Production of a xylose-stimulated beta-glucosidase and a cellulase-free thermostable xylanase by the thermophilic fungus Humicola brevis var. thermoidea under solid state fermentation

Humicola brevis var. thermoidea cultivated under solid state fermentation in wheat bran and water (1:2 w/v) was a good producer of beta-glucosidase and xylanase. After optimization using response surface methodology the level of xylanase reached 5,791.2 +/- A 411.2 U g(-1), while beta-glucosidase production was increased about 2.6-fold, reaching 20.7 +/- A 1.5 U g(-1). Cellulase levels were negligible. Biochemical characterization of H. brevis beta-glucosidase and xylanase activities showed that they were stable in a wide pH range. Optimum pH for beta-glucosidase and xylanase activities were 5.0 and 5.5, respectively, but the xylanase showed 80 % of maximal activity when assayed at pH 8.0. Both enzymes presented high thermal stability. The beta-glucosidase maintained about 95 % of its activity after 26 h in water at 55 A degrees C, with half-lives of 15.7 h at 60 A degrees C and 5.1 h at 65 A degrees C. The presence of xylose during heat treatment at 65 A degrees C protected beta-glucosidase against thermal inactivation. Xylanase maintained about 80 % of its activity after 200 h in water at 60 A degrees C. Xylose stimulated beta-glucosidase activity up to 1.7-fold, at 200 mmol L-1. The notable features of both xylanase and beta-glucosidase suggest that H. brevis crude culture extract may be useful to compose efficient enzymatic cocktails for lignocellulosic materials treatment or paper pulp biobleaching.

Induction of cellulase and hemicellulase activities of Thermoascus aurantiacus by xylan hydrolyzed products

Thermoascus aurantiacus is able to secrete most of the hemicellulolytic and cellulolytic enzymes. To establish the xylanase inducers of T. aurantiacus, the mycelia were first grown on glucose up until the end of the exponential growth phase, followed by washing and re-suspension in a basal medium without a carbon source. Pre-weighed amounts of xylose (final concentration of 3.5 mg/ml), xylobiose (7 mg/ml) and hydrolyzed xylan from sugarcane bagasse (HXSB) which contained xylose, xylobiose and xylotriose (6.8 mg/ml) were evaluated as inducers of xylanase. It was observed that xylose did not suppress enzyme induction of T. aurantiacus when used in low concentrations, regardless of whether it was inoculated with xylobiose. Xylobiose promoted fast enzyme production stopping after 10 h, even at a low consumption rate of the carbon source; therefore xylobiose appears to be the natural inducer of xylanase. In HXSB only a negligible xylanase activity was determined. Xylose present in HXSB was consumed within the first 10 h while xylobiose was partially hydrolyzed at a slow rate. The profile of alpha-arabinofuranosidase induction was very similar in media induced with xylobiose or HXSB, but induction with xylose showed some positive effects as well. The production profile for the xylanase was accompanied by low levels of cellulolytic activity. In comparison, growth in HXSB resulted in different profiles of both xylanase and cellulase production, excluding the possibility of xylanase acting as endoglucanases.

delta-Lactam derivative from thermophilic soil fungus exhibits in vitro anti-allergic activity

From cultures of thermophilic soil fungus Humicola grisea var thermoidea, a delta-lactam derivative (3-(2-(4-hydroxyphenyl)-2-oxoethyl)-5,6-dihydropyridin-2( 1H)-one) that displayed anti-allergic activity was isolated, which was predicted by in silico computational chemistry approaches. The in vitro anti-allergic activity was investigated by beta-hexosaminidase release assay in rat basophilic leukaemia RBL-2H3 cells. The delta-lactam derivative exhibited similar anti-allergic activity (IC50 = 18.7 +/- 6.7 mu M) in comparison with ketotifen fumarate (IC50 = 15.0 +/- 1.3 mu M) and stronger anti-allergic activity than azelastine (IC50 = 32.0 mu M). Also, the MTT cytotoxicity assay with RBL-2H3 cells showed that delta-lactam does not display cytotoxicity at concentrations lower than 50 mu M. This study suggests that the delta-lactam derivative has the potential to be used as a lead compound in the development of anti-allergic drugs for clinical use in humans.

Revision of the fungus-growing ant genera Mycetophylax Emery and Paramycetophylax Kusnezov rev. stat., and description of Kalathomyrmex n. gen. (Formicidae: Myrmicinae: Attini)

Based on the morphology of workers, gynes and males, we revise the taxonomy of nominal taxa traditionally included by authors in the fungus-growing ant genus Mycetophylax. Our results indicate that Mycetophylax Emery (Myrmicocrypta brittoni Wheeler, 1907, type species, by designation of Emery, 1913; junior synonym of Cyphomyrmex conformis Mayr, 1884 by Kempf, 1962) includes M. conformis, M. simplex (Emery, 1888), and M. morschi (Emery, 1888) new combination (formerly in Cyphomyrmex), with several synonymies. Mycetophylax bruchi (Santschi, 1916) does not belong to the same genus and is diagnosed, in addition to other characters, by a psammophore arising at the anterior margin of the clypeus. For this species we are resurrecting from synonymy Paramycetophylax Kusnezov, 1956 (Mycetophylax bruchi as type species, by original designation, with M. cristulatus as its new synonym). Myrmicocrypta emeryi Forel, 1907 is the only attine in which females lack the median clypeal seta and have the antennal insertion areas very much enlarged and anteriorly produced, with the psammophore setae arising from the middle of the clypeus and not at its anterior margin as in Paramycetophylax. Notwithstanding its inclusion in Mycetophylax by recent authors, it is here recognized as belonging to a hitherto undescribed, thus far monotypic genus, Kalathomyrmex new genus (Myrmicocrypta emeryi as its type species, here designated). We redescribe workers, gynes and males of all species in the three genera and describe for the first time gynes of Mycetophylax conformis and M. simplex, males of M. simplex and M. morschi, and gynes of P. bruchi. Furthermore we present a key to the workers of the taxa treated here (most formerly included under the name Mycetophylax), a key to workers of the Mycetophylax in the revised sense, SEM pictures and high resolution AutoMontage(C) photographs of the species, along with maps of collection records and a summary of biological observations.

Parabiosis between basal fungus-growing ants (Formicidae, Attini)

We describe the first observation of parabiosis between two Attini ants (Apterostigma urichii Forel and Cyphomyrmex faunulus Wheeler) found in northern Manaus, AM, Brazil. Complete, mature colonies of both species were found in a single cavity inside a rotten log, sharing and tending a single combined fungus garden, made up of two distinct halves, each cultivated by one species. Workers of one species often antennated workers of the other species and showed no aggression toward each other or toward each other`s workers, queens, or immatures. Laboratory observations suggest that immatures of both species feed on hyphae from either half of the fungus garden. We were not able to find other parabiotic pairs involving the same species in the same locality, although we found colonies of both species sharing trails and foraging territories.

Genetic transformation of Diaporthe phaseolorum, an endophytic fungus found in mangrove forests, mediated by Agrobacterium tumefaciens

We describe the genetic transformation of the mycelial tissue of Diaporthe phaseolorum, an endophytic fungus isolated from the mangrove species Laguncularia racemosa, using Agrobacterium tumefaciens-mediated transformation (ATMT). ATMT uses both the hygromycin B resistant (hph) gene and green fluorescent protein as the selection agents. The T-DNA integration into the fungal genome was assessed by both PCR and Southern blotting. All transformants examined were mitotically stable. An analysis of the T-DNA flanking sequences by thermal asymmetric interlaced PCR (TAIL-PCR) demonstrated that the disrupted genes in the transformants had similarities with conserved domains in proteins involved in antibiotic biosynthesis pathways. A library of 520 transformants was generated, and 31 of these transformants had no antibiotic activity against Staphylococcus aureus, an important human pathogen. The protocol described here, using ATMT in D. phaseolorum, will be useful for the identification and analysis of fungal genes controlling pathogenicity and antibiotic pathways. Moreover, this protocol may be used as a reference for other species in the Diaporthe genus. This is the first report to describe Agrobacterium-mediated transformation of D. phaseolorum as a tool for insertional mutagenesis.

Antibacterial compound from the endophytic fungus Phomopsis longicolla isolated from the tropical red seaweed Bostrychia radicans

Endophytic fungi isolated from the red seaweed Bostrychia radicans were studied to identify their molecularly diverse and biologically active natural chemical products. According to 28S ribosomal DNA-based identification, the strain named C81 was 98% identical to Phomopsis longicolla. This strain was cultivated in solid rice medium and produced three major metabolites identified as 18-deoxycytochalasin H (1), mycophenolic acid (2), and dicerandrol C (3). The chemical structures of these compounds were elucidated by 1D and 2D nuclear magnetic resonance as well as by mass spectrometry. Dicerandrol C had significant antimicrobial activity against Staphylococcus aureus (ATCC 6538) and Staphylococcus saprophyticus (ATCC 15305), with minimum inhibitory concentrations of 1 and 2 mu g ml(-1) (1.33 and 2.66 mu M), respectively. These results show the presence of promising metabolites and indicate that these natural products should be considered in the development of new antibiotics.

USE OF ADDITIVES IN THE WOOD BIODEGRADATION BY THE FUNGUS Ceriporiopsis subvermispora: EFFECT IN THE MANGANESE PEROXIDASE-DEPENDENT LIPID PEROXIDATION

USE OF ADDITIVES IN THE WOOD BIODEGRADATION BY THE FUNGUS Ceriporiopsis subvermispora: EFFECT IN THE MANGANESE PEROXIDASE-DEPENDENT LIPID PEROXIDATION. Ceriporiopsis subvermispora is a selective fungus in the wood delignification and the most promising in biopulping. Through the lipid peroxidation initiated by manganese peroxidase (MnP), free radicals can be generated, which can act in the degradation of lignin nonphenolic structures. This work evaluated the prooxidant activity (based in lipid peroxidation) of enzymatic extracts from wood biodegradation by this fungus in cultures containing exogenous calcium, oxalic acid or soybean oil. It was observed that MnP significant activity is required to promote lipid peroxidation and wood delignification. Positive correlation between prooxidant activity x MnP was observed up to 300 IU kg(-1) of wood.

Amycolatopsis thermophila sp nov and Amycolatopsis viridis sp nov., thermophilic actinomycetes isolated from arid soil

The taxonomic positions of two thermophilic actinomycetes isolated from an arid Australian soil sample were established based on an investigation using a polyphasic taxonomic approach. The organisms had chemical and morphological properties typical of members of the genus Amycolatopsis and formed distinct phyletic lines in the Amycolatopsis methanolica 16S rRNA subclade. The two organisms were distinguished from one another and from the type strains of related species of the genus Amycolatopsis using a range of phenotypic properties. Based on the combined genotypic and phenotypic data, it is proposed that the two isolates be classified in the genus Amycolatopsis as Amycolatopsis thermophila sp. nov. (type strain GY088(T)=NCIMB 14699(T)=NRRL B-24836(T)) and Amycolatopsis viridis sp. nov. (type strain GY115(T)=NCIMB 14700(T)= NRRL B-24837(T)).

Meroterpenes isolated from the endophytic fungus Guignardia mangiferae

Two new guignardones and one tricycloalternarene derivatives, named guignardone D, E (2-3) and tricycloalternarene F (4), and the known guignardone A (1) were isolated from Guignardia mangiferae, an endophytic fungus from the leaves of Viguiera arenaria (Asteraceae), after fermentation in Czapek medium. Structures were established on the basis of their spectroscopic data, including H-1 NMR, C-13 NMR, HMQC, HMBC and HRESI-MS. (C) 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

Amycolatopsis granulosa sp nov., Amycolatopsis ruanii sp nov and Amycolatopsis thermalba sp nov., thermophilic actinomycetes isolated from arid soils

The taxonomic positions of three thermophilic actinomycetes isolated from arid soil samples were established by using a polyphasic approach. The organisms had chemical and morphological features that were consistent with their classification in the genus Amycolatopsis. 16S rRNA gene sequence data supported the classification of the isolates in the genus Amycolatopsis and showed that they formed distinct branches in the Amycolatopsis methanolica subclade. DNA-DNA relatedness studies between the isolates and their phylogenetic neighbours showed that they belonged to distinct genomic species. The three isolates were readily distinguished from one another and from the type strains of species classified in the A. methanolica subclade based on a combination of phenotypic properties and by genomic fingerprinting. Consequently, it is proposed that the three isolates be classified in the genus Amycolatopsis as representatives of Amycolatopsis granulosa sp. nov. (type strain GY307(T)=NCIMB 14709(T)=NRRL B-24844(T)), Amycolatopsis ruanii sp. nov. (type strain NMG112(T)=NCIMB 14711(T)=NRRL B-24848(T)) and Amycolatopsis thermalba sp. nov. (type strain SF45(T)=NCIMB 14705(T)=NRRL B-24845(T)).

Conformational stability of recombinant manganese superoxide dismutase from the filamentous fungus Trichoderma reesei

Superoxide dismutases (SODS; EC 1.15.1.1) are part of the antioxidant system of aerobic organisms and are used as a defense against oxidative injury caused by reactive oxygen species (ROS). The cloning and sequencing of the 788-bp genomic DNA from Trichoderma reesei strain QM9414 (anamorph of Hypocrea jecorina) revealed an open reading frame encoding a protein of 212 amino acid residues, with 65-90% similarity to manganese superoxide dismutase from other filamentous fungi. The TrMnSOD was purified and shown to be stable from 20 to 90 degrees C for 1 h at pH from 8 to 11.5, while maintaining its biological activity. (C) 2011 Elsevier B.V. All rights reserved.

Influence of caffeine on the survival of leaf-cutting ants Atta sexdens rubropilosa and in vitro growth of their mutualistic fungus

BACKGROUND: Leaf-cutting ants collect plant fresh material for the cultivation of their mutualistic fungus. Atta sexdens rubropilosa Forel (Hymenoptera: Formicidae) cause great economic losses through their foraging activity, mainly in agriculture. The main control method is the application of granulated toxic baits incorporated with an active ingredient (AI). The present goal is to evaluate the effect of caffeine on in vitro growth of the mutualistic fungus and on the survival of the leaf-cutting ants, aiming to verify the potential toxicity of this secondary metabolite over these organisms. RESULTS: Three distinct patterns of fungal growth correlated with caffeine concentration were observed: (1) no effect (0.01% caffeine); (2) intermediate growth reduction (0.05% caffeine); (3) drastic growth reduction (0.10 and 0.50% caffeine). The highest caffeine concentration causes fungus death in the first week. As for insect survival, caffeine does not seem to exert any effect. The treatments with diet containing caffeine showed similar values of M50, irrespective of caffeine concentration. CONCLUSION: As caffeine was shown to reduce growth of the mutualistic fungus of Atta sexdens rubropilosa, but with no conclusive effect on insect survival, a hypothetical explanation for the selection of different Coffea species by this leaf-cutting ant species might be associated with caffeine toxicity to the fungus. Copyright (C) 2011 Society of Chemical Industry

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.

Effects of exogenous calcium or oxalic acid on Pinus taeda treatment with the white-rot fungus Ceriporiopsis subvermispora

Pinus taeda wood chips were treated with the biopulping fungus Ceriporiopsis subvermispora in calcium-or oxalic acid-amended cultures. The secretion of hydrolytic and oxidative enzymes was inhibited only in the cultures having the highest concentration of calcium (1400 mg kg(-1) wood). Calcium decreased the availability of free oxalic acid, inhibited fungal growth, and reduced lignin mineralization and transformations. Oxalic acid amendment in the cultures was found not to affect the lignin mineralization and transformations; however, it did inhibit the depolymerization reactions detectable in the residual lignin that was retained in the biotreated wood. C. subvermispora presented catabolic activity for oxalic acid in the cultures amended with 1660 mg acid kg(-1) wood, whereas oxalic acid was synthesized when it was amended at low amounts or initially absent in the cultures. These data suggest one ideal ratio of oxalic acid in C. subvermispora cultures and indicate that its exogenous addition does not necessarily accompany the further degradation of lignin. Crown Copyright (C) 2012 Published by Elsevier Ltd. All rights reserved.