Extracellular invertase from Aspergillus flavus

An extracellular invertase was induced in cultures of Aspergillus flavus Link during growth in liquid medium that contained sucrose as the sole carbon source. Synthesis of this enzyme was repressed by the addition of glucose or fructose to sucrose-metabolizing cells, and was induced in a glucose or fructose-metabolizing culture by the addition of sucrose. A. flavus invertase had a pH optimum of 6.0 and an apparent Km of approximately 133 mM for sucrose. The enzyme required potassium phosphate for maximum activity, optimum concentration being 250 mM. The enzyme was partially purified by ammonium sulphate precipitation followed by dialysis and separated by molecular exclusion into three components with molecular weights ranging from approximately 40,000 to 55,000.     

New Toxin from Aspergillus flavus

Two nonaflatoxin-producing isolates of Aspergillus flavus produced a new nonfluorescent nitrogen-containing metabolite that was highly toxic to 1-day-old cockerels. The oral mean lethal dose of toxin was 19 mg/kg. Chemical and physical data obtained on the purified toxin demonstrated that it was not one of the previously reported metabolites of A. flavus. The common name "flavutoxin" has been assigned to the toxin.     

Synthesis of chitin by particulate preparations from Aspergillus flavus

Cell-free extracts from Aspergillus flavus catalyzed the synthesis of chitin from UDP-GlcNAc. Most of the activity was associated with membrane-rich fractions whereas no activity was detected in the cell walls. Chitin synthetase was activated by fungal acid proteases; animal and plant proteases destroyed it. Upon incubation at 0 C and 28 C chitin synthetase was inactivated, probably by the action of proteases present in the particulate preparations. Maximal activity was obtained at pH 6.6–7.1 and 15 C. Arrhenius plot showed a biphasic curve with the transition at 7 C. E values were 3300 Kcal/mole above this temperature and 15500 Kcal/mole below it. The enzyme was activated by GlcNAc and required a divalent metal, the most active being Mg⁺⁺. By plotting v vs UDP-GlcNAc concentration a sigmoidal curve was obtained. Km calculated at high substrate concentrations was 20mm. Chitin synthetase was competitively inhibited by polyoxin D (Ki 6.5 m) and UDP (Ki 1.35mm), the latter giving complex kinetics.This work was supported by grants No. 020 and 847 of the Consejo Nacional de Ciencia y Tecnología, México.Part of this work was carried out while the authors were research visitors at the Department of Plant Pathology, University of California, Riverside.     

Isolation of aminopeptidase from Aspergillus flavus

A mixture of aminopeptidase and neutral protease from the Aspergillus flavus mould obtained by chromatography on DEAE-Sephadex was fractionated by chromatography on the hydroxyalkyl methacrylate gel with chemically bonded 1,6 hexamethylene diamine and d-leucine. Aminopeptidase thus obtained was electrophoretically homogeneous. Conditions for chromatography were worked out allowing a one stage isolation of a highly active aminopeptidase sample directly from the alcoholic precipitate of the culture medium of the Aspergillus flavus mould.     

Nitrification of Aspartate by Aspergillus flavus

Heterotrophic conversion of l-aspartic acid to nitrification products by Aspergillus flavus was studied in a replacement incubation system. Numerous amino acids supported nitrification; aspartate and glutamate were about equivalent as the best sources of nitrate. Addition of sodium bicarbonate to the incubation system substantially enhanced nitrate formation for all nitrifiable amino acids except aspartic acid, but the basis for the bicarbonate effect is obscure. The yield of nitrate from l-aspartate was not approached by forms of aspartic acid resulting from substitution on the beta carbon, the amino nitrogen, or the gamma carboxyl group or by aspartate presented as the d-configuration. There was no relationship between nitrate formation and the occurrence of such possible intermediates as nitrite, bound hydroxylamine, ammonia, aspergillic acid, and beta-nitropropionic acid. Uniformly labeled (14)C-l-aspartate that was nitrified in replacement incubation led to no accumulation of label in possible nitrification products in the culture filtrate. Label was found in components of the mycelium after acid hydrolysis, with heaviest accumulation in what appeared to be glucosamine and an unidentified compound, possibly acetylglucosamine. Detectable label was redistributed into serine, glycine, and threonine.     

Degradation of aflatoxin by Aspergillus flavus

Aflatoxin degradative activity was demonstrated in 6- to 12-d-old intact mycelium and cell-free extracts of Aspergillus flavus. The addition of cycloheximide, SKF 525-A or metyrapone to cultures of A. flavus prevented subsequent degradation of the aflatoxins, while in cell-free extracts degradation was inhibited by SKF 525-A, metyrapone and cytochrome c but not by KCN. In cell-free extracts, aflatoxin degradation was enhanced by NADPH and NaIO4. The results suggest the involvement of cytochrome P-450 monooxygenases in the aflatoxin degradative activity of A. flavus.     

The Inhibition of Cow-Liver Catalase by 3-Nitropropionate

The cow-liver, catalase was inhibited by 3-nitropropionate (3-NPA) at every pH tested. The 50% inhibition degree, ?, of 3-NPA under experimental condition was 8.7 × 10^?4 M, which was almost that of phenol (1.0 × 10^?3 M). Among 3-NPA, 1-nitropropane, and propionate, 3-NPA had the most effective inhibitory effect. The maximum absorption spectrum of free catalase in the Soret region was not shifted clearly by an addition of 3-NPA, but the intensity of it was decreased. The inhibition type of 3-NPA was riot definitely classified as the cyanide type. Results indicate that this inhibition essentially be due to reaction between the prosthetic group and anion form of nitro group in 3-NPA, and that the inhibitory action of nitro group may be stimulated by carboxylic group. Finally, this inhibition could be thought as a partial toxic cause in vivo.     

Malic acid accumulation by Aspergillus flavus

Summary ¹³C Nuclear magnetic resonance and fumarase and NAD-malate dehydrogenase isoenzyme studies were carried out in a strain of A. flavus which produces relatively high levels of l-malic acid from glucose. The results of the ¹³C NMR showed that the ¹³C label from [1-¹³C] glucose was incorporated only to C-3 (-CH₂-) of l-malic acid and indicated that this acid must be synthesized from pyruvate mainly via oxaloacetate. Electrophoretic analysis has established the presence of unique mitochondrial and cytosolic isoenzymes for fumarase and malate dehydrogenase. Changes in the isoenzyme pattern were observed for malate dehydrogenase but not for fumarase during acid production. Cycloheximide inhibited profoundly both l-malic acid production and the increase in the major isoenzyme of malate dehydrogenase, without affecting either the total activity of fumarase or its isoenzyme pattern. The results suggested that de novo protein synthesis is involved in the increase in the activity of the major isoenzyme of malate dehydrogenase and that this isoenzyme is essential for l-malic acid production and accumulation.     

Analysis of secreted proteins from Aspergillus flavus

MS/MS techniques in proteomics make possible the identification of proteins from organisms with little or no genome sequence information available. Peptide sequences are obtained from tandem mass spectra by matching peptide mass and fragmentation information to protein sequence information from related organisms, including unannotated genome sequence data. This peptide identification data can then be grouped and reconstructed into protein data. In this study, we have used this approach to study protein secretion by Aspergillus flavus, a filamentous fungus for which very little genome sequence information is available. A. flavus is capable of degrading the flavonoid rutin (quercetin 3-O-glycoside), as the only source of carbon via an extracellular enzyme system. In this continuing study, a proteomic analysis was used to identify secreted proteins from A. flavus when grown on rutin. The growth media glucose and potato dextrose were used to identify differentially expressed secreted proteins. The secreted proteins were analyzed by 1- and 2-DE and MS/MS. A total of 51 unique A. flavus secreted proteins were identified from the three growth conditions. Ten proteins were unique to rutin-, five to glucose- and one to potato dextrose-grown A. flavus. Sixteen secreted proteins were common to all three media. Fourteen identifications were of hypothetical proteins or proteins of unknown functions. To our knowledge, this is the first extensive proteomic study conducted to identify the secreted proteins from a filamentous fungus.     

Malic acid accumulation by Aspergillus flavus

Summary Scanning electron microscopy revealed that Aspergillus flavus produced unusual crystals and hair-like processes during its l-malic acid production phase. Crystallinic dendritic aggregates were formed on the hyphae growing as pellets. The size and number of crystal aggregates increased during the fermentation in parallel with l-malic acid accumulation. The crystals (composed of calcium malate as well as small amounts of calcium succinate and calcium fumarate) were removed from the hyphae, after incubation with 6N HCl. On day 5 of the fermentation, about 9% of the total amount of l-malic acid produced was accounted for by the attached crystals. In addition to crystal formation we observed the appearance of hair-like processes during the early phase (2 days) of malic acid production only.     

Aflatoxigenic Isolates of Aspergillus flavus from Pecans

Of 120 isolates of the Aspergillus flavus group from pecans used in bakery products, 85 were shown to produce aflatoxin on yeast extract sucrose medium. Extracts from moldy sections of raw pecans obtained commercially at the retail level showed aflatoxin-like spots on thin-layer chromatography. Cooked (autoclaved) pecans inoculated with toxigenic isolates of A. flavus were also good substrates for aflatoxin production.     

Role of 3-Nitropropanoic acid in nitrate formation by Aspergillus flavus

Doxtader, K. G. (Cornell University, Ithaca, N.Y.), and M. Alexander. Role of 3-nitropropanoic acid in nitrate formation by Aspergillus flavus. J. Bacteriol. 91:1186-1191. 1966.-Aspergillus flavus formed nitrate, 3-nitropropanoic acid (3-NPA), kojic acid, and a substance tentatively identified as N-formyl-N-hydroxy-glycine during growth in a medium with ammonium as sole nitrogen source. The concentration of the nitro compound reached a maximum prior to the appearance of nitrate; the 3-NPA level subsequently decreased with a concomitant increase in nitrate concentration. Replacement cultures of A. flavus produced nitrate from culture filtrates containing 3-NPA or from synthetic 3-NPA but not when supplied with fresh ammonium-sucrose medium, the nitrate-nitrogen formed being equivalent to 50% of the quantity of the 3-NPA-nitrogen initially present. Neither nitrate nor 3-NPA was synthesized by the fungus during growth in media with low pH or low ammonium concentrations. It is proposed that 3-NPA is either an intermediate or is in equilibrium with an intermediate in nitrification by the fungus.     

DNA from Aspergillus flavus contains 5-methylcytosine

DNA from Aspergillus sp. has been reported not to contain 5-methylcytosine. However, it has been found that Aspergillus nidulans responds to 5-azacytidine, a drug that is a strong inhibitor of DNA methyltransferases. Therefore, we have re-examined the occurrence of 5-methylcytosine in DNA from Aspergillus flavus by using a highly sensitive and specific method for detection of modified bases in genomic DNA comprising high-performance liquid chromatography separation of nucleosides, labeling of the nucleoside with deoxynucleoside kinase and two-dimensional thin-layer chromatography. Our results show that 5-methylcytosine is present in DNA from A. flavus. We estimate the relative amounts of 5-methylcytosine to cytosine to be approximately 1/400.     

Decolorization of Synthetic Dyes by Aspergillus flavus

A problem of paramount importance that has attracted the attention of environmental biologists is the discharge of highly colored effluents into the environment by various industries, which use a wide range of synthetic dyes. The existing chemical methods for dye degradation are not only expensive but also contributes to secondary pollution due to high dose of the chemicals used. Hence an alternative is to exploit the potential of microorganisms to alleviate this problem. The current paper deals with the isolation, characterization, and sugar utilization for better growth of Aspergillus flavus, a marine fungus from the Bay of Bengal. The goal is to assess the bioremediation potential of a variety of synthetic, paper mill, and color photography dyes. A correlation between the amount of sugar used, biomass, and quality of protein produced was observed. This fungus is capable of reducing between 80% and 90% of synthetic dyes and 100% color photography effluents within 3 to 7 days, and 8 days, respectively. Significant effect of carbon sources was observed in the decolorization of the synthetic dye crystal violet, up to 90% in 3 to 7 days, by Aspergillus flavus. The organism showed better growth with fructose as the sole carbon source for the least sugar consumption. Therefore, this fungus can be used as an economical and eco-friendly tool to minimize the pollution by industries to a significant extent.