Preparations of Extracellular Proteinases from Aspergillus ochraceus 513 and Aspergillus alliaceus 7 dN1

Preparations of extracellular proteolytic enzymes with high anticoagulant activity resembling protein C activators were isolated from the culture liquids of Aspergillus ochraceus 513 and Aspergillus alliaceus 7 dN1 by precipitation with ammonium sulfate and subsequent purification from ammonium ions by gel filtration on a column with Sephadex G-25. The pH and temperature activity optima and stability of the proteolytic enzymes from A. ochraceus 513 and A. alliaceus 7 dN1 were determined.     

Ochratoxin production by the Aspergillus ochraceus group and Aspergillus alliaceus

Ochratoxin A is a toxic and carcinogenic fungal secondary metabolite; its presence in foods is increasingly regulated. Various fungi are known to produce ochratoxins, but it is not known which species produce ochratoxins consistently and which species cause ochratoxin contamination of various crops. We isolated fungi in the Aspergillus ochraceus group (section Circumdati) and Aspergillus alliaceus from tree nut orchards, nuts, and figs in California. A total of 72 isolates were grown in potato dextrose broth and yeast extract-sucrose broth for 10 days at 30 degrees C and tested for production of ochratoxin A in vitro by high-pressure liquid chromatography. Among isolates from California figs, tree nuts, and orchards, A. ochraceus and Aspergillus melleus were the most common species. No field isolates of A. ochraceus or A. melleus produced ochratoxin A above the level of detection (0.01 microg/ml). All A. alliaceus isolates produced ochratoxin A, up to 30 microg/ml. We examined 50,000 figs for fungal infections and measured ochratoxin content in figs with visible fungal colonies. Pooled figs infected with A. alliaceus contained ochratoxin A, figs infected with the A. ochraceus group had little or none, and figs infected with Penicillium had none. These results suggest that the little-known species A. alliaceus is an important ochratoxin-producing fungus in California and that it may be responsible for the ochratoxin contamination occasionally observed in figs.     

Ochratoxin production by Aspergillus species.

Ochratoxin production was tested in 172 strains representing species in sections Fumigati, Circumdati, Candidi, and Wentii of the genus Aspergillus by an immunochemical method using a monoclonal antibody preparation against ochratoxin A. Ochratoxin A was detected in Aspergillus ochraceus, A. alliaceus, A. sclerotiorum, A. sulphureus, A. albertensis, A. auricomus, and A. wentii strains. This is the first report of production of ochratoxins in the latter three species. Ochratoxin production by these species was confirmed by high-performance thin-layer chromatography and by high-performance liquid chromatography. The chemical methods also indicated the production of ochratoxin B by all of the Aspergillus strains mentioned above.     

DNA fingerprinting analysis of Petromyces alliaceus (Aspergillus section Flavi)

The objective of this study was to evaluate the ability of the Aspergillus flavus pAF28 DNA probe to produce DNA fingerprints for distinguishing among genotypes of Petromyces alliaceus (Aspergillus section Flavi), a fungus considered responsible for the ochratoxin A contamination that is occasionally observed in California fig orchards. P. alliaceus (14 isolates), Petromyces albertensis (one isolate), and seven species of Aspergillus section Circumdati (14 isolates) were analyzed by DNA fingerprinting using a repetitive sequence DNA probe pAF28 derived from A. flavus. The presence of hybridization bands with the DNA probe and with the P. alliaceus or P. albertensis genomic DNA indicates a close relationship between A. flavus and P. alliaceus. Twelve distinct DNA fingerprint groups or genotypes were identified among the 15 isolates of Petromyces. Conspecificity of P. alliaceus and P. albertensis is suggested based on DNA fingerprints. Species belonging to Aspergillus section Circumdati hybridized only slightly at the 7.0-kb region with the repetitive DNA probe, unlike the highly polymorphic hybridization patterns obtained from P. alliaceus and A. flavus, suggesting very little homology of the probe to Aspergillus section Circum dati genomic DNA. The pAF28 DNA probe offers a tool for typing and monitoring specific P. alliaceus clonal populations and for estimating the genotypic diversity of P. alliaceus in orchards, vineyards, or crop fields.     

The separation of true coagulases from other enzymes of the extracellular proteolytic complex of Aspergillus ochraceus by using affinity chromatography]

A complex of proteases has been isolated from the cultural broth of fungi Aspergillus ochraceus HP-19 by precipitation ammonium sulfate. Using the method of affinity chromatography on the biospecific sorbents the conditions for separation of proteases into ones with coagulase activity and with fibrinolytic activity have been found.     

Plasma-coagulating and fibrinolytic activities of fungi in the genus Aspergillus

The exocellular plasmocoagulating and fibrinolytic activities were studied in 100 cultures of the Aspergillus genus belonging to 29 species during their submerged cultivation in three media. It has been found that 38 cultures can coagulate human plasma, 75 cultures can cause lysis of fibrin platelets, and 22 cultures are capable of dissolving standard plasma clots within 6 hours. The cultures synthesize three types of proteolytic enzymes according to the specificity toward blood proteins: (1) proteases with the predominant fibrinolytic action; (2) proteases which possess both the fibrinolytic and plasmocoagulating activities; (3) proteases manifesting only the plasmocoagulating action. A. ochraceus 19 producing individual plasmocoagulases and fibrinolytic enzymes at a high rate was isolated. The composition of the enzyme complex synthesized by the culture depended on the composition of the medium and on the cultivation conditions.     

Characterization and population analysis of the mating-type genes in Aspergillus flavus and Aspergillus parasiticus

We characterize the mating-type genes in Aspergillus flavus,Aspergillus parasiticus and Petromyces alliaceus. A single MAT1-1 or MAT1-2 gene was detected in the genomes of A. flavus and A. parasiticus, which is consistent with a potential heterothallic organization of MAT genes in these species. In contrast, the only known, functionally homothallic species in Aspergillus section Flavi, P. alliaceus, has tightly linked (<2kb) MAT1-1 and MAT1-2 genes, typical of other self-fertile homothallic euascomycetes. This is the first example of linked MAT genes within a homothallic species of Aspergillus. We tested the null hypothesis of no significant difference in the frequency of MAT1-1 and MAT1-2 in A. flavus and A. parasiticus sampled from a single peanut field in Georgia. For each species, mating-type frequencies were determined for the total population samples and for samples that were clone-corrected based on vegetative compatibility groups (VCGs) and aflatoxin gene cluster haplotypes. There was no significant difference in the frequency of the two mating types for A. flavus and A. parasiticus in either VCG or haplotype clone-corrected samples. The existence of both mating-type genes in equal proportions in A. flavus and A. parasiticus populations, coupled with their expression at the mRNA level and the high amino acid sequence identity of MAT1-1 (77%) and MAT1-2 (83%) with corresponding homologs in P. alliaceus, indicates the potential functionality of these genes and the possible existence of a sexual state in these agriculturally important species.     

Culture media and sources of nitrogen promoting the formation of stromata and ascocarps in Petromyces alliaceus (Aspergillus section Flavi)

Petromyces alliaceus Malloch and Cain is the only known sexually reproducing fungus classified in Aspergillus section Flavi. The goal of this research was to identify culture media and sources of nitrogen that best support the formation of stromata with ascocarps. Three cultures of P. alliaceus isolated from crop field soils were grown on selected agar media in Petri dishes for 7 months at 30 degrees C in darkness. The largest numbers of stromata were recorded for cultures grown on Czapek's agar (CZA) and a mixed cereal agar (MCA), while the percentage of stromata containing ascocarps was greatest (P相似文献   

Isolation, purification, and separation of the complex preparation of extracellular proteinases with fibrinolytic and anticoagulant properties from Aspergillus ochraceus 513]

The extracellular proteinase complex of the microscopic fungus Aspergillus ochraceus 513 was isolated, purified, and separated by affinity chromatography on bacillichin-silochrom and subsequent column chromatography on DEAE-Toyopearl 650 M. The extracellular enzyme of the protein C activator type had a molecular mass of 36.5 kDa and activity close to that of the Agkistrodon snake venom protein C activator. The fibrinolytic and anticoagulant activities of the enzyme were investigated.     

Transformation of Aspergillus alliaceus using the Aspergillus niger niaD gene encoding nitrate reductase

Abstract A heterologous transformation system for Aspergillus alliaceus based on the Aspergillus niger nitrate reductase structural gene ( niaD ) has been developed. Two mutants of A. alliaceus (M3 and M17), each carrying an niaD mutation were isolated by screening UV-irradiated cells for the inability to grow on nitrate as sole nitrogen source. Using plasmid pSTA 10, transformation frequencies of 4 and 200 per μg DNA respectively were obtained for these two strains. All the niaD ⁺ transformants tested were mitotically stable. Southern hybridisation analyses showed that the vector DNA sequences were present.     

Production of xylanolytic enzymes during growth on pulverized grass by Aspergillus ochraceus-42

Xylanase and β-xylosidase with activity of 6.46 U mg^-1 and 0.500 U mg^-1, respectively, were produced extracellularly by Aspergillus ochraceus during growth on pulverized grass in liquid state fermentation, compared to 9.3 U mg^-1 and 0.74 U mg^-1 when pure xylan was used. The culture filtrate was devoid of any cellulase activity. Xylanolytic enzymes were produced optimally in 144 h of incubation on 1% pulverized grass, pH 6.5. About 8.43% (w/w) sugars were liberated from alkali-treated grass in 6 h by the synergistic effect of xylanolytic enzymes. The half-lives for xylanase and β-xylosidase at 50°C were 210 min and 300 min, respectively, and half-life increased with the increase in protein concentration. Both mono- and divalent cations, especially K⁺ and Zn²⁺, exhibited a profound effect on the rate of enzyme saccharification.     

Molecular typing of Aspergillus ochraceus and construction of species specific SCAR-primers based on AFLP

70 strains of Aspergillus ochraceus mainly isolated from Brazilian coffee related sources were investigated for genetic relatedness using automated laser fluorescence analysis of AFLP fragments. Cluster analysis of fingerprints revealed a very close relationship among most of the strains. Based on these results, a sub-set of characteristic A. ochraceus strains was chosen for the detection of marker sequences. These sequences were obtained from silver stained AFLPs separated on polyacrylamide gels. A number of bands characteristic for A. ochraceus were detected and cut out from the gels. DNA was reamplified, cloned and fragments were sequenced. Based on these sequences a set of SCAR PCR-primers was constructed. PCRs were optimised for specificity and subsequently tested against a panel of Aspergillus species. Using this approach a PCR specific for Aspergillus ochraceus was developed.     

The use of micromycetes for cleaning parts of aircraft

The mycelial Fungi Penicillium funiculosum, P. citrinum, P. expansum, P. chrysogenum, Aspergillus ochraceus, A. alliaceus, A. luchaensis, A. flavus, and A. niger were isolated from enrichment cultures. These fungi actively destruct carbon deposits formed during exploitation of aircraft. A biotechnological method for removing fouling from parts of aircraft engines (PAE) was developed. This method is less laborious, more rapid and ecologically clean than contemporary chemical methods. Scanning microscopy was suggested to use for estimating the degree of decarbonization of PAE surfaces.     

Immunochemical identification of fungi using polyclonal antibodies raised in rabbits to exoantigens from Aspergillus ochraceus

Aspergillus ochraceus Wilhelm is a producer of highly toxic mycotoxin, ochratoxin A, and is known to contribute to the damage of grain on storage. Rabbit antibodies were produced against the exoantigens of A. ochraceus , and ELISA and immunoblotting procedures were used to characterize their reactivity towards fungal exoantigens of four Aspergillus , seven Penicillium , three Fusarium , one Mucor and one Alternaria species. The antibodies were specific for A. ochraceus and essentially cross-reacted neither with other fungi nor with the water-soluble grain components. These antibodies, therefore, have the potential of being utilized for the discriminative monitoring of A. ochraceus in foodstuffs and feeds.     

Detection and quantification of Aspergillus ochraceus in green coffee by PCR

AIMS: The aim of this study was to detect and quantify DNA of the ochratoxinogenic fungus Aspergillus ochraceus in green coffee and to compare the results with the ochratoxin A content of naturally contaminated samples. METHODS AND RESULTS: A DNA extraction protocol based on a combination of ultrasonification and a commercial kit was tested for the recovery of fungal DNA. PCR and real-time PCR protocols were established for the detection of A. ochraceus. Sensitivity of the PCR was checked by the addition of inoculated green coffee and pure fungal DNA to uncontaminated green coffee samples. The A. ochraceus DNA content of 30 naturally contaminated green coffee samples was determined and compared with the ochratoxin A concentrations. CONCLUSIONS: Aspergillus ochraceus can be rapidly and specifically detected in green coffee by PCR. A positive correlation between the ochratoxin A content and the DNA quantity was established. Significance and Impact of the Study: This work offers a quick alternative to the conventional mycological detection and quantification of A. ochraceus in green coffee.     

Glucoamylase of Aspergillus niger

Aspergillus niger produces two extracellular glucoamylases (GAI of Mr 85 300 and GAII of Mr 77 600) separable on DEAE-cellulose. The enzymes differes in electrophoretic mobility, thermostability and substrate specificity. The GAI/GAII ratio depends on the concentration and form of nitrogen (nitrate or ammonium) in the culture medium. Proteinase VIII from Bacillus subtilis converts GAI to a form showing properties similar to those of GAII. Possible proteolytic degradation of GAI to GAII by Asp. niger endogenous proteinase(s) is suggested.     

Microbiological hydroxylation of estradiol: formation of 2- and 4-hydroxyestradiol by Aspergillus alliaceus

Microorganisms known to hydroxylate alkaloids, amino acids, and aromatic substrates were examined for their potential to hydroxylate 17 beta-estradiol and estrone. Thin-layer chromatography of fermentation extracts revealed a wide range of steroid products. Aspergillus alliaceus (UI 315) was the only culture capable of producing good yields of catechol estrogens with 17 beta-estradiol. The organism also transformed estrone but not to catechol products. Analytical experiments with high-performance liquid chromatography revealed that A. alliaceus formed 4- and 2-hydroxyestradiol with yields of 45 and 16%, respectively. A preparative-scale incubation was conducted in 2 liters of medium containing 1 g of 17 beta-estradiol as substrate. 4-Hydroxyestradiol was isolated and identified by proton nuclear magnetic resonance and high-resolution mass spectrometry. Ascorbic acid was added to microbial reaction mixtures as an antioxidant to prevent the decomposition of unstable catechol estrogen metabolites. The microbial transformation of 17 beta-estradiol by A. alliaceus provides an efficient one-step method for the preparation of catechol estrogens.     

Microbiological hydroxylation of estradiol: formation of 2- and 4-hydroxyestradiol by Aspergillus alliaceus.

Microorganisms known to hydroxylate alkaloids, amino acids, and aromatic substrates were examined for their potential to hydroxylate 17 beta-estradiol and estrone. Thin-layer chromatography of fermentation extracts revealed a wide range of steroid products. Aspergillus alliaceus (UI 315) was the only culture capable of producing good yields of catechol estrogens with 17 beta-estradiol. The organism also transformed estrone but not to catechol products. Analytical experiments with high-performance liquid chromatography revealed that A. alliaceus formed 4- and 2-hydroxyestradiol with yields of 45 and 16%, respectively. A preparative-scale incubation was conducted in 2 liters of medium containing 1 g of 17 beta-estradiol as substrate. 4-Hydroxyestradiol was isolated and identified by proton nuclear magnetic resonance and high-resolution mass spectrometry. Ascorbic acid was added to microbial reaction mixtures as an antioxidant to prevent the decomposition of unstable catechol estrogen metabolites. The microbial transformation of 17 beta-estradiol by A. alliaceus provides an efficient one-step method for the preparation of catechol estrogens.     

Phytase activity in Aspergillus fumigatus isolates

Extracellular phytase from Aspergillus fumigatus isolates was characterized and their genes were cloned and sequenced. Based on their banding pattern in SDS-PAGE all phytases were found to be glycosylated and have similar molecular mass. A correlation between lower optimum pH (4.0) and a higher optimum temperature (70 degrees C) was found in these enzymes. All enzymes characterized displayed a lower specific activity for phytic acid and were more susceptible to proteolytic degradation than the Aspergillus niger phytase that is now commercially available. DNA sequencing established almost no sequence variation in any of the genes and no correlation is evident between a specific amino acid sequence and any physicochemical and catalytic properties of the enzymes. Despite two of the isolates having identical deduced amino acid sequence, characterization of the enzymes encoded by these two identical genes revealed differences in both pH and temperature optimum. This suggests that differences in pH and temperature optimum in these four isolates of A. fumigatus may be due in part to subtle differences in posttranslational modification.     

Biosynthesis of diaporthin and orthosporin by Aspergillus ochraceus

Diaporthin and orthosporin were characterised from the fungus Aspergillus ochraceus D2306. Diaporthin was identified by high-resolution electron impact mass spectrometry and 1H and 13C NMR spectroscopy, from which new spectroscopic assignments were made. Orthosporin was also identified by mass spectrometry and both fungal metabolites are reported for the first time as co-metabolites and also as products of A. ochraceus. The methylation inhibitor ethionine affected production of both diaporthin and orthosporin in spite of no obvious methylation step in the biosynthesis of orthosporin, implying that extracellular orthosporin may arise by de-O-methylation of diaporthin. The biosynthetic origin of diaporthin was demonstrated by incorporation of [1-14C]acetate and [methyl-14C]methionine administered in early idiophase.