum, however, their extremely slow growth rate and low yield of epothilone is the challenge. So, screening for a novel fungal endophyte dwelling medicinal plants, with higher epothilone productivity and feasibility of growth manipulation was the objective. Aspergillus niger EFBL-SR OR342867, an endophyte of Latania loddegesii, has been recognized as the heady epothilone producer (140.2 µg/L). The chemical structural identity of the TLC-purified putative sample of A. niger was resolved from the HPLC, FTIR and LC-ESI-MS/MS analyses, with an identical molecular structure of the authentic epothilone B. The purified A. niger epothilone B showed a resilient activity against MCF-7 (0.022 µM), HepG-2 (0.037 µM), and HCT-116 (0.12 µM), with selectivity indices 21.8, 12.9 and 4, respectively. The purified epothilone B exhibited a potential anti-wound healing activity to HepG-2 and MCF-7 cells by ~ 54.07 and 60.0%, respectively, after 24 h, compared to the untreated cells. The purified epothilone has a significant antiproliferative effect by arresting the cellular growth of MCF-7 at G2/M phase by ~ 2.1 folds, inducing the total apoptosis by ~ 12.2 folds, normalized to the control cells. The epothilone B productivity by A. niger was optimized by the response surface methodology, with ~ 1.4 fold increments (266.9 µg/L), over the control. The epothilone productivity by A. niger was reduced by ~ 2.4 folds by 6 months storage as a slope culture at 4 °C, however, the epothilone productivity was slightly restored with ethylacetate extracts of L. loddegesii, confirming the plant-derived chemical signals that partially triggers the biosynthetic genes of A. niger epothilones. So, this is the first report emphasizing the metabolic potency of A. niger, an endophyte of L. loddegesii, to produce epothilone B, that could be a new platform for industrial production of this drug.
Assuntos
Antineoplásicos , Aspergillus niger , Endófitos , Epotilonas , Cicatrização , Epotilonas/farmacologia , Epotilonas/biossíntese , Epotilonas/química , Epotilonas/metabolismo , Humanos , Endófitos/metabolismo , Endófitos/química , Aspergillus niger/efeitos dos fármacos , Aspergillus niger/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/química , Cicatrização/efeitos dos fármacos , Células MCF-7 , Células Hep G2 , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacosRESUMO
AIMS: In this study, the antifungal efficacy and phytotoxicity of silica coated porous zinc oxide nanoparticle (SZNP) were analyzed as this nanocomposite was observed to be a suitable platform for slow release fungicides and has the promise to bring down the dosage of other agrochemicals as well. METHODS AND RESULTS: Loading and release kinetics of tricyclazole, a potent fungicide, were analyzed by measuring surface area (SBET) using Brunauer-Emmett-Teller (BET) isotherm and liquid chromatography tandem mass spectrometry (LC-MS/MS), respectively. The antifungal efficacy of ZnO nanoparticle (ZNP) and SZNP was investigated on two phytopathogenic fungi (Alternaria solani and Aspergillus niger). The morphological changes to the fungal structure due to ZNP and SZNP treatment were studied by field emission-scanning electron microscopy. Nanoparticle mediated elevation of reactive oxygen species (ROS) in fungal samples was detected by analyzing the levels of superoxide dismutase, catalase, thiol content, lipid peroxidation, and by 2,7-dichlorofluorescin diacetate assay. The phytotoxicity of these two nanostructures was assessed in rice plants by measuring primary plant growth parameters. Further, the translocation of the nanocomposite in the same plant model system was examined by checking the presence of fluorescein isothiocyanate tagged SZNP within the plant tissue. CONCLUSIONS: ZNP had superior antifungal efficacy than SZNP and caused the generation of more ROS in the fungal samples. Even then, SZNP was preferred as an agrochemical delivery vehicle because, unlike ZNP alone, it was not toxic to plant system. Moreover, as silica in nanoform is entomotoxic in nature and nano ZnO has antifungal property, both the cargo (agrochemical) and the carrier system (silica coated porous nano zinc oxide) will have a synergistic effect in crop protection.
Assuntos
Antifúngicos , Nanocompostos , Dióxido de Silício , Óxido de Zinco , Óxido de Zinco/farmacologia , Nanocompostos/toxicidade , Dióxido de Silício/farmacologia , Dióxido de Silício/química , Antifúngicos/farmacologia , Agroquímicos/farmacologia , Aspergillus niger/efeitos dos fármacos , Aspergillus niger/crescimento & desenvolvimento , Oryza/microbiologia , Oryza/crescimento & desenvolvimento , Oryza/efeitos dos fármacos , Fungicidas Industriais/farmacologia , Porosidade , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Preparações de Ação Retardada , Espécies Reativas de Oxigênio/metabolismoRESUMO
Disposal and recycling of heavy metal-enriched biomass is the key to measure the success of phytoremediation. This study employed innovative approach to use Aspergillus niger (A. niger) for the treatment of Cd-contaminated Helianthus annuus L. (sunflower) stalk after phytoremediation. Single-factor results showed that the removal of Cd at an initial pH of 3 was superior to sucrose and inoculation amount. 67.67% of Cd was removed by A. niger leaching system after 11 days based on response surface methodology optimum conditions (sucrose: 76.266 g L-1; inoculation amount: 10%; initial pH: 3), while the concentrations of nitrogen, phosphorus and potassium (N, P and K) of sunflower stalk were unaffected. While physicochemical pretreatment effectively enhanced the bioleaching efficiency, it also resulted in significant loss of P and K elements, thereby reducing the value of biomass for recycling and utilization. Therefore, the direct A. niger leaching method without pretreatment is more advantageous for the safe treatment and recycling of Cd-contaminated sunflower stalks.
Assuntos
Aspergillus niger , Biodegradação Ambiental , Cádmio , Helianthus , Helianthus/metabolismo , Aspergillus niger/metabolismo , Cádmio/metabolismo , Poluentes do Solo/metabolismo , BiomassaRESUMO
Oxygen is crucial for converting glucose to gluconic acid catalyzed by glucose oxidase (Gox). However, industrial gluconic acid production faces oxygen supply limitations. To enhance Gox efficiency, Vitreoscilla hemoglobin (VHb) has been considered as an efficient oxygen transfer carrier. This study identified GoxA, a specific isoform of Gox in the industrial gluconic acid-producing strain of Aspergillus niger. Various forms of VHb expression in A. niger were tested to improve GoxA's catalytic efficiency. Surprisingly, the expression of free VHb, both intracellularly and extracellularly, did not promote gluconic acid production during shake flask fermentation. Then, five fusion proteins were constructed by linking Gox and VHb using various methods. Among these, VHb-GS1-GoxA, where VHb's C-terminus connected to GoxA's N-terminus via the flexible linker GS1, demonstrated a significantly higher Kcat/Km value (96% higher) than GoxA. Unfortunately, the expression of VHb-GS1-GoxA in A. niger was limited, resulting in a low gluconic acid production of 3.0 g/L. To overcome the low expression problem, single- and dual-strain systems were designed with tools of SpyCatcher/SpyTag and SnoopCatcher/SnoopTag. In these systems, Gox and VHb were separately expressed and then self-assembled into complex proteins. Impressively, the single-strain system outperformed the GoxA overexpression strain S1971, resulting in 23% and 9% higher gluconic acid production under 0.6 vvm and 1.2 vvm aeration conditions in the bioreactor fermentation, respectively. The successful construction of Gox and VHb fusion or complex proteins, as proposed in this study, presents promising approaches to enhance Gox catalytic efficiency and lower aerodynamic costs in gluconic acid production. KEY POINTS: ⢠Overexpressing free VHb in A. niger did not improve the catalytic efficiency of Gox ⢠The VHb-GS1-GoxA showed an increased Kcat/Km value by 96% than GoxA ⢠The single-strain system worked better in the gluconic acid bioreactor fermentation.
Assuntos
Aspergillus niger , Glucose Oxidase , Aspergillus niger/genética , Glucose Oxidase/genética , Catálise , OxigênioRESUMO
Alternative splicing (AS) greatly expands the protein diversity in eukaryotes. Although AS variants have been frequently reported existing in filamentous fungi, it remains unclear whether lignocellulose-degrading enzyme genes in industrially important fungi undergo AS events. In this work, AS events of lignocellulose-degrading enzymes genes in Aspergillus niger under two carbon sources (glucose and wheat straw) were investigated by RNA-Seq. The results showed that a total of 23 out of the 56 lignocellulose-degrading enzyme genes had AS events and intron retention was the main type of these AS events. The AS variant enzymes from the annotated endo-ß-1,4-xylanase F1 gene (xynF1) and the endo-ß-1,4-glucanase D gene (eglD), noted as XYNF1-AS and EGLD-AS, were characterized compared to their normal splicing products XYNF1 and EGLD, respectively. The AS variant XYNF1-AS displayed xylanase activity whereas XYNF1 did not. As for EGLD-AS and EGLD, neither of them showed annotated endo-ß-1,4-glucanase activity. Instead, both showed lytic polysaccharide monooxygenase (LPMO) activity with some differences in catalytic properties. Our work demonstrated that the AS variants in A. niger were good sources for discovering novel lignocellulose-degrading enzymes. KEY POINTS: ⢠AS events were identified in the lignocellulose-degrading enzyme genes of A. niger. ⢠New ß-1,4-xylanase and LPMO derived from AS events were characterized.
Assuntos
Processamento Alternativo , Aspergillus niger , Aspergillus niger/metabolismo , Lignina/metabolismoRESUMO
Aspergillus oryzae PrtR is an ortholog of the transcription factor PrtT, which positively regulates the transcription of extracellular peptidase genes in Aspergillus niger and Aspergillus fumigatus. To identify the genes under the control of PrtR and elucidate its regulatory mechanism in A. oryzae, prtR gene disruption mutants were generated. The control strain clearly showed a halo on media containing skim milk as the nitrogen source, whereas the ΔprtR strain formed a smaller halo. Measurement of acid peptidase activity revealed that approximately 84% of acidic endopeptidase and 86% of carboxypeptidase activities are positively regulated by PrtR. As the transcription of the prtR gene varied depending on culture conditions, especially with or without a protein substrate, it was considered that its transcription would be regulated in response to a nitrogen source. In addition, contrary to previous expectations, PrtR was found to act both in promoting and repressing the transcription of extracellular peptidase genes. The mode of regulation varied from gene to gene. Some genes were regulated in the same manner in both liquid and solid cultures, whereas others were regulated in different ways depending on the culture conditions. Furthermore, PrtR has been suggested to regulate the transcription of peptidase genes that are closely associated with other transcription factors. KEY POINTS: ⢠Almost all peptidase genes in Aspergillus oryzae are positively regulated by PrtR ⢠However, several genes are regulated negatively by PrtR ⢠PrtR optimizes transcription of peptidase genes in response to culture conditions.
Assuntos
Aspergillus oryzae , Aspergillus oryzae/genética , Aspergillus fumigatus , Aspergillus niger , Endopeptidases , Nitrogênio , Fatores de Transcrição/genéticaRESUMO
Enzyme immobilized on magnetic nanomaterials is a promising biocatalyst with efficient recovery under applied magnets. In this study, a recombinant extracellular lipase from Aspergillus niger GZUF36 (PEXANL1) expressed in Pichia pastoris GS115 was immobilized on ionic liquid-modified magnetic nano ferric oxide (Fe3O4@SiO2@ILs) via electrostatic and hydrophobic interaction. The morphology, structure, and properties of Fe3O4@SiO2@ILs and immobilized PEXANL1 were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, x-ray diffraction, vibration sample magnetometer, and zeta potential analysis. Under optimized conditions, the immobilization efficiency and activity recovery of immobilized PEXANL1 were 52 ± 2% and 122 ± 2%, respectively. The enzymatic properties of immobilized PEXANL1 were also investigated. The results showed that immobilized PEXANL1 achieved the maximum activity at pH 5.0 and 45 °C, and the lipolytic activity of immobilized PEXANL1 was more than twice that of PEXANL1. Compared to PEXANL1, immobilized PEXANL1 exhibited enhanced tolerance to temperature, metal ions, surfactants, and organic solvents. The operation stability experiments revealed that immobilized PEXANL1 maintained 86 ± 3% of its activity after 6 reaction cycles. The enhanced catalytic performance in enzyme immobilization on Fe3O4@SiO2@ILs made nanobiocatalysts a compelling choice for bio-industrial applications. Furthermore, Fe3O4@SiO2@ILs could also benefit various industrial enzymes and their practical uses. KEY POINTS: ⢠Immobilized PEXANL1 was confirmed by SEM, FT-IR, and XRD. ⢠The specific activity of immobilized PEXANL1 was more than twice that of PEXANL1. ⢠Immobilized PEXANL1 had improved properties with good operational stability.
Assuntos
Líquidos Iônicos , Estabilidade Enzimática , Líquidos Iônicos/química , Aspergillus niger/genética , Aspergillus niger/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Dióxido de Silício/química , Lipase/metabolismoRESUMO
Beer is a beverage that contains gluten and cannot be consumed by people with celiac disease. In this context, the enzyme prolyl endoprotease (PEP) can be used to reduce the gluten content in beer. The present study aimed to produce the PEP from Aspergillus sp. FSDE 16 using solid-state fermentation with 5 conditions and comparing with a similar commercial enzyme produced from Aspergillus niger in the production of a gluten-free beer. The results of the performed cultures showed that during the culture, the most increased protease activity (54.46 U/mL) occurred on the 4th day. In contrast, for PEP, the highest activity (0.0356 U/mL) was obtained on the 3rd day of culture in condition. Regarding beer production, cell growth, pH, and total soluble solids showed similar behavior over the 7 days for beers produced without enzyme addition or with the addition of commercial enzyme and with the addition of the enzyme extract produced. The addition of the enzyme and the enzyme extract did not promote changes, and all the beers produced showed similar and satisfactory results, with acid pH between 4 and 5, total soluble solids ranging from 4.80 to 5.05, alcohol content ranging from 2.83% to 3.08%, and all beers having a dark character with deep amber and light copper color. Gluten removal was effectively using the commercial enzyme and the enzyme produced according to condition (v) reaching gluten concentrations equal to 17 ± 5.31 and 21.19 ± 11.28 ppm, respectively. In this way, the production of the enzyme by SSF and its application in the removal of gluten in beer was efficient.
Assuntos
Cerveja , Serina Endopeptidases , Humanos , Cerveja/análise , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Prolil Oligopeptidases , Fermentação , Glutens/análise , Glutens/metabolismo , Aspergillus niger , Extratos VegetaisRESUMO
Genes flbA-E are involved in sporulation and vegetative growth in Aspergillus nidulans. Inactivation of either of these genes results in a fluffy phenotype with delayed or even abolished sporulation. Previously, a non-sporulating phenotype was obtained by inactivating flbA in Aspergillus niger, which was accompanied by lysis, thinner cell walls, and an increased secretome complexity. Here, we further studied the role of the flb genes of A. niger. Strains ΔflbA, ΔflbB and ΔflbE showed increased biomass formation, while inactivation of flbA-D reduced, or even abolished, formation of conidia. Strain ΔflbA was more sensitive to H2O2, DTT, and the cell wall integrity stress compounds SDS and Congo Red (CR). Also, ΔflbC was more sensitive to SDS, while ΔflbB, ΔflbD, and ΔflbE were more sensitive to CR. On the other hand, inactivation of flbE increased resistance to H2O2. Enzyme secretion was impacted when the Δflb strains were grown on xylose. Strain ΔflbE showed reduced xylanase, cellulase and amylase secretion. On the other hand, amylase secretion at the periphery of the ΔflbA colony was reduced but not in its center, while secretion of this enzyme was increased in the center of the ΔflbB colony but not at its periphery. Inactivation of flbC and flbD also impacted zonal cellulase and amylase activity. Together, the Flb protein family of A. niger function in biomass formation, sporulation, stress response, and protein secretion.
Assuntos
Aspergillus niger , Celulases , Animais , Aspergillus niger/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Peróxido de Hidrogênio/metabolismo , Estágios do Ciclo de Vida , Celulases/metabolismo , Amilases/metabolismo , Esporos FúngicosRESUMO
Agricultural waste valorisation provides a sustainable solution to waste management, and combining waste utilisation with commodity production allows for responsible production processes. Recombinant Aspergillus niger D15 strains expressing fungal endoglucanases (Trichoderma reesei eg1 and eg2 and Aspergillus carneus aceg) were evaluated for their ability to utilise lactose as a carbon source to determine whether dairy waste could be used as a feedstock for enzyme production. The recombinant A. niger D15[eg1]PyrG, D15[eg2]PyrG, and D15[aceg]PyrG strains produced maximum endoglucanase activities of 34, 54, and 34 U/mL, respectively, on lactose and 23, 27, and 22 U/mL, respectively, on whey. The A. niger D15[eg2]PyrG strain was used to optimise the whey medium. Maximum endoglucanase activity of 46 U/mL was produced on 10% whey medium containing 0.6% NaNO3. The results obtained indicate that dairy whey can be utilised as a feedstock for recombinant enzyme production. However, variations in enzyme activities were observed and require further investigation.