Production of thermophilic endo-ß-1,4-xylanases by Aspergillus fumigatus FBSPE-05 using agro-industrial by-products.

In the present paper, endo-ß-1,4-xylanase production by Aspergillus fumigatus was evaluated in solid-state fermentation using low-cost substrates such as sugarcane bagasse (SCB), brewer's spent grain (BSG), and wheat bran (WB). The partial characterization of the crude enzyme was also performed. In the experimental conditions, the highest levels of endo-ß-1,4-xylanase production by A. fumigatus FBSPE-05 occurred within 8 days incubation when using SCB/liquid medium at 1:2 ratio (219.5 U g(-1)) and 4 days incubation when using WB/liquid medium at 1:1 ratio (215.6 U g(-1)). Crude enzyme from this last condition was used to enzyme characterization, showing best enzyme activity at 60 °C and pH 6.0, which suggests a thermophilic endoxylanase. The crude enzyme retained 73% of its activity after 1 h at 60 °C, and zymogram has shown three bands of endo-ß-1,4-xylanase activity, with different molecular masses. A. fumigatus FBSPE-05 was able to grow and produce good levels of endo-ß-1,4-xylanase using agro-industrial by-products, making this strain worthy for further investigation. To our knowledge, this is the first study reporting the use of SCB and/or BSG as sole substrates for endoxylanase production by solid-state fermentation using A. fumigatus.

Brewer's spent grain and corn steep liquor as substrates for cellulolytic enzymes production by Streptomyces malaysiensis.

AIMS: To evaluate cellulase production by Streptomyces malaysiensis in submerged fermentation using brewer's spent grain (BSG) and wheat bran (WB) as carbon source, and corn steep liquor (CSL) as nitrogen source, as compared to yeast extract (YE), and partial characterization of the crude enzyme. METHODS AND RESULTS: Maximum cellulase production by Streptomyces malaysiensis (720 U l(-1)) occurred within 4 days incubation when using a growth medium containing BSG 0.5% (w/v) and CSL1.2% (w/v). CMCases activity showed to be stable over an acidic pH range (2.0-7.0) and in temperatures of 40-60 degrees C. Zymogram indicated three bands of CMCase activity, with different molecular masses. CONCLUSION: S. malaysiensis was able to grow and produce good levels of CMCases using solely brewer's spent grain and corn steep liquor as low-cost substrates, making this strain and these low cost by-product worthy for further investigation, and potentially feasible for biotechnological applications in different areas. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first study reporting the use of the low-cost by-products brewer's spent grain and corn steep liquor, as sole substrates for microbial enzyme production.

Aspergillus fumigatus thermophilic and acidophilic endoglucanases.

This study evaluated the production of cellulolytic enzymes by an Aspergillus fumigatus strain, isolated from sugar cane bagasse, according to its ability to grow on microcrystalline cellulose as the sole carbon source. The effect of the carbon source (brewer's spent grain, sugarcane bagasse, and wheat bran) and of the nitrogen source (corn steep liquor and sodium nitrate) on cellulase production was studied using submerged and solid state cultivations at 30 degrees C. The highest levels of endoglucanase (CMCase) corresponded to 365 U L(-1) and was obtained using sugarcane bagasse (1%) and corn steep liquor (1.2%) in submerged fermentation within 6 days of cultivation. This supernatant was used to run a sodium dodecyl sulfate polyacrylamide gel electrophoresis that showed six bands with endoglucanase activity. CMCase activity was higher at 65 degrees C and pH 2.0, indicating that this microorganism produces a thermophilic and acid endoglucanase. Solid state cultivation favored FPase production, that reached 47 U g(-1) of dry substrate (wheat bran and sugarcane bagasse) within 3 days.

A low-cost fermentation medium for thermophilic protease production by Streptomyces sp. 594 using feather meal and corn steep liquor.

Protease production by Streptomyces sp. 594 was obtained after submerged fermentation (SF) and solid-state fermentation (SSF) using feather meal (FM) and corn steep liquor (CSL) as sole sources of carbon and nitrogen. Enzyme productions were 13.4 U ml(-1) in SF and 21.5 U g(-1) in SSF; these values were approximately 86% and 39% higher, respectively, than those obtained previously when yeast extract was used in place of CSL. The proteases, which belong to the serine and metalloproteinase classes, were active at high temperatures (55 degrees C to 90 degrees C) and over a wide range of pH values (5.0 to 10.0). Thus, these thermophilic proteases have shown interesting properties for industrial purposes. As far as we are concerned, this is the first contribution toward the microbial production of thermophilic proteases by a streptomycete using a low-cost medium composed of industrial poultry (FM) and corn processing by-products (CSL).

Thermophilic protease production by Streptomyces sp. 594 in submerged and solid-state fermentations using feather meal.

AIMS: Protease production by Streptomyces sp. 594 in submerged (SF) and solid-state fermentation (SSF) using feather meal, an industrial poultry residue, and partial characterization of the crude enzyme. METHODS AND RESULTS: Streptomyces sp. 594 produced proteases in SF (7.2 +/- 0.2 U ml(-1)) and SSF (15.5 +/- 0.41 U g(-1)), with pH increase in both media. Considering protease activity, values obtained in the liquid extract after SSF (6.3 +/- 0.17 U ml(-1)) were lower than those from SF. The proteases, which belong to serine and metalloproteinase classes, were active over a wide range of pH (5.0-10.0) and high temperatures (55-80 degrees C). Strain 594 was also able to degrade feather in agar and liquid media. Keratinase activity (80 U l(-1)) also confirmed the keratin degrading capacity of this streptomycete. CONCLUSIONS: Proteases produced using residues from poultry industry have shown interesting properties for industrial purposes. SIGNIFICANCE AND IMPACT OF THE STUDY: As far as we are concerned, this is the first contribution towards the production of thermophilic protease by a streptomycete in SSF using a keratinous waste.

Effect of different carbon sources on endochitinase production by Colletotrichum gloeosporioides.

The present work analyzes the production of endochitinase by Colletotrichum gloeosporioides, a phytopathogenic fungus, using six different carbon sources and two pH values. For quantitative assay of endochitinase activity in solution, the synthetic substrate 4-methylumbelliferyl-beta-D-N,N',N"-triacetylchitotrioside was used. The major productions were obtained at pH 7.0 and 9.0, when colloidal chitin and glucose were used, whereas xylose and lactose were not good carbon sources. When testing different concentrations of colloidal chitin, glucose and glucosamine, colloidal chitin 0.5% was the best substrate, giving values of 2.4 U at the fifth day. When using glucose, best production occurred at 0.3% concentration, after 5 days growth, with values of 1.31 U. Endochitinase production was markedly decreased in high levels of glucose and in all glucosamine concentrations tested. SDS-PAGE co-polymerized with glycol-chitin analysis showed three major activity bands of 200, 100, and 95 kDa, when incubated at 50 degrees C.

Streptomyces drozdowiczii sp. nov., a novel cellulolytic streptomycete from soil in Brazil.

An actinomycete strain, isolated from a Mata Atlântica soil sample, showing cellulolytic activity was subjected to polyphasic taxonomic characterization to determine its identity. Strain M7aT presented morphological and chemotaxonomic characteristics consistent with its assignment to the genus Streptomyces. Phylogenetic analysis of its 16S rDNA sequence revealed that the strain differed from described streptomycetes available in the public databases; the most closely related species was Streptomyces laceyi, with 98.4% nucleotide similarity. It also differed from other cellulolytic strains in its phenotypic characteristics. It is therefore proposed that strain M7aT, a cellulolytic strain with biotechnological potential, represents a novel species, named Streptomyces drozdowiczii sp. nov. The type strain is M7aT (=CBMAI 0498T=CIP 107837T=NRRL B-24297T).

Protein synthesis inhibitory activity in culture filtrates from new strains of Streptomyces isolated from Brazilian tropical soils.

AIMS: To investigate the effect of the culture supernatants from three newly isolated Streptomyces strains, 221, 235 and 606 on eukaryotic cells. METHODS AND RESULTS: Cell lines were treated with the culture filtrates and assayed for protein synthesis by metabolic labelling, followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis. RNA synthesis was investigated by [5-3H]uridine incorporation. The three culture filtrates presented a strong inhibitory activity, reducing total protein synthesis of different eukaryotic cell lines by more than 85%. No effect on cellular RNA synthesis was detected. The culture filtrates did not affect the growth of the prokaryotic cells tested. CONCLUSIONS: These new Streptomyces strains, recently isolated from Brazilian tropical soils, produce molecule(s) with inhibitory activity specific to eukaryote protein synthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: Streptomyces strains 221, 235 and 606, probably representing new species, might produce new bioactive compound(s), and can be used as valuable tools to study the protein synthesis pathway in eukaryotes.

Purification and characterization of an endochitinase produced by Colletotrichum gloeosporioides.

The phytopathogenic fungus Colletotrichum gloeosporioides was analyzed for chitinase activity, the best production occurring at the fourth day. A 43 kDa endochitinase with specific activity of 413 U microg(-1) protein was purified corresponding to a 75% yield. The optima of temperature and pH for the enzyme were 50 degrees C and pH 7.0, respectively. The enzyme showed a high stability at 50 degrees C and pH 7.0. Values of pH from 5.0 up to 7.0 gave, at least, 50% of maximum activity, suggesting a biotechnological application. Further studies are in progress to determine the possible use of this endochitinase in biological control.

Proteases from actinomycetes interfere in solid media plate assays of hyaluronidase activity.

Four hundred and fifteen actinomycete strains were screened for hyaluronidase activity in two plate assays media. In the first one, using hyaluronic acid as substrate and bovine serum albumin (BSA) to help precipitation of the nondegraded substrate, only strain 594 and hyaluronidase control were positive. In the second assay, plates with hyaluronic acid, but not BSA, gave the same results. For plates containing only BSA, proteinase activity was detected in strain 594. When hyaluronic acid was treated with pronase, the only clear zones, in the second assay without BSA, were those around hyaluronidase controls. Protease activity, commonly found in actinomycetes, was detected only in strain 594, among the 415 studied, when tested in hyaluronidase assay using hyaluronate plus BSA. This may be due to the composition of the growth medium, since media with different composition gave different results for protease activity in each of the 15 strains analyzed. These data suggest that proteases can affect an accurate detection of hyaluronidase in media containing proteins, not only from hyaluronate preparations, but also from other medium ingredients. Thus, for a correct interpretation of the method, they must be excluded. Commercial Hyaluronidase used as controls must be also tested for the presence of protease contamination.

Purification of a thermostable endochitinase from Streptomyces RC1071 isolated from a cerrado soil and its antagonism against phytopathogenic fungi.

AIMS: The chitinolytic activity of an actinomycete, isolated from a tropical acidic ferrasol (FAO) under cerrado (savanna) vegetation, is reported. METHODS AND RESULTS: Selection of the strain was based on spot inoculation on solid colloidal chitin medium. The use of chemotaxonomic, morphological and physiological procedures placed it in the Streptomyces genus, but identification to species level could not be achieved. A protein with endochitinase activity was isolated and purified from the supernatant fluid by concentration, precipitation, hydrophobic interaction, gel filtration and adsorption procedures. The molecular size of the purified chitinase was estimated by gel filtration to be 70 kDa, and its pI was 6.1. The enzyme had temperature and pH optima of 40 degrees C and 8.0, respectively, and showed thermal (30-70 degrees C) and pH (4-9) stabilities. Antifungal activity of the selected strain was observed following in vitro experiments using growing cells, crude extract or the purified endochitinase, and by detecting growth inhibition of the tested phytopathogenic fungi. CONCLUSION: Strain Streptomyces RC 1071 could not be placed into any known species, suggesting a new taxon. The purified endochitinase presented similar molecular weight, optimum temperature and pH activity, and stability of other endochitinolytic enzymes reported in the literature. In all three in vitro experiments performed, inhibition of growth of the phytopathogenic fungi used as test organisms was observed. SIGNIFICANCE AND IMPACT OF THE STUDY: Some of the endochitinase characteristics such as thermal stability, as well as pH tolerance, are very interesting for biotechnological purposes. In addition, due to its antifungal activity, Streptomyces RC 1071 seems promising for use in biological control.

Isolation and characterization of actinomycetes from Brazilian tropical soils.

Actinomycetes have been isolated from three Brazilian tropical soils. The dispersion and differential centrifugation procedure revealed count values 1.5 to 5.0 times greater than those obtained by the conventional dilution plate technique for all soils and media tested. Eighteen strains, promising for biotechnological applications, were submitted to chemotaxonomic procedures and numerical taxonomy for identification. Two were identified as Amycolatopsis orientalis, one as Streptomyces misakiensis, and two tentatively included or associated to S. chromofuscus and S. griseoruber. The others, all belonging to the Streptomyces genus, could not be fitted into any known species, and were arranged by the UPGMA analysis for classification, as an isolated group. This suggests that the actinomycetes in tropical soils may represent a vast unexplored resource for biotechnology.

Effects of Angular Leaf Spot and Rust on Yield Loss of Phaseolus vulgaris.

ABSTRACT Three field experiments were conducted in 1997, 1998, and 1999 to investigate the effects of angular leaf spot and rust, separately or combined, on host growth and yield of individual bean plants (Phaseolus vulgaris). In each experiment, three treatments were established by inoculating cv. Carioca with Phaeoisariopsis griseola, Uromyces appendiculatus, or with both pathogens. An additional control treatment was not inoculated, but was sprayed with a fungicide. In the 1997 and 1999 experiments, angular leaf spot reached higher disease levels than rust, whereas in 1998, rust was more severe than angular leaf spot. Host growth, expressed as healthy leaf area duration (HAD), and yield were the highest in 1997 and lowest in 1998. In each experiment, the treatments did not differ significantly to the area under leaf area progress curve, HAD, and healthy leaf area absorption (HAA). All inoculated treatments had significantly more severe disease and less yield than the control treatment. Based on the analysis of 60 plants in each experiment, yield was not related to the areas under disease progress curve for either or both diseases. In 1997 and 1999, yield was related to HAD (R(2) = 0.57 and 0.43) and HAA(R(2) = 0.60 and 0.55). Based on the combined analysis of all 36 plots, angular leaf spot reduced the leaf area because of defoliation, whereas rust did not affect the leaf area. Rust reduced yield more than four times that of angular leaf spot, although the decrease in photosynthesis to angular leaf spot was twice that of rust.

Endocellulase and exocellulase activities of two Streptomyces strains isolated from a forest soil.

Two Streptomyces strains, M7a and M23, from a Brazilian forest soil were evaluated for the cellulase production of their supernatants after growth in a microcrystalline cellulose medium, using carboxymethylcellulose and filter paper as substrates at different temperatures and pH values. Endoglucanase and exoglucanase activities were compared to a commercial Trichoderma reesei cellulase using fluorogenic conjugated substrates. Similar specific activities were observed for the enzyme preparations of strain M23 and T. reesei. For M7a the activities were about seven times higher than those obtained for T. reesei. Extracellular or cell-associated cellobiase activities were not detected in both strains.

Comparative effects of niflumic acid and nifedipine on 5-hydroxytryptamine- and acetylcholine-induced contraction of the rat trachea.

The effects of niflumic acid, an inhibitor of Ca(2+)-activated Cl(-) (Cl((Ca))) channels, were compared with those of the voltage-dependent Ca(2+) channel (VDCC) blocker nifedipine on 5-hydroxytryptamine (5-HT)- and acetylcholine-induced contractions of the rat isolated trachea. Niflumic acid (3-100 microM) induced a concentration-dependent inhibition of 5-HT (10 microM)-induced contractions, with a reduction to 37.0+/-9.5% of the control at the highest concentration. One micromolar nifedipine, which completely blocked 60 mM KCl-induced contractions, reduced the response to 5-HT similarly to 39.2+/-11.5% of the control. The inhibition of the 5-HT response was not significantly different from that produced by the combined presence of nifedipine (1 microM) and niflumic acid (100 microM), suggesting that their effects were not additive. In contrast, neither niflumic acid (3-100 microM) nor nifedipine (1 microM) inhibited acetylcholine-induced contractions. The contraction to 5-HT (10 microM) in Cl(-)-free solution was decreased by more than approximately 85% of the control, whilst that of acetylcholine was reduced only by approximately 36%. Our data show that niflumic acid exerts selective inhibitory effects on 5-HT-induced contraction, and suggest that activation of Cl((Ca)) channels may be a mechanism whereby 5-HT (but not acetylcholine) induces Ca(2+) entry via VDCCs to elicit contraction.

Chitinolytic activity of actinomycetes from a cerrado soil and their potential in biocontrol.

The crude enzyme extracts from five actinomycetes selected from a cerrado soil presented very good endochitinolytic activity when compared to a commercial chitinase. Exochitinase and chitobiase activities were also detected. They were identified as Streptomyces, but could not be characterized to species level, probably corresponding to new ones. The crude extracts, obtained from growth on fungal mycelium plus chitin of three of the strains, have shown a very pronounced activity against phytopathogenic fungi. In tests using growing cells, all five strains were active. These data suggest that these strains are potential biocontrol agents.

Extracellular serine-proteinases isolated from Streptomyces alboniger: partial characterization and effect of aprotinin on cellular structure.

Streptomyces alboniger ATCC 12461 grown in brain heart infusion (BHI) medium produced two extracellular serine-proteinases, denoted SP I and SP II, which were purified by ammonium sulfate precipitation and aprotinin-agarose affinity chromatography. SP I was purified 88,9-fold and SP II 66,7- fold, with 33.4% and 10.4% yield, respectively. The optimum pH for the proteinases activity, using a-N-p-tosyl-L-arginine-methyl ester (TAME) as substrate, was 9-10 and the optimum temperature was 37 degrees C. The proteolytic activity of SP I and SP II was inhibited by aprotinin and SP I was partially inhibited by leupeptin, both serine-proteinase inhibitors. S. alboniger growth in BHI-liquid medium decreased when 5 mg/ml, 10 mg/ml of aprotinin was used, being completely inhibited with 20 mg/ml and 40 mg/ml. At the ultrastructural level, aprotinin-treated S. alboniger cells showed swelling of the bacterial body and condensation of the genetic material, probably related to the inhibition of its growth.