Isoelectrophoretic characterization of extracellular polygalacturonases of variousAspergillus alliaceus strains

The composition of pectin hydrolase complexes produced by variousAspergillus alliaceus strains was studied under the conditions of induction, catabolite repression, or constitutive synthesis. The strains were found similar in terms of the polygalacturonase spectrum and different with regard to the levels of endo- and exoenzyme activities. The analysis of the zymograms of inducible polygalacturonases revealed that all tested cultures contained at least 24 molecular forms of polygalacturonase. Taking into account only the three molecular forms typical of all analyzed strains ofA. alliaceus with pI values of 5.7, 5.9, and 6.3, one can use the spectrum of constitutive, catabolite repression-resistant polygalacturonases as an additional taxonomic species criterion.     

One-step Concentration and Partial Purification of Aspergillus kawachii Non-acidic Polygalacturonases by Adsorption to Glass Fiber Microfilters

The non-acidic polygalacturonases produced by Aspergillus kawachii in a glucose/tryptone medium were adsorbed to a glass fiber microfilter that was used to clarify the fermentation broth. Maximum adsorption occurred at pH 3 under low ionic strength conditions. The adsorbed activity could be readily released with a buffer solution at pH 5. Based upon these observations, a separation process was developed which enabled the broth to be clarified and, at the same time, the non-acidic polygalacturonases to be concentrated 20-fold and purified 100-fold in a unique filtration step. The practical advantage of recovering polygalacturonases by a filtration process lies in the simplicity and efficiency of the operation involved. Received 27 July 2005; Revisions requested 24 August 2005; Revisions received 16 November 2005; Accepted 16 November 2005     

Microbial hydroxylation. I. Hydroxylation of aniline byAspergillus alliaceus,A. albertensis andA. terreus

Summary Six hundred and seventy microorganisms were screened for the ability to perform stereoselective aromatic hydroxylation reactions of industrial significance, using aniline as a model substrate. TLC and HPLC analyses with diode array detection were used to identify and characterize hydroxylase activities. Of 79 cultures belonging to the speciesAspergillus alliaceus, A. albertensis, andA. terreus, 26 strains produced 2-aminophenol. Thirty strains were able to hydroxylate aniline in thepara position. Five strains ofA. terreus produced an unidentified phenolic compound in high yield.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

Patterns of oligosaccharide production by polygalacturonases

The products of hydrolytic action of 18 enzyme preparations at pH 3·5 and 5·5 on pectate were analyzed by gel-filtration chromatography early in the course of reaction (8–15% hydrolysis), and at a time 10 times that required for 10% hydrolysis. The degree of hydrolysis at the latter time varied from 25 to 74%. Three patterns of oligosaccharide production could be distinguished: endo-hydrolysis, exo-hydrolysis, and that due to S-polygalacturonase. The initial products of endo-hydrolysis were mixed oligosaccharides 5–30 units long; monomer and dimer appeared early but represented less than 2% of the products until late in the reaction. exo-Polygalacturonase (not entirely free of endo-) showed predominant production of the monomer and was clearly evident when mixed with four parts of endo-polygalacturonase. The time course of reducing group production by highly purified S-polygalacturonase could be reproduced by the above mixture of exo- and endo-polygalacturonases, but the pattern of products and the pH relations could not. The initial products of S-polygalacturonase were monomer, dimer and pentamer with lesser amounts of trimer and tetramer. After the hydolysis of the polymer and large oligomers, the pentamer was attacked by S-polygalacturonase, in the same way that the accumulated hexamer, etc. were finally hydrolysed by the endo-polygalacturonase.     

Maxillary sinusitis from Microascus cinereus and Aspergillus repens

Microascus was associated with Aspergillus repens in a left maxillary sinus. Tissue contained septale filaments of two types, conidia, ostiolate perithecia containing ascospores corresponding to Microascus cinereus which was identified by culture. The abundance of sexual fructifications in the tissue indicates that pathogenicity is due to Microascus cinereus.     

Biodegradation of lignocellulosic waste by Aspergillus terreus

Biodegradation of lignocellulosic waste by Aspergillus terreus is reported for the first time. This isolate produced 250 CMCase (carboxymethyl cellulase or endoglucanase) U.ml^-1 and biodegraded hay and straw during 3 days and the biomass production on straw was 5g.L^-1dry weight from 0.25 cm² inoculated mycellium. This strain secreted endocellulases and exocellulases in the culture medium, but some of the enzymes produced, remained cell membrane bound. Cell bound enzymes were released by various treatments. The highest amount of endoglucanase and exoglucanase was released when the cells were treated with sonication. Aspergillus terreus was added to two tanks containing sugar wastewater and pulp manufacturing waste, as a seed for COD removal. This fungus reduced the COD by 40–80 percent, also, ammonia was reduced from 14.5 mM to 5.6 mM in sugar beet wastewater. The effects of crude enzyme of this fungus for COD removal was studied.     

Improvement of trypanocidal metabolites production by Aspergillus fumigatus using neural networks

An optimization procedure using artificial neural networks was developed to determine the optimal combination of parameters, such as medium culture, initial pH, temperature and time of fermentation for maximal trypanocidal metabolites production by Aspergillus fumigatus. A data set of 81 experiments was carried out and an artificial neural network was trained to identify the optimal conditions for this process. Good correlation was obtained between the experimental and predicted values of lysis of the trypomastigote forms of Trypanosoma cruzi (r2 = 0.9990). The simulations of fermentation performance were undertaken on combinations of input variables and the highest level of activity against T. cruzi was obtained from the chloroform extract of the modified Jackson medium culture, initial pH of 6.0, incubated at 40 degrees C for 144 h. It displayed lysis of 95% of the trypomastigote forms of T. cruzi and the red blood cells remained normal.     

Promotion of conidial head formation in Aspergillus oryzae by a salt

Addition of KCl to medium at 0.1 M or higher promoted the formation of conidial heads in Aspergillus oryzae. When higher concentrations of KCl were added, a larger number of conidial heads were formed. NaCl and MgCl₂ were slightly less effective than KCl. The effect of salt on the formation of conidial heads on a minimal medium was as high as on a potato/dextrose medium but slightly higher than that on a complex medium when 1 M KCl was added.     

Cytochemical localization and ultrastructure of Aspergillus flavus in cottonseed

Cottonseeds having fluorescent fibers were harvested from fields in Arizona and examined utilizing light microscopy and transmission electron microscopy. The occurrence of fluorescent fibers indicated that seeds had been infected by Aspergillus flavus during development. Presence of A. flavus was verified by plating portions of seeds with fluorescent fibers. Hyphae, conidial heads, and conidia were identified readily in differentially-stained cotyledon tissue processed for light microscopy. Utilization of transmission electron microscopy permitted observations on lignified seed coats and cotyledons of mature cottonseeds. Hyphae were located throughout the cotyledon and in the nonlignified layers of the seed coat. The identification of hyphae in cross sections of vessel elements within the seed coat provided ultrastructural evidence supporting the hypothesis that A. flavus may enter seeds via the vascular tissue. Controls for the microscopy studies included observations on cottonseeds with no visual signs of infection and on laboratory-grown cultures of A. flavus. These observations demonstrated that the hyphae localized within fluorescent seeds had features characteristic of A. flavus and that fungal-like structures do not occur within uninfected seeds.     

Isolation and characterization of phytase isozymes produced by Aspergillus oryzae

A mutant strain (KL-38) of Aspergillus oryzae was obtained by UV irradiation. Phytase activity of KL-38 in molded rice (koji rice) was about 2.7-fold of that obtained from the parent strain (BP-1). Phytase activity of KL-38 in the submerged culture was similar to that of BP-1. Two types of phytase were produced from koji culture: phytase I (Phy I) was produced during incubation of both koji and submerged cultures, and phytase II (Phy II) was obtained only from koji culture. Phy II production was increased in KL-38 compared with BP-1, whereas the production of Phy I was similar for both KL-38 and BP-1. This finding indicates that A. oryzae has at least two types of phytase isozyme.     

Susceptibility of Larvae of Galleria mellonella to Infection by Aspergillus fumigatus is Dependent upon Stage of Conidial Germination

The ability of conidia of the human pathogenic fungus Aspergillus fumigatus to kill larvae of the insect Galleria mellonella was investigated. Conidia at different stages of the germination process displayed variations in their virulence as measured using the Galleria infection model. Non-germinating (‘resting’) conidia were avirulent except when an inoculation density of 1 × 10⁷ conidia per insect was used. Conidia that had been induced to commence the germination process by pre-culturing in growth medium for 3 h were capable of killing larvae at densities of 1 × 10⁶ and 1 × 10⁷ per insect. An inoculation density of 1 × 10⁵ conidia per insect remained avirulent. Conidia in the outgrowth phase of germination (characterised as the formation of a germ tube) were the most virulent and were capable of killing 100% of larvae after 5 or 24 h when 1 × 10⁷ or 1 × 10⁶ conidia, that had been allowed to germinate for 24 h, were used. Examination of the response of insect haemocytes to conidia at different stages of the germination process established that haemocytes could engulf non-germinating conidia and those in the early stages of the germination process but that conidia, which had reached the outgrowth stages of germination were not phagocytosed. The results presented here indicate that haemocytes of G. mellonella are capable of phagocytosing A. fumigatus conidia less than 3.0 μm in diameter but that conidia greater than this are too large to be engulfed. The virulence of A. fumigatus in G. mellonella larvae can be ascertained within 60–90 h if infection densities of 1 × 10⁶ or 1 × 10⁷ activated conidia (pre-incubated for 2–3 h) per insect are employed.     

Decolourization of Black Oxidized Olive-Mill Wastewater by a New Tannase-Producing Aspergillus flavus Strain Isolated from Soil

Summary By contaminating a Tunisian soil with black oxidized and sterilized olive-mill wastewaters (OMW), 30 new indigenous fungal soil strains able to overcome the OMW toxicity could be directly selected. Ten of the fungal strains previously isolated were screened for their capability to grow in a liquid culture medium containing oxidized OMW as the only source of carbon and energy. According to these preliminary tests, strain F2 showed the best capability of removing black colour and COD (chemical oxygen demand) and was further identified as Aspergillus flavus. After optimization of batch-liquid culture conditions in the presence of oxidized OMW, the time course of biomass and enzyme production by A. flavus F2 was followed in relation to colour and COD removal. A. flavus F2 could efficiently decolourize and detoxify the black oxidized OMW (58 and 46% of colour and COD removal, respectively, after 6 days of cultivation), concomitantly with the production of tannase (8000 UI/l on day 3).     

Production of sterigmatocystin by Aspergillus versicolor isolated from roughage

A total of 69 samples of hay and straw collected during the winter period of 1984/85 were surveyed for their contamination by Aspergillus versicolor. The percentage of A. versicolor-positive samples was 14.5%. Nineteen A. versicolor strains mainly isolated from roughage were tested for the production of sterigmatocystin. All of the isolates examined were capable of producing different levels of sterigmatocystin on a cracked corn substrate. The majority of these strains were highly toxigenic; 53% of the isolates produced more than 500 mg/kg of sterigmatocystin. These findings suggest that corn is a very suitable substrate for sterigmatocystin production and that particularly in the surface layers of feed stocks and corn silos such toxigenic strains of A. versicolor can produce considerable growth and possibly sterigmatocystin, too.     

Immunochemical detection of ochratoxin A in black Aspergillus strains

One hundred and fifty-seven strains belonging to Aspergillus section Nigri were tested for ochratoxin A production using three different methods: a relatively new immunochemical method based on an enzyme-linked immunosorbent assay (ELISA), thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The monoclonal antibody-based ELISA technique was successfully used to screen for low levels of ochratoxin A in the black Aspergilli without concentrating the culture filtrates. The results were confirmed by TLC and HPLC analysis and chemical derivatization. These latter methods required concentrated filtrates. Ochratoxin A was detected in the culture filtrates of five of the 12 A. carbonarius strains, none of the 45 A.japonicus strains and three of the 100 isolates in the A. niger aggregate (A. foetidus, A. awamori and A. niger).Abbreviations ELISA enzyme-linked immunosorbent assay - HPLC high-performance liquid chromatography - OA ochratoxin A - TLC thin-layer chromatography     

Effect of citrate on radial growth and conidiation of the mould Aspergillus nidulans

A mutation of the ctsA locus of Aspergillus nidulans affects both the radial growth and conidiation of the mould when grown in the presence of citrate. The ctsA locus was allocated to linkage group IV but it recombines freely with inoB2 and pyroA4 (which are also in linkage group IV). It is recessive in heterozygous diploids. A possible role for this gene in maintaining membrane integrity is discussed.The authors are with the Departamento de Genética, FMRP-USP, Av. Bandeirantes, 3900, 14049 Ribeirão Preto, SP., Brazil.     

Sugarcane molasses and yeast powder used in the Fructooligosaccharides production by Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611

Different concentrations of sucrose (3–25% w/v) and peptone (2–5% w/v) were studied in the formulation of media during the cultivation of Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611. Moreover, cane molasses (3.5–17.5% w/v total sugar) and yeast powder (1.5–5% w/v) were used as alternative nutrients for both strains’ cultivation. These media were formulated for analysis of cellular growth, β-Fructosyltransferase and Fructooligosaccharides (FOS) production. Transfructosylating activity (U _t ) and FOS production were analyzed by HPLC. The highest enzyme production by both the strains was 3% (w/v) sucrose and 3% (w/v) peptone, or 3.5% (w/v) total sugars present in cane molasses and 1.5% (w/v) yeast powder. Cane molasses and yeast powder were as good as sucrose and peptone in the enzyme and FOS (around 60% w/w) production by studied strains.     

Short communication: Influence of inoculum preparation on citric acid production by Aspergillus niger

The type of sporulation medium and time of incubation had an effect on spore viability and citric acid production by mycelia grown from Aspergillus niger spores. Shu & Johnson agar (SJA) and potato dextrose agar gave higher citric acid titres than malt-extract agar. SJA also gave better germinability than the other media. Viability increased with time of incubation, but higher production of citric acid was achieved with spores incubated for less than 7 days.