Bio-oxidation of progesterone by Penicillium oxalicum CBMAI 1185 and evaluation of the cytotoxic activity.

We report the biotransformation of progesterone 1 by whole cells of Brazilian marine-derived fungi. A preliminary screening with 12 fungi revealed that the strains Penicillium oxalicum CBMAI 1996, Mucor racemous CBMAI 847, Cladosporium sp. CBMAI 1237, Penicillium oxalicum CBMAI 1185 and Aspergillus sydowii CBMAI 935 were efficient in the biotransformation of progesterone 1 in the first days of the reaction, with conversion values ranging from 75 % to 99 %. The fungus P. oxalicum CBMAI 1185 was employed in the reactions in quintuplicate to purify and characterize the main biotransformation products of progesterone 1. The compounds testololactone 1a, 12ß-hydroxyandrostenedione 1b and 1ß-hydroxyandrostenedione 1c were isolated and characterized by NMR, MS, [α]D and MP. In addition, the chromatographic yield of compound 1a was determined by HPLC-PDA in the screening experiments. In this study, we show a biotransformation pathway of progesterone 1, suggesting the presence of several enzymes such as Baeyer-Villiger monooxygenases, dehydrogenases and cytochrome P450 monooxygenases in the fungus P. oxalicum CBMAI 1185. In summary, the results obtained in this study contribute to the synthetic area and have environmental importance, since the marine-derived fungi can be employed in the biodegradation of steroids present in wastewater and the environment. The cytotoxic results demonstrate that the biodegradation products were inactive against the cell lines, in contrast to progesterone.

Effect of the oscillating magnetic field on airborne fungal.

This study shows that some species of fungi are affected by the magnetic field, which should be taken into account in studies of airborne fungal and air quality. The aim of this paper was to evaluate the effect of the oscillating magnetic field (OMF) on the behavior of colonies of three fungi genus growth in different culture mediums. The stains were: Aspergillus niger, Cladosporium cladosporioides and Penicillium citrinum and were inoculated in 90 mm Petri dishes with: Malt Extract Agar (MEA), Sabouraud Dextrose Agar (SDA) and Czapek-Dox Agar (CDA). Was applied them OMF of 60 Hz/220 V between 1 and 5 mT during 2 h and then they were incubated 7 days to 28 °C. Colonies size (mm) every day was measured. Stimulation in the colonies size of all experimental conditions was showed; the greatest size of A. niger in MEA was notorious. It was demonstrated by statist analyze that only colonies size with 1 mT was significance respect to the control. The effect of OMF on the cellular metabolism was evidenced, as well as: less exudation and major pigmentation of P. citrinum in MEA; variation of pigmentation of A. niger and C. cladosporioides in CDA and increase of conidiogenesis of A. niger in SDA. Was concluded that the applied OMF had a major influence on size colony and mycelia pigmentation of A. niger that C. cladosporioides and P. citrinum, independently of the nutritional state according to the culture medium employed in this study.

Synthesis of Knoevenagel Adducts Under Microwave Irradiation and Biocatalytic Ene-Reduction by the Marine-Derived Fungus Cladosporium sp. CBMAI 1237 for the Production of 2-Cyano-3-Phenylpropanamide Derivatives.

The organic synthesis has been driven by the need of sustainable processes, which also requires efficiency and cost-effectiveness. In this work, we described the synthesis of nine Knoevenagel adducts between cyanoacetamide and aromatic aldehydes ((E)-2-cyano-3-(phenyl)acrylamide derivatives), employing triethylamine as catalyst under microwave irradiation in 30 min with excellent yields (93-99% yield). Then, these adducts were employed in the C-C double bond bioreduction by the marine-derived fungus Cladosporium sp. CBMAI 1237 for obtention of 2-cyano-3-phenylpropanamide derivatives in mild conditions and short reaction time for a whole-cells reduction (phosphate buffer pH 7.0, 32 °C, 130 rpm, 8 h) with good yields (48-90%). It is important to emphasize that the experimental conditions, especially the reaction time, should be carefully evaluated for the obtention of high yields. Since a biodegradation process consumed the obtained product in extended periods, probably due to the use of the substrate as carbon and nitrogen source. This approach showed that the use of coupled and greener catalysis methods such as microwave irradiation and biocatalytic reduction, which employs unique biocatalysts like marine-derived fungi, can be an interesting tool for the obtention of organic molecules.

Mold burden in house dust and its relationship with asthma control.

OBJECTIVE: Some evidences indicate that exposure to molds or their products can be relevant for the loss of asthma control. Thus, we measured the mold burden present inside houses of subjects with asthma, and evaluated its relationship with asthma control. METHODS: Markers of asthma control in adult patients residing in Mexico City were evaluated through questionnaires and spirometry. Dust was collected from the patients' houses and its fungal content was determined by mold specific quantitative PCR (MSQPCR) for 36 fungal species. RESULTS: Forty-two patients with asthma (12 males, 30 females) with a mean age of 45 years (18-76 years) were included in the study. The level of asthma control measured through the Asthma Control Test ranged from 9 to 25 (mean 20.9). The FEV1/FVC ratio fluctuated from 38 to 106 %predicted (mean, 87.4 %predicted). Associations between mold burden and asthma control differed between males and females. Thus, concentrations of some molds, particularly Aspergillus fumigatus, Aureobasidium pullulans, Stachybotrys chartarum, Alternaria alternata, Cladosporium cladosporioides 2, Cladosporium herbarum, and Epicoccum nigrum, were negatively associated with parameters of asthma control in male subjects, but not in female patients. CONCLUSION: Our results showed that potential indoor exposure to some molds is associated with less asthma control in male subjects.

Heterologous expression, purification and characterization of a highly thermolabile endoxylanase from the Antarctic fungus Cladosporium sp.

Numerous endoxylanases from mesophilic fungi have been purified and characterized. However, endoxylanases from cold-adapted fungi, especially those from Antarctica, have been less studied. In this work, a cDNA from the Antarctic fungus Cladosporium sp. with similarity to endoxylanases from glycosyl hydrolase family 10, was cloned and expressed in Pichia pastoris. The pure recombinant enzyme (named XynA) showed optimal activity on xylan at 50 °C and pH 6-7. The enzyme releases xylooligosaccharides but not xylose, indicating that XynA is a classical endoxylanase. The enzyme was most active on xylans with high content of arabinose (rye arabinoylan and wheat arabinoxylan) than on xylans with low content of arabinose (oat spelts xylan, birchwood xylan and beechwood xylan). Finally, XynA showed a very low thermostability. After 20-30 min of incubation at 40 °C, the enzyme was completely inactivated, suggesting that XynA would be the most thermolabile endoxylanase described so far in filamentous fungi. This is one of the few reports describing the heterologous expression and characterization of a xylanase from a fungus isolated from Antarctica.

Fungi in spices and mycotoxigenic potential of some Aspergilli isolated.

The aim of this study was to identify fungal species present in 200 samples of rosemary, fennel, cinnamon, clove, pepperoni, black and white pepper and oregano and evaluate the mycotoxigenic potential of the some Aspergilli isolated. Clove, black and white peppers were analyzed by direct plating. For rosemary, cinnamon, fennel, pepperoni pepper and oregano samples were used spread plate. Mycotoxigenic capacity was verified by the agar plug method. With the exception of clove, all the spices showed high fungal contamination, especially by Aspergillus sp., Penicillium sp. and Cladosporium sp. Frequency of toxigenic Aspergillus spp. was intense in white and black peppers, with presence of Aspergillus flavus (up to 32%), Aspergillus nomius (up to 12%), Aspergillus parasiticus (up to 4%), Aspergillus niger complex (up to 52%), Aspergillus ochraceus (up 12%) and Aspergillus carbonarius (up to 4%). 14,2% of A. flavus isolated from black pepper were aflatoxins producers. In the white pepper, 66.7% of A. flavus isolates and 100% of A. nomius were aflatoxigenic. Oregano showed the highest number of A. niger complex isolates (49), however, only 2.04% produced ochratoxin A. This study showed a huge fungal presence in spices, which could compromise the sensorial quality of these products and represent a hazard for consumers.

Biodegradation of anthracene and several PAHs by the marine-derived fungus Cladosporium sp. CBMAI 1237.

The biodegradation of polycyclic aromatic hydrocarbons (PAHs) by marine-derived fungi was reported in this work. Marine-derived fungi (Trichoderma harzianum CBMAI 1677, Cladosporium sp. CBMAI 1237, Aspergillus sydowii CBMAI 935, Penicillium citrinum CBMAI 1186 and Mucor racemosus CBMAI 847) biodegraded anthracene (14days, 130rpm, 50mgmL-1 initial concentration in malt 2% medium). Cladosporium sp. CBMAI 1237 was the most efficient strain and biodegraded more anthracene in the presence (42% biodegradation) than in the absence (26%) of artificial seawater, suggesting that the biodegradation of PAHs may be faster in seawater than in non-saline environment. After 21days, Cladosporium sp. CBMAI 1237 biodegraded anthracene (71% biodegradation), anthrone (100%), anthraquinone (32%), acenaphthene (78%), fluorene (70%), phenanthrene (47%), fluoranthene (52%), pyrene (62%) and nitropyrene (64%). Previous undocumented metabolites were identified and, anthraquinone was a common product of different PAHs biodegradation. The marine-derived fungus Cladosporium sp. CBMAI 1237 showed potential for bioremediation of PAHs.

Degradation of keratin substrates by keratinolytic fungi

Background: The hydrolysis of keratin wastes by microorganisms is considered a biotechnological alternative for recycling and valorization through keratinolytic microorganisms. Despite their resistant structure, keratin wastes can be efficiently degraded by various microorganisms through the secretion of keratinases, which are promising enzymes for several applications, including detergents, fertilizers, and leather and textile industry. In an attempt to isolate keratinolytic microorganisms that can reach commercial exploitation as keratinase producers, the current work assesses the dynamics of keratin biodegradation by several keratinolytic fungal strains isolated from soil. The activity of fungal strains to degrade keratin substrates was evaluated by SEM, FTRIR-ATR spectra and TGA analysis. Results: SEM observations offered relevant information on interactions between microorganism and structural elements of hair strands. FTIR spectra of the bands at 1035­1075 cm-1 assigned to sulfoxide bond appeared because of S­S bond breaking, which demonstrated the initiation of keratin biodegradation. According to TGA, in the second zone of thermal denaturation, where keratin degradation occurs, the highest weight loss of 71.10% was obtained for sample incubated with Fusarium sp. 1A. Conclusions: Among the tested strains, Fusarium sp. 1A was the most active organism in the degradation process with the strongest denaturation of polypeptide chains. Because keratinolytic microorganisms and their enzymes keratinases represent a subject of scientific and economic interest because of their capability to hydrolyze keratin, Fusarium sp. 1A was selected for further studies.

Biodegradation of the Pyrethroid Pesticide Esfenvalerate by Marine-Derived Fungi.

Esfenvalerate biodegradation by marine-derived fungi is reported here. Esfenvalerate (S,S-fenvalerate) and its main metabolites [3-phenoxybenzaldehyde (PBAld), 3-phenoxybenzoic acid (PBAc), 3-phenoxybenzyl alcohol (PBAlc), and 2-(4-chlorophenyl)-3-methylbutyric acid (CLAc)] were quantitatively analyzed by a validated method in triplicate experiments. All the strains (Penicillium raistrickii CBMAI 931, Aspergillus sydowii CBMAI 935, Cladosporium sp. CBMAI 1237, Microsphaeropsis sp. CBMAI 1675, Acremonium sp. CBMAI 1676, Westerdykella sp. CBMAI 1679, and Cladosporium sp. CBMAI 1678) were able to degrade esfenvalerate, however, with different efficiencies. Initially, 100 mg L(-1) esfenvalerate (Sumidan 150SC) was added to each culture in 3 % malt liquid medium. Residual esfenvalerate (64.8-95.2 mg L(-1)) and the concentrations of PBAc (0.5-7.4 mg L(-1)), ClAc (0.1-7.5 mg L(-1)), and PBAlc (0.2 mg L(-1)) were determined after 14 days. In experiments after 7, 14, 21, and 28 days of biodegradation with the three most efficient strains, increasing concentrations of the toxic compounds PBAc (2.7-16.6 mg L(-1), after 28 days) and CLAc (6.6-13.4 mg L(-1), after 28 days) were observed. A biodegradation pathway was proposed, based on HPLC-ToF results. The biodegradation pathway includes PBAld, PBAc, PBAlc, ClAc, 2-hydroxy-2-(3-phenoxyphenyl)acetonitrile, 3-(hydroxyphenoxy)benzoic acid, and methyl 3-phenoxy benzoate. Marine-derived fungi were able to biodegrade esfenvalerate in a commercial formulation and showed their potential for future bioremediation studies in contaminated soils and water bodies.

Fungi associated with rocks of the Atacama Desert: taxonomy, distribution, diversity, ecology and bioprospection for bioactive compounds.

This study assessed the diversity of cultivable rock-associated fungi from Atacama Desert. A total of 81 fungal isolates obtained were identified as 29 Ascomycota taxa by sequencing different regions of DNA. Cladosporium halotolerans, Penicillium chrysogenum and Penicillium cf. citrinum were the most frequent species, which occur at least in four different altitudes. The diversity and similarity indices ranged in the fungal communities across the latitudinal gradient. The Fisher-α index displayed the higher values for the fungal communities obtained from the siltstone and fine matrix of pyroclastic rocks with finer grain size, which are more degraded. A total of 23 fungal extracts displayed activity against the different targets screened. The extract of P. chrysogenum afforded the compounds α-linolenic acid and ergosterol endoperoxide, which were active against Cryptococcus neoformans and methicillin-resistance Staphylococcus aureus respectively. Our study represents the first report of a new habitat of fungi associated with rocks of the Atacama Desert and indicated the presence of interesting fungal community, including species related with saprobes, parasite/pathogen and mycotoxigenic taxa. The geological characteristics of the rocks, associated with the presence of rich resident/resilient fungal communities suggests that the rocks may provide a favourable microenvironment fungal colonization, survival and dispersal in extreme conditions.

Stereoselective Bioreduction of α-Azido Ketones by Whole Cells of Marine-Derived Fungi.

Seven strains of marine-derived fungi (Aspergillus sclerotiorum CBMAI 849, Cladosporium cladosporioides CBMAI 857, Penicillium raistrickii CBMAI 931, Penicillium citrinum CBMA 1186, Mucor racemosus CBMAI 847, Beauveria felina CBMAI 738, and Penicillium oxalicum CBMAI 1185) and terrestrial fungus Penicillium chrysogenum CBMA1199 were screened as catalysts for the asymmetric reduction of α-keto azides 5-8 to their corresponding ß-azidophenylethanols 9-12. The marine fungi showed Prelog and anti-Prelog selectivities to the reduction α-keto azides 5-8. The fungi A. sclerotiorum CBMAI 849, C. cladosporioides CBMAI 857, P. raistrickii CBMAI 931, and P. citrinum CBMA 1186 catalyzed the reduction of azido ketone 6 to the corresponding (R)-2-azido-1-(4-methoxyphenyl)ethanol (10) with good conversions (68-100 %) and excellent enantiomeric excesses (>99 % ee) according to Prelog rule.

Cold-active xylanase produced by fungi associated with Antarctic marine sponges.

Despite their potential biotechnological applications, cold-active xylanolytic enzymes have been poorly studied. In this work, 38 fungi isolated from marine sponges collected in King George Island, Antarctica, were screened as new sources of cold-active xylanases. All of them showed xylanase activity at 15 and 23 °C in semiquantitative plate assays. One of these isolates, Cladosporium sp., showed the highest activity and was characterized in detail. Cladosporium sp. showed higher xylanolytic activity when grown on beechwood or birchwood xylan and wheat bran, but wheat straw and oat bran were not so good inducers of this activity. The optimal pH for xylanase activity was 6.0, although pH stability was slightly wider (pH 5-7). On the other hand, Cladosporium sp. showed high xylanase activity at low temperatures and very low thermal stability. Interestingly, thermal stability was even lower after culture media were removed and replaced by buffer, suggesting that low molecular component(s) of the culture media could be important in the stabilization of cold-active xylanase activity. To the best of our knowledge, this study is the first report on extracellular xylanase production by fungi associated with Antarctic marine sponges.

Cnidarian-derived filamentous fungi from Brazil: isolation, characterisation and RBBR decolourisation screening.

Marine-derived fungi represent a valuable source of structurally novel and biologically active metabolites of industrial interest. They also have drawn attention for their capacity to degrade several pollutants, including textile dyes, organochlorides and polycyclic aromatic hydrocarbons (PAHs), among others. The fungal tolerance to higher concentrations of salt might be considered an advantage for bioremediation processes in the marine environment. Therefore, filamentous fungi were isolated from cnidarians (scleractinian coral and zoanthids) collected from the north coast of São Paulo State, Brazil. A total of 144 filamentous fungi were morphologically and molecularly characterised. Among them there were several species of Penicillium and Aspergillus, in addition to Cladosporium spp., Eutypella sp., Fusarium spp., Khuskia sp., Mucor sp., Peacilomyces sp., Phoma sp. and Trichoderma spp. These fungi were tested regarding their decolourisation activity for Remazol Brilliant Blue R (RBBR), a textile dye used as an initial screening for PAH-degrading fungi. The most efficient fungi for RBBR decolourisation after 12 days were Penicillium citrinum CBMAI 853 (100%), Aspergillus sulphureus CBMAI 849 (95%), Cladosporium cladosporioides CBMAI 857 (93%) and Trichoderma sp. CBMAI 852 (89%). Besides its efficiency for dye decolourisation within liquid media, C. cladosporioides CBMAI 857 also decolourised dye on solid media, forming a decolourisation halo. Further research on the biotechnological potential, including studies on PAH metabolism, of these selected fungi are in progress.

Microbiologic oxidation of isosafrole into piperonal.

The biotransformation of isosafrole by Cladosporium sphaerospermum yielded piperonal, which is a compound of great commercial importance in the flavor and fragrance industries. The experiments were performed in 500-mL conical flasks containing 100 mL of Czapek-modified medium in an orbital shaker with controlled agitation and temperature. Spores of C. sphaerospermum were used as inocula, and after 96 h of incubation the substrate was added to the culture. Samples of 2 mL were withdrawn at 24-h intervals and analyzed by gas chromatography, (GC) and/or GC/MS spectroscopy.

Post-harvest storage of corn: effect of beginning moisture content on mycoflora and fumonisin contamination.

The effect of storage on mycoflora profile was monitored bimonthly in 36 corn (Zea mays L.) samples, dividing the same sample into groups dried to 11 and 14% moisture content (1008 analysis). These groups were further subdivided based on the initial total count (moulds and yeasts) up to 10(4) CFU g(-1) (12 samples, range 1.6 x 10(4) to 9.0 x 10(4), mean 3.8 x 10(4) CFU g(-1)) and up to 10(5) CFU g(-1) (24 samples, range 1.0 x 10(5) to 5.0 x 10(5), mean 2.7 x 10(5) CFU g(-1)). In the corn group dried to 11%, the fumonisin content was analysed at the initial stage (freshly harvested) and at the end of 12-month storage. Fusarium spp. and Penicillium spp. prevailed at the freshly harvested stage (100%), maintaining this profile throughout 12 months, in corn dried to both 11 and 14%. Cladosporium spp., Aspergillus spp. and Phoma spp. were also detected at lower frequencies during the storage. Fusarium spp. and the total fungal colony count during 12-month storage carried out with samples dried to 11 or 14% moisture content were statistically evaluated using ANOVA for randomized complete block design. The correlation between storage time and Fusarium spp. and total fungal colony count data was analysed by Pearson's correlation test. There was no difference in Fusarium spp. and total counts in the 10(4) CFU g(-1) initial total count group throughout the storage time (p < 0.05). There was a negative correlation between fungal population and storage time (p < 0.05) in the 10(5) CFU g(-1) initial total count group. Fumonisins were detected in all freshly harvested corn, at a mean concentration of 9.9 +/- 6.0 micro g g(-1) (range 0.74-22.6 micro g g(-)1). These values did not change in the 12-month stored corn (mean of 9.9 +/- 5.8 micro g g(-1), range 0.81-23.7 micro g g(-1)). These post harvest data indicated the importance of moisture content at the crop harvesting/predrying stage to control fungal growth and further fumonisin production.

Cladosporium carrionii and Hormoconis resinae (C. resinae): cell wall and melanin studies.

Two phaeoid strains of the fungus Cladosporium carrionii (SR3 from a xerophyte species and PP8201 from a patient), and one strain of Hormoconis resinae (Cladosporium resinae), isolated from oil-impregnated soil, were analyzed for their cell wall composition by colorimetric methods, X-ray diffraction, infrared spectroscopy, and solid-state 13C-nuclear magnetic resonance. Results suggested that the cell walls were composed mainly of hexoses (34%-47%) as beta-1,3-glucan (some galactose and mannose were also present) and melanin, chitin being absent. Electron microscopic observations suggested that melanin was found not only in the cell wall but also in intracellular bodies resembling melanosomes.