Biotransformation of a tetrahydrofuran lignan by the endophytic fungus Phomopsis Sp.

The biotrasformation of the tetrahydrofuran lignan, (-)-grandisin, by the endophitic fungus Phomopsis sp, obtained from Viguiera arenaria, led to the formation of a new compound determined as 3,4-dimethyl-2-(4'-hydroxy-3',5'-dimethoxyphenyl)-5-methoxy-tetrahydrofuran. The metabolite was evaluated against the parasite Trypanosoma cruzi, the causative agent of Chagas's disease, and showed a trypanocidal activity (IC50 9.8 μmol L-1) similar to the natural precursor (IC50 3.7 μmol L-1).A biotransformação da lignana tetraidrofurânica, (-)-grandisina, pelo fungo endofítico Phomopsis sp, obtido de Viguiera arenaria, conduziu à formação de um novo metabólito caracterizado como 3,4-dimetil-2-(4'-hidróxi-3',5'-dimetóxifenil)-5-metóxi-tetraidrofurano. O metabólito foi analisado contra o parasita Trypanosoma cruzi, o agente causador da doença de Chagas, e mostrou uma atividade tripanocida (IC50 9,8 μmol L-1) similar ao precursor natural (IC50 3,7 μmol L-1).Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Microbial transformation of the sesquiterpene lactone tagitinin C by the fungus Aspergillus terreus

The biotransformation of the sesquiterpene lactone tagitinin C by the fungus Aspergillus terreus MT 5.3 yielded a rare derivative that was elucidated by spectrometric methods. The fungus led to the formation of a different product through an unusual epoxidation reaction between C4 and C5, formation of a C3,C10 ether bridge, and a methoxylation of the C1 of tagitinin C. The chemical structure of the product, namely 1 beta-methoxy-3 alpha-hydroxy-3,10 beta-4,5 alpha-diepoxy-8 beta-isobutyroyloxygermacr-11(13)-en-6 alpha,12-olide, is the same as that of a derivative that was recently isolated from the flowers of a Brazilian population of Mexican sunflower (Tithonia diversifolia), which is the source of the substrate tagitinin C. The in vitro cytotoxic activity of the substrate and the biotransformed product were evaluated in HL-60 cells using an MTT assay, and both compounds were found to be cytotoxic. We show that soil fungi may be useful in the biotransformation of sesquiterpene lactones, thereby leading to unusual changes in their chemical structures that may preserve or alter their biological activities, and may also mimic plant biosynthetic pathways for production of secondary metabolites.Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP-Biopro-specta/Biota-process) [04/07935-6]Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq

Phosphatidylinositol phospholipase C mediates carbon sensing and vegetative nuclear duplication rates in Aspergillus nidulans

In this work, we disrupted one of three putative phosphatidylinositol phospholipase C genes of Aspergillus nidulans and studied its effect on carbon source sensing linked to vegetative mitotic nuclear division. We showed that glucose does not affect nuclear division rates during early vegetative conidial germination (6-7 h) in either the wild type or the plcA-deficient mutant. Only after 8 h of cultivation on glucose did the mutant strain present some decrease in nuclear duplication. However, decreased nuclear division rates were observed in the wild type when cultivated in media amended with polypectate, whereas our plcA-deficient mutant did not show slow nuclear duplication rates when grown on this carbon source, even though it requires induction and secretion of multiple pectinolytic enzymes to be metabolized. Thus, plcA appears to be directly linked to high-molecular-weight carbon source sensing.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[2002/11874-7]CAPESFungal Genetics Laborator

Partial purification and characterization of exopolygalacturonase II and III of Penicillium frequentans Purificação parcial e caracterização das exopoligalacturonases II e III de Penicillium frequentans

Previous studies from our laboratory have demonstrated that the fungus Penicillium frequentans produces high levels of polygalacturonase and pectinesterase. Endopolygalacturonase I (Endo-PG I) and Exopolygalacturonase I (Exo-PG I) were previously purified and characterized. In the present study two extracellular polygalacturonases were separated, partially purified and biochemically characterized. Both were characterized as exopolygalacturonases so they were named exopolygalacturonase II (Exo-PG II) and exopolygalacturonase III (Exo-PG III) which had a molecular mass of 63 kDa (Exo-PG II) and 79 kDa (Exo-PG III). The Km values were 1.6 and 0.059 g/L and the Vmax values were 2571 and 185 U/mg, respectively. The optimum temperature was 50ºC for both enzymes, while the optimum pH was 5.0 for Exo-PG II and 5.8 for Exo-PG III.<br>Estudos prévios realizados em nosso laboratório demonstraram que o fungo Penicillium frequentans produz altos níveis de poligalacturonase e pectinesterase. Endopoligalacturonase I (Endo-PG I) e Exopoligalacturonase I (Exo-PG I) já foram previamente purificadas e caracterizadas. No presente trabalho foram separadas, purificadas parcialmente e caracterizadas bioquimicamente duas poligalacturonases extracelulares. Ambas foram caracterizadas como exopoligalacturonases e então foram designadas exopoligalacturonase II (Exo-PG II) e exopoligalacturonase III (Exo-PG III) as quais apresentaram massa molecular de 63 kDa (Exo-PG II) e 79 kDa (Exo-PG III). Os valores de Km foram 1,6 e 0,059 g/L e os de Vmáx = 2571 e 185 U/mg, respectivamente. A temperatura ótima de ambas foi 50ºC e enquanto o pH ótimo da Exo-PG II foi 5,0 o da Exo-PG III foi 5,8

Lipase Production by Endophytic Fungus Cercospora kikuchii: Stability of Enzymatic Activity after Spray Drying in the Presence of Carbohydrates

The present work deals with improving the production and stabilization of lipases from Cercospora kikuchii. Maximum enzyme production (9.384 U/ml) was obtained after 6 days in a medium supplemented with 2% soybean oil. The lipases were spray dried with different adjuvants, and their stability was studied. The residual enzyme activity after drying with 10% (w/v) of lactose, b- cyclodextrin, maltodextrin, mannitol, gum arabic, and trehalose ranged from 63 to 100%. The enzyme activity was lost in the absence of adjuvants. Most of the adjuvants used kept up at least 50% of the enzymatic activity at 5 degrees C and 40% at 25 degrees C after 8 months. The lipase dried with 10% of beta-cyclodextrin retained 72% of activity at 5 degrees C. Lipases were separated by butyl-sepharose column into 4 pools, and pool 4 was partially purified (33.1%; 269.5 U/mg protein). This pool was also spray dried in maltodextrin DE10, and it maintained 100% of activity.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[04/07935-6]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[08/52732-7]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[Proc. 06/50955-3]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[Proc. 08/52732-7

Antifungal Activity against Filamentous Fungi of Ts1, a Multifunctional Toxin from Tityus serrulatus Scorpion Venom

Antimicrobial peptides (AMPs) are ubiquitous and multipotent components of the innate immune defense arsenal used by both prokaryotic and eukaryotic organisms. The search for new AMPs has increased in recent years, due to the growing development of microbial resistance to therapeutical drugs. In this work, we evaluate the effects of Tityus serrulatus venom (Tsv), its fractions and its major toxin Ts1, a beta-neurotoxin, on fungi growth. The fractions were obtained by ion-exchange chromatography of Tsv. The growth inhibition of 11 pathogenic and non-pathogenic filamentous fungi (Aspergillus fumigatus, A. nidulans, A. niger, A. terreus, Neurospora crassa, Penicillium corylophilum, P. ochrochloron, P. verrucosum, P. viridicatum, P. waksmanii, and Talaromyces flavus) was evaluated by quantitative microplate reader assay. Tsv (100 and 500 μg/well, which correspond to 1 and 5 mg/mL, respectively, of total soluble protein) was active in inhibiting growth of A. nidulans, A. terreus, P. corylophilum, and P. verrucosum, especially in the higher concentration used and at the first 30 h. After this period, fungi might have used Tsv components as alternative sources of nutrients, and therefore, increased their growth tax. Only fractions IX, X, XI, XIIA, XIIB (3 and 7.5 μg/well, which correspond to 30 and 75 μg/mL, respectively, of total soluble protein) and Ts1 (1.5, 3, and 6 μg/well, which correspond to 2.18, 4.36, and 8.72 μM, respectively) showed antifungal activity. Ts1 showed to be a non-morphogenic toxin with dose-dependent activity against A. nidulans, inhibiting 100% of fungal growth from 3 μg/well (4.36 μM). The inhibitory effect of Ts1 against A. nidulans growth was accompanied by fungistatic effects and was not amended by 1 mM CaCl2 or tetrodotoxin (46.98 and 93.96 μM). The structural differences between Ts1 and drosomycin, a potent cysteine-rich antifungal peptide, are discussed here. Our results highlight the antifungal potential of the first cysteine-containing scorpion toxin. Since Ts1 is a multifunctional toxin, we suggest that it could be used as a template in the design of engineered scorpion AMPs and in the search for new mechanisms of action of antifungal drugs