Purification and characterization of the extracellular aspartyl protease APSm1 from the phytopathogen fungus Stenocarpella maydis.

The extracellular protease APSm1 was purified to homogeneity from Stenocarpella maydis that was grown in acidic minimal media with glucose and ammonium sulfate. The purification procedure consisted of ion exchange chromatography coupled to an FPLC (Fast Protein Liquid Chromatography) system, resulting in a 15.3% recovery and a 2.3-fold increase in specific activity. The molecular weight of the purified enzyme was estimated to be 56.8 kDa by SDS-PAGE. Enzymatic activity toward hemoglobin was optimal at pH 2.0 and at 25 °C. The effects of six protease inhibitors on APSm1 activity were tested. Pepstatin A inhibited APSm1 activity, as the protein is in fact an aspartyl protease. The pure enzyme degraded hemoglobin, albumin and proteins obtained from corn germ at pH 3 but did not have any milk-clotting activities. The Km and Vmax values obtained were 0.514 mg/mL and 0.222 µmol/min, respectively, using hemoglobin as the substrate. This work is the first to report the purification of a secreted aspartyl protease from S. maydis.

Biochemical study of the extracellular aspartyl protease Eap1 from the phytopathogen fungus Sporisorium reilianum.

In this work, the extracellular protease Eap1 from Sporisorium reilianum was characterized in solid and liquid cultures using different culture media. The results showed that Eap1 was produced in all media and under all culture conditions, with the most activity in solid culture at an acidic pH of 3-5. Following purification, the 41 kDa protease demonstrated aspartyl protease activity. The enzyme was stable at a wide range of temperatures and pH values, but 45°C and pH 3 were optimal. The K(m) and V(max( values obtained were 0.69 mg/mL and 0.66 µmol/min, respectively, with albumin as the substrate. Eap1 degraded hemoglobin as well as proteins obtained from corn germ, roots, stems and slides at pH 3 and also had milk-clotting activity. Sequencing analysis showed that this protein has 100% similarity to the peptide sequence theoretically obtained from the sr11394 gene, which encodes an aspartyl protease secreted by S. reilianum.

Isolation of bacteria with antifungal activity against the phytopathogenic fungi Stenocarpella maydis and Stenocarpella macrospora.

Stenocarpella maydis and Stenocarpella macrospora are the causal agents of ear rot in corn, which is one of the most destructive diseases in this crop worldwide. These fungi are important mycotoxin producers that cause different pathologies in farmed animals and represent an important risk for humans. In this work, 160 strains were isolated from soil of corn crops of which 10 showed antifungal activity against these phytopathogens, which, were identified as: Bacillus subtilis, Pseudomonas spp., Pseudomonas fluorescens, and Pantoea agglomerans by sequencing of 16S rRNA gene and the phylogenetic analysis. From cultures of each strain, extracellular filtrates were obtained and assayed to determine antifungal activity. The best filtrates were obtained in the stationary phase of B. subtilis cultures that were stable to the temperature and extreme pH values; in addition they did not show a cytotoxicity effect against brine shrimp and inhibited germination of conidia. The bacteria described in this work have the potential to be used in the control of white ear rot disease.

Use of water hyacinth as a substrate for the production of filamentous fungal hydrolytic enzymes in solid-state fermentation.

The objective of the present work was to evaluate the water hyacinth (WH) as a substrate for the production of hydrolytic enzymes (cellulases and hemicellulases) of 100 strains of filamentous fungi under conditions of solid growth. Five fungal strains, identified as Trichoderma harzianum, Trichoderma atroviride, Penicillium griseofulvum, Penicillium commune and Aspergillus versicolor, were selected and studied for their ability to grow on water hyacinth as a substrate and carbon source only, evaluating hydrolytic enzymatic activities (α-l-arabinofuranosidase, cellulase, xylanase and ß-d-xylopyranosidase) and extracellular protein per g of water hyacinth dry matter (gdm). The five strains selected were able to produce the four enzymes studied; however, T. harzianum strain PBCA produces the highest xylanase (149.3 ± 14.3 IU/gdm at 108 h), cellulase (16.4 ± 0.6 IU/gdm at 84 h) and ß-d-xylopyranosidase (127.7 ± 14.8 IU/gdm at 48 h). In contrast, the fungus with the highest α-l-arabinofuranosidase activity was A. versicolor, with 129.8 ± 13.3 IU/gdm after 108 h. In conclusion, T. harzianum showed the best production of the hydrolytic enzymes studied, using as a matrix and carbon source, water hyacinth. In addition, catalytic activities of arabinofuranosidase and xylopyranosidase were reported for the first time in T. versicolor and T. harzianum.

Melanoidin-containing wastewaters induce selective laccase gene expression in the white-rot fungus Trametes sp. I-62.

Wastewaters generated from the production of ethanol from sugar cane molasses may have detrimental effects on the environment due to their high chemical oxygen demand and dark brown color. The color is mainly associated with the presence of melanoidins, which are highly recalcitrant to biodegradation. We report here the induction of laccases by molasses wastewaters and molasses melanoidins in the basidiomycetous fungus Trametes sp. I-62. The time course of effluent decolorization and laccase activity in the culture supernatant of the fungus were correlated. The expression of laccase genes lcc1 and lcc2 increased as a result of the addition of complete molasses wastewater and its high molecular weight fraction to fungal cultures. This is the first time differential laccase gene expression has been reported to occur upon exposure of fungal cultures to molasses wastewaters and their melanoidins.

Comparative analysis of laccase-isozymes patterns of several related Polyporaceae species under different culture conditions.

The study of isozymes has to date been successfully applied in the identification of a number of different microorganisms, particularly fungi. Trametes sp. I-62 (CECT20197) is a white-rot fungus with potential biotechnological applications. It has been identified as Trametes on the basis of the mycelium morphology, but the features observed are not sufficient for its classification at the species level. Given that this strain produces a very characteristic laccase isozyme pattern, in the present work we compared its isozyme profile with the profiles produced by ten other very closely phylogenetically related fungi from the Polyporaceae family. The data indicates that Trametes sp. I-62 could not be included in any of the taxonomically related species which were analysed in the present work. Extracellular laccases were detected in nine of the fungi studied and in four of them namely Trametes subectypus, Trametes pavonia, Trametes ochracea and Trametes gallica, this is the first report of such activity. Given that phylogenetically related fungi growing in the same culture conditions exhibit different laccase patterns, these isozymes could be used as an additional criterion for fungal identification.

Structural close-related aromatic compounds have different effects on laccase activity and on lcc gene expression in the ligninolytic fungus Trametes sp. I-62.

Nine phenolic compounds (p-coumaric acid, ferulic acid, guaiacol, syringol, p-methoxyphenol, pyrocatechol, phloroglucinol, 3,5-dihydroxybenzoic acid, and syringaldazine) were tested for their ability to increase laccase production in the ligninolytic basidiomycete Trametes sp. I-62. All these compounds resulted in increases in laccase activity, with the highest levels being detected in the presence of p-coumaric acid (273-fold) and guaiacol (73-fold). The three laccase isozyme genes in this fungus lcc1, lcc2, and lcc3 are differentially expressed in the presence of some of these aromatics with total lcc transcript levels differing markedly depending on the aromatic compound tested. Guaiacol (the best inducer of lcc gene transcription) and p-coumaric acid selectively induced expression of lcc1 and lcc2; ferulic acid induced lcc3 expression, while 3,5-dihydroxybenzoic acid had no marked effect on laccase gene transcription. The results demonstrate that close-related aromatic compounds appear to have different effects on both laccase activity levels and lcc gene expression in this basidiomycete.