Partial purification and some properties of a T2 ribonuclease from Aspergillus flavipes

A ribonuclease was partially purified from the culture medium of Aspergillus flavipes (IZ:1501), after 96 h of cultivation by chromatography on DEAE-cellulose and Sephadex G100 columns. The molecular weight of the RNase was estimated to be 40 kD by gel filtration using Sephadex G100, and the optimum pH and temperature were 4.0 and 50-55 degrees C, respectively. Catalytic activity was inhibited by Zn+2, Fe+3, Hg+2 and Ag+ ions. The enzyme did not show an exact base specificity and produced four kinds of 3'-nucleotides from yeast RNA.

Solubilização de fosfatos de rocha por Aspergillus niger em diferentes tipos de vinhaça

Aspergillus niger was inoculated into flasks containing mixed of different origins and fluorapatite as a source of phosphorus, or alternatively rock phosphates of different compositions. There was no difference in fungal growth or fluorapatite solubilization when sterilized or unsterilized vinasse was used. Total and soluble solid content was at least two times higher in 65/35 vinasse than in 10/1 vinasse. The higher total sugar content causing higher titratable acidity levels, or the lower fungal growth, may possibly have favored the greater accumulation of soluble phosphate in 10/1 than in 65/10 vinasse. No appreciable differences in residual soluble phosphate levels were detected with increasing fluorapatite concentrations. Rock phosphates of different origins and with different phosphorus concentrations affected the solubilizing ability of the fungus. Whereas crude concentrated apatite phosphorus favored the greatest accumulation of soluble phosphate in the culture medium (1.08 mg/ml), the highest solubilization (72% total phosphate) was achieved with Patos de Minas material obtained from the first crushing.

Mastite subclínica bubalina por Aspergillus fumigatus

This research aimed to report the occurrence of subclinical mastitis in a buffalo from a study carried out with 548 milk samples of 137 Murrah and Mediterranean buffalos from seven milk properties, located in the cities of Jau, Botucatu and Sorocaba, State of São Paulo. The animals of the study were submitted to a clinical examination of the mammary gland by the inspection and to the diagnosis of clinical and subclinical mastitis by California Mastitis Test (CMT), being the milk samples later directed to the laboratory for microbiological studies and also to the test of Whiteside Modified (WSM). The isolated agents were identified by the morphological characteristics of its colonies and through microcultive staining with blue cotton. Two (02) pure fungi samples were isolated representing 2.86% of the total isolated microorganism, corresponding to two mammary rooms in one animal, and were classified as pertaining to the Aspergillus fumigatus specie. The animal in question showed reaction of ++ to the CMT in both affected rooms and negative reaction to the WSM. In this way it is concluded that the Aspergillus fumigatus participates in a discrete way as a determinant agent of bubaline subclinical mastitis, however it is important because the affected animals can act as potential reservoirs and may be able to generate the infection in human beings.

Biomass production and secretion of hydrolytic enzymes are influenced by the structural complexity of the nitrogen source in Fusarium oxysporum and Aspergillus nidulans

The structural complexity of the nitrogen sources strongly affects biomass production and secretion of hydrolytic enzymes in filamentous fungi. Fusarium oxysporum and Aspergillus nidulans were grown in media containing glucose or starch, and supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids), peptides (peptone) and protein (gelatin). In glucose, when the initial pH was adjusted to 5.0, for both microorganisms, higher biomass production occurred upon supplementation with a nitrogen source in the peptide form (peptone and gelatin). With a close to neutrality pH, biomass accumulation was lower only in the presence of the ammonium salt. When grown in starch, biomass accumulation and secretion of hydrolytic enzymes (amylolytic and proteolytic) by Fusarium also depended on the nature of the nitrogen supplement and the pH. When the initial pH was adjusted to 5.0, higher growth and higher amylolytic activities were detected in the media supplemented with peptone, gelatin and casamino acids. However, at pH 7.0, higher biomass accumulation and higher amylolytic activities were observed upon supplementation with peptone or gelatin. Ammonium sulfate and casamino acids induced a lower production of biomass, and a different level of amylolytic enzyme secretion: high in ammonium sulfate and low in casamino acids. Secretion of proteolytic activity was always higher in the media supplemented with peptone and gelatin. Aspergillus, when grown in starch, was not as dependent as Fusarium on the nature of nitrogen source or the pH. The results described in this work indicate that the metabolism of fungi is regulated not only by pH, but also by the level of structural complexity of the nitrogen source in correlation to the carbon source.

Improvement of Aspergillus niger glucoamylase thermostability by directed evolution

Directed evolution was used to improve the thermostability of Aspergillus niger glucoamylase (GA) expressed in Saccharomyces cerevisiae. A starch-plate assay developed to screen GA mutants for thermostability gave results consistent with those of irreversible thermoinactivation kinetic analysis. Several thermostable multiply-mutated GAs were isolated and characterized by DNA sequencing and kinetic analysis. Three new GA mutations, T62A, T290A and H391Y, have been identified that encode GAs that are more thermostable than wild-type GA, and that improve thermostability cumulatively. These individual mutations were combined with the previously constructed thermostable site-directed mutations D20C/A27C (forming a disulficle bond), S30P, and G137A to create a multiply-mutated GA designated THS8. THS8 GA is substantially more thermostable than wild-type GA at 8OoC, with a 5.1 kJ/mol increase in the free energy of therrnoinactivation, making it the most thermostable Aspergillus niger GA mutant characterized to date. THS8 GA and the singly-mutated GAs have specific activities and catalytic efficiencies (k(cat)/K-m) similar to those of wild-type GA.

Purification and characterization of beta-fructosidase with inulinase activity from Aspergillus niger-245

Aspergillus niger - 245 a strain isolated from soil samples showed good beta -fructosidase activity when inoculated in medium formulated with dahlia extract tubers. The enzyme was purified by precipitation in ammonium sulphate and percolated in DEAE-Sephadex A-50 and CM-cellulose columns, witch showed a single peack in all the purification steps, maintaining the I/S ratio between 0.32 to, 0.39. Optimum pH for inulinase activity (I) was between 4.0 - 4.5 and for invertase activity (S) between 2.5 and 50. The optimum temperature was 60 degrees .C for both activities and no loss in activity was observed when it was maintained at this temperature for 30 min. The K-m value was 1.44 and 5.0 respectively, for I and S and V-m value 10.48 and 30.55 respectively. The I activity was strongly inhibited by Hg2+ and Ag+ and 2 x 10(-3) M of glucose, but not by fructose at the same concentration. The enzyme showed an exo-action mechanism acting on the inulin of different origins. In assay conditions total hydrolysis of all the frutans was obtained although it has shown larger activity on the chicory inulin than that one from artichoke Jerusalem and dahlia, in the first 30 min. The obtained results suggested that the enzyme presented good potential for industrial application in the preparing the fructose syrups.

Production of extracellular alkaline proteases by Aspergillus clavatus

The production of extracellular alkaline proteases from Aspergillus clavatus was evaluated in a culture filtrate medium, with different carbon and nitrogen sources. The fungus was cultivated at three different temperatures during 10 days. The proteolytic activity was determined on casein pH 9.5 at 37degreesC. The highest alkaline proteolytic activity (38 U/ml) was verified for culture medium containing glucose and casein at 1% (w/v) as substrates, obtained from cultures developed at 25degreesC for 6 days. Cultures developed in Vogel medium with glucose at 2% (w/v) and 0.2% (w/v) NH4NO3 showed higher proteolytic activity (27 U/ml) when compared to the cultures with 1% of the same sugar. Optimum temperature was 40degreesC and the half-lives at 40, 45 and 50degreesC were 90, 25 and 18 min, respectively. Optimum pH of enzymatic activity was 9.5 and the enzyme was stable from pH 6.0 to 12.0.

Antioxidant activity of the melanin pigment extracted from Aspergillus nidulans

Melanins are pigments of high molecular weight formed by oxidative polymerization of phenolic or indolic compounds. A number of fungi, including Aspergillus nidulans, produce pigments related or identical to melanin, which are located on cell walls or exist as extracellular polymers. The aim of the present study was to assess the antioxidant activity of synthetic melanin and of the pigment extracted from the mycelium and culture medium after growth of the highly melanized strain (MEL1) from A. nidulans. The ability of the melanin pigment to scavenge the oxidants HOCl and H2O2 was evaluated by inhibition of the oxidation of 5-thio-2-nitrobenzoic acid (TNB) using several melanin concentrations. The results showed that the pigment of the MEL1 strain competes with TNB for H2O2 and HOCl, inhibiting TNB oxidation in a concentration-dependent manner. For the HOCl oxidant, this inhibition was comparable to that of synthetic melanin, whose IC50 values were quite close for both pigments. Thus, our results suggest that the melanin from A. nidulans is a potential HOCl scavenger and may be considered a promising material for the cosmetic industry for the formulation of creams that protect the skin against possible oxidative damage.

Isolation and characterisation of cycloheximide sensitive mutants of Aspergillus nidulans

Aspergillus nidulans is a non-pathogenic fungus with well-developed genetics which provides an excellent model system for studying different aspects of drug resistance in filamentous fungi. As a preliminary step to characterizing genes that confer pleiotropic drug resistance in Aspergillus, we isolated cycloheximide-sensitive mutants of A. nidulans, which is normally resistant to this: drug. The rationale for this approach is to identify gents whose products are important for drug resistance by analysing mutations that alter the resistance/sensitivity status of the cell. Fifteen cycloheximide-sensitive (named scy for sensitive to cycloheximide) mutants of A, nidulans were isolated and genetically characterised. Each scy mutant was crossed with the wild-type strain and five of the crosses gave 50% cycloheximide-sensitive progeny suggesting that they carry a single mutation required for cycloheximide sensitivity. We examined ten sep mutants for resistance/sensitivity to other drugs or stress agents with different and/or the same mechanism of action, Sis of these mutants exhibited other altered resistance/sensitivity phenotypes which were linked to the cycloheximide sensitivity, These six mutants were analyzed by pairwise crosses and found to represent six linkage groups, named scyA-F. One of the mutants showed fragmentation of its vacuolar system and, in addition, its growth was osmotic, low-pi-II and oxidative-stress sensitive.

Pectin lyase from Aspergillus giganteus: Comparative study of productivity of submerged fermentation on citrus pectin and orange waste

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Bioremediation of direct dyes in simulated textile effluents by a paramorphogenic form of Aspergillus oryzae

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Characterization of a thermostable extracellular tannase produced under submerged fermentation by Aspergillus ochraceus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)