Biosynthesis and uptake of copper nanoparticles by dead biomass of Hypocrea lixii isolated from the metal mine in the Brazilian Amazon Region

A biological system for the biosynthesis of nanoparticles (NPs) and uptake of copper from wastewater, using dead biomass of Hypocrea lixii was analyzed and described for the first time. The equilibrium and kinetics investigation of the biosorption of copper onto dead, dried and live biomass of fungus were performed as a function of initial metal concentration, pH, temperature, agitation and inoculum volume. The high biosorption capacity was observed for dead biomass, completed within 60 min of contact, at pH 5.0, temperature of 40 °C and agitation speed of 150 rpm with a maximum copper biosorption of 19.0 mg g(-1). The equilibrium data were better described using the Langmuir isotherm and kinetic analysis indicated that copper biosorption follows a pseudo-second-order model. The average size, morphology and location of NPs biosynthesized by the fungus were determined by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS) and transmission electron microscopy (TEM). NPs were mainly spherical, with an average size of 24.5 nm, and were synthesized extracellularly. The X-ray diffraction (XRD) analysis confirms the presence of metallic copper particles. Infrared spectroscopy (FTIR) study revealed that the amide groups interact with the particles, which was accountable for the stability of NPs. This method further confirmed the presence of proteins as stabilizing and capping agents surrounding the copper NPs. These studies demonstrate that dead biomass of Hypocrea lixii provides an economic and technically feasible option for bioremediation of wastewater and is a potential candidate for industrial-scale production of copper NPs.

Comparison of β-1,3-glucanase production by Botryosphaeria rhodina MAMB-05 and Trichoderma harzianum Rifai and its optimization using a statistical mixture-design

Botryosphaeria rhodina MAMB-05 produced β-1,3-glucanases and botryosphaeran when grown on glucose, while Trichoderma harzianum Rifai only produced the enzyme. A comparison of long-term cultivation (300h) by B. rhodina demonstrated a correlation between the formation of botryosphaeran (48h) and its consumption (after 108h), and de-repression of β-1,3-glucanase synthesis when glucose was depleted from the nutrient medium, whereas for T. harzianum enzyme production commenced during exponential growth. Growth profiles and levels of β-1,3-glucanases produced by both fungi on botryosphaeran also differed, as well as the production of β-1,3-glucanases and β-1,6-glucanases on glucose, lactose, laminarin, botryosphaeran, lasiodiplodan, curdlan, Brewer's yeast powder and lyophilized fungal mycelium, which were dependent upon the carbon source used. A statistical mixture-design used to optimize β-1,3-glucanase production by both fungi evaluated botryosphaeran, glucose and lactose concentrations as variables. For B. rhodina, glucose and lactose promoted enzyme production at the same levels (2.30UmL -1), whereas botryosphaeran added to these substrates exerted a synergic effect favorable for β-glucanase production by T. harzianum (4.25UmL -1). © 2010 Elsevier B.V.

Agaricus blazei as a Substrate for the Production of β-1,3-Glucanase by Trichoderma harzianum Rifai

Extracellular β-1,3-glucanase was produced by Trichoderma harzianum Rifai cultivated in the Agaricus blazei (Agaricus brasiliensis) extract as a substrate in submerged fermentation. A 22-central composite factorial design was developed using the time of culture (x1/day) and Agaricus blazei extract concentration (x2/(g/L)) as variables, and the results were analyzed using response surface methodology (RSM). The results showed that the Agaricus blazei extract concentration was the most important variable in the production of β-1,3-glucanase, and the maximum β-1,3-glucanase activity (0.77 U/mL) was obtained in one day of cultivation. The β-glucan present in the cell wall of Agaricus blazei mushroom proved to be a good substrate for inducing the production of specific β-1,3-glucanase by Trichoderma harzianum Rifai.

Xylo-oligosaccharides from lignocellulosic materials: Chemical structure, health benefits and production by chemical and enzymatic hydrolysis

Currently, there is worldwide interest in the technological use of agro-industrial residues as a renewable source of food and biofuels. Lignocellulosic materials (LCMs) are a rich source of cellulose and hemicellulose. Hemicellulose is rich in xylan, a polysaccharide used to develop technology for producing alcohol, xylose, xylitol and xylo-oligosaccharides (XOSs). The XOSs are unusual oligosaccharides whose main constituent is xylose linked by β 1-4 bonds. The XOS applications described in this paper highlight that they are considered soluble dietary fibers that have prebiotic activity, favoring the improvement of bowel functions and immune function and having antimicrobial and other health benefits. These effects open a new perspective on potential applications for animal production and human consumption. The raw materials that are rich in hemicellulose include sugar cane bagasse, corncobs, rice husks, olive pits, barley straw, tobacco stalk, cotton stalk, sunflower stalk and wheat straw. The XOS-yielding treatments that have been studied include acid hydrolysis, alkaline hydrolysis, auto-hydrolysis and enzymatic hydrolysis, but the breaking of bonds present in these compounds is relatively difficult and costly, thus limiting the production of XOS. To obviate this limitation, a thorough evaluation of the most convenient methods and the opportunities for innovation in this area is needed. Another challenge is the screening and taxonomy of microorganisms that produce the xylanolytic complex and enzymes and reaction mechanisms involved. Among the standing out microorganisms involved in lignocellulose degradation are Trichoderma harzianum, Cellulosimicrobium cellulans, Penicillium janczewskii, Penicillium echinulatu, Trichoderma reesei and Aspergillus awamori. The enzyme complex predominantly comprises endoxylanase and enzymes that remove hemicellulose side groups such as the acetyl group. The complex has low β-xylosidase activities because β-xylosidase stimulates the production of xylose instead of XOS; xylose, in turn, inhibits the enzymes that produce XOS. The enzymatic conversion of xylan in XOS is the preferred route for the food industries because of problems associated with chemical technologies (e.g., acid hydrolysis) due to the release of toxic and undesired products, such as furfural. The improvement of the bioprocess for XOS production and its benefits for several applications are discussed in this study. © 2012 Elsevier Ltd.

Comparing submerged and solid-state fermentation of agro-industrial residues for the production and characterization of lipase by Trichoderma harzianum

Lipase production by Trichoderma harzianum was evaluated in submerged fermentation (SF) and solid-state fermentation (SSF) using a variety of agro-industrial residues. Cultures in SF showed the highest activity (1.4 U/mL) in medium containing 0.5 % (w/v) yeast extract, 1 % (v/v) olive oil and 2.5 C:N ratio. This paper is the first to report lipase production by T. harzianum in SSF. A 1:2 mixture of castor oil cake and sugarcane bagasse supplemented with 1 % (v/w) olive oil showed the best results among the cultures in SSF (4 U/g ds). Lipolytic activity was stable in a slightly acidic to neutral pH, maintaining 50 % activity after 30 min at 50 C. Eighty percent of the activity remained after 1 h in 25 % (v/v) methanol, ethanol, isopropanol or acetone. Activity was observed with vegetable oils (olive, soybean, corn and sunflower) and long-chain triacylglycerols (triolein), confirming the presence of a true lipase. The results of this study are promising because they demonstrate an enzyme with interesting properties for application in catalysis produced by fermentation at low cost. © 2012 Springer-Verlag and the University of Milan.

Effects of endophyte colonization of Vicia faba (Fabaceae) plants on the life

The effects of fungal endophytes Beauveria bassiana isolates ICIPE 279, G1LU3, S4SU1 and Hypocrea lixii isolate F3ST1 on the life-history of Phaedrotoma scabriventris and Diglyphus isaea, parasitoids of the pea leafminer Liriomyza huidobrensis, were studied in the laboratory. Parasitoids were allowed to parasitize 2nd-3rd L. huidobrensis larvae reared on endophytically-inoculated Vicia faba. In the control, parasitoids were reared on non-inoculated host plants. Parasitism, pupation, adult emergence and survival were recorded. No significant difference was observed between the control and the endophyte-inoculated plants in terms of parasitism rates of P. scabriventris (p = 0.68) and D. isaea (p = 0.45) and adult' survival times (p = 0.06). The survival period of the F1 progeny of P. scabriventris was reduced (p < 0.0001) in B. bassiana S4SU1 to 28 days as compared to more than 40 days for B. bassiana G1LU3, ICIPE 279 and H. lixii F3ST1. However, no significant difference (p = 0.54) was observed in the survival times of the F1 progeny of D. isaea. This study has therefore demonstrated the beneficial effects of both endophytes and parasitoids in L. huidobrensis population suppression.

Colonization of Onions by Endophytic Fungi and Their Impacts on the Biology of Thrips tabaci

Endophytic fungi, which live within host plant tissues without causing any visible symptom of infection, are important mutualists that mediate plant-herbivore interactions. Thrips tabaci (Lindeman) is one of the key pests of onion, Allium cepa L., an economically important agricultural crop cultivated worldwide. However, information on endophyte colonization of onions, and their impacts on the biology of thrips feeding on them, is lacking. We tested the colonization of onion plants by selected fungal endophyte isolates using two inoculation methods. The effects of inoculated endophytes on T. tabaci infesting onion were also examined. Seven fungal endophytes used in our study were able to colonize onion plants either by the seed or seedling inoculation methods. Seed inoculation resulted in 1.47 times higher mean percentage post-inoculation recovery of all the endophytes tested as compared to seedling inoculation. Fewer thrips were observed on plants inoculated with Clonostachys rosea ICIPE 707, Trichoderma asperellum M2RT4, Trichoderma atroviride ICIPE 710, Trichoderma harzianum 709, Hypocrea lixii F3ST1 and Fusarium sp. ICIPE 712 isolates as compared to those inoculated with Fusarium sp. ICIPE 717 and the control treatments. Onion plants colonized by C. rosea ICIPE 707, T. asperellum M2RT4, T. atroviride ICIPE 710 and H. lixii F3ST1 had significantly lower feeding punctures as compared to the other treatments. Among the isolates tested, the lowest numbers of eggs were laid by T. tabaci on H. lixii F3ST1 and C. rosea ICIPE 707 inoculated plants. These results extend the knowledge on colonization of onions by fungal endophytes and their effects on Thrips tabaci.

Molecular evidence that the asexual industrial fungus Trichoderma reesei is a clonal derivative of the ascomycete Hypocrea jecorina.

The relationship of the important cellulase producing asexual fungus Trichoderma reesei to its putative teleomorphic (sexual) ancestor Hypocrea jecorina and other species of the Trichoderma sect. Longibrachiatum was studied by PCR-fingerprinting and sequence analyses of the nuclear ribosomal DNA region containing the internal transcribed spacers (ITS-1 and ITS-2) and the 5.8S rRNA gene. The differences in the corresponding ITS sequences allowed a grouping of anamorphic (asexual) species of Trichoderma sect. Longibrachiatum into Trichoderma longibrachiatum, Trichoderma pseudokoningii, and Trichoderma reesei. The sexual species Hypocrea schweinitzii and H. jecorina were also clearly separated from each other. H. jecorina and T. reesei exhibited identical sequences, suggesting close relatedness or even species identity. Intraspecific and interspecific variation in the PCR-fingerprinting patterns supported the differentiation of species based on ITS sequences, the grouping of the strains, and the assignment of these strains to individual species. The variations between T. reesei and H. jecorina were at the same order of magnitude as found between all strains of H. jecorina, but much lower than the observed interspecific variations. Identical ITS sequences and the high similarity of PCR-fingerprinting patterns indicate a very close relationship between T. reesei and H. jecorina, whereas differences of the ITS sequences and the PCR-fingerprinting patterns show a clear phylogenetic distance between T. reesei/H. jecorina and T. longibrachiatum. T. reesei is considered to be an asexual, clonal line derived from a population of the tropical ascomycete H. jecorina.

Computationally-guided thermostabilization of the primary endoglucanase from Hypocrea jecorina for cellulosic biofuel production

The creation of thermostable enzymes has wide-ranging applications in industrial, scientific, and pharmaceutical settings. As various stabilization techniques exist, it is often unclear how to best proceed. To this end, we have redesigned Cel5A (HjCel5A) from Hypocrea jecorina (anamorph Trichoderma reesei) to comparatively evaluate several significantly divergent stabilization methods: 1) consensus design, 2) core repacking, 3) helix dipole stabilization, 4) FoldX ΔΔG approximations, 5) Triad ΔΔG approximations, and 6) entropy reduction through backbone stabilization. As several of these techniques require structural data, we initially solved the first crystal structure of HjCel5A to 2.05 Å. Results from the stabilization experiments demonstrate that consensus design works best at accurately predicting highly stabilizing and active mutations. FoldX and helix dipole stabilization, however, also performed well. Both methods rely on structural data and can reveal non-conserved, structure-dependent mutations with high fidelity. HjCel5A is a prime target for stabilization. Capable of cleaving cellulose strands from agricultural waste into fermentable sugars, this protein functions as the primary endoglucanase in an organism commonly used in the sustainable biofuels industry. Creating a long-lived, highly active thermostable HjCel5A would allow cellulose hydrolysis to proceed more efficiently, lowering production expenses. We employed information gleaned during the survey of stabilization techniques to generate HjCel5A variants demonstrating a 12-15 °C increase in the temperature at which 50% of the total activity persists, an 11-14 °C increase in optimal operating temperature, and a 60% increase over the maximal amount of hydrolysis achievable using the wild type enzyme. We anticipate that our comparative analysis of stabilization methods will prove useful in future thermostabilization experiments.

Engineering enzyme systems by recombination

Homologous recombination is a source of diversity in both natural and directed evolution. Standing genetic variation that has passed the test of natural selection is combined in new ways, generating functional and sometimes unexpected changes. In this work we evaluate the utility of homologous recombination as a protein engineering tool, both in comparison with and combined with other protein engineering techniques, and apply it to an industrially important enzyme: Hypocrea jecorina Cel5a.

Chapter 1 reviews work over the last five years on protein engineering by recombination. Chapter 2 describes the recombination of Hypocrea jecorina Cel5a endoglucanase with homologous enzymes in order to improve its activity at high temperatures. A chimeric Cel5a that is 10.1 °C more stable than wild-type and hydrolyzes 25% more cellulose at elevated temperatures is reported. Chapter 3 describes an investigation into the synergy of thermostable cellulases that have been engineered by recombination and other methods. An engineered endoglucanase and two engineered cellobiohydrolases synergistically hydrolyzed cellulose at high temperatures, releasing over 200% more reducing sugars over 60 h at their optimal mixture relative to the best mixture of wild-type enzymes. These results provide a framework for engineering cellulolytic enzyme mixtures for the industrial conditions of high temperatures and long incubation times.

In addition to this work on recombination, we explored three other problems in protein engineering. Chapter 4 describes an investigation into replacing enzymes with complex cofactors with simple cofactors, using an E. coli enolase as a model system. Chapter 5 describes engineering broad-spectrum aldehyde resistance in Saccharomyces cerevisiae by evolving an alcohol dehydrogenase simultaneously for activity and promiscuity. Chapter 6 describes an attempt to engineer gene-targeted hypermutagenesis into E. coli to facilitate continuous in vivo selection systems.

Descrição da diversidade de fungos associados a formigas Attini por método independente de cultivo

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Trichoderma harzianum expressed sequence tags for identification of genes with putative roles in mycoparasitism against Fusarium solani

The plant pathogen Fusarium solani causes a disease root rot of common bean (Phaseolus vulgaris) resulting in great losses of yield in irrigated areas of the Southeast and Midwest regions of Brazil. Species of the genus Trichoderma have been used in the biological control of this pathogen as an alternative to chemical control. To gain new insights into the biocontrol mechanism used by Trichoderma harzianum against the phytopathogenic fungus, Fusarium solani, we performed a transcriptome analysis using expressed sequence tags (ESTs) and quantitative real-time PCR (RT-qPCR) approaches. A cDNA library from T. harzianum mycelium (isolate ALL42) grown on cell walls of F. solani (CWFS) was constructed and analyzed. A total of 2927 high quality sequences were selected from 3845 and 37.7% were identified as unique genes. The Gene Ontology analysis revealed that the majority of the annotated genes are involved in metabolic processes (80.9%), followed by cellular process (73.7%). We tested twenty genes that encode proteins with potential role in biological control. RT-qPCR analysis showed that none of these genes were expressed when T. harzianum was challenged with itself. These genes showed different patterns of expression during in vitro interaction between T. harzianum and F. solani. (C) 2012 Elsevier Inc. All rights reserved.

The fungal metabolite eugenitin as additive for Aspergillus niveus glucoamylase activation

Endophytic microorganisms live inside tissues of host plants apparently do not causing warning to them, and area promising source of bioactive molecules as antimicrobial and antitumoral drugs. In this work, we report the isolation of eugenitin from cultures of the endophyte Mycoleptodiscus indicus and its potential as additive for Aspergillus niveus glucoamylase activation. The glucoamylase hydrolytic activity increased twofold using 5 mM of eugenitin and this activation could be explained by the binding mode of eugenitin with the three-dimensional structure of glucoamylase. The in silica prediction of ligand binding sites revealed at least 9 possible interaction sites able to accommodate eugenitin on glucoamylase from Hypocrea jecorina. Besides, we evaluated the effect of pH and temperature on activity and stability, as well as in the hydrolysis of different substrates and kinetic parameters either in presence or absence of eugenitin. The results displayed by eugenitin as additive to glucoamylase activation are promising and provide novel perspectives for applications of fungal metabolites. (C) 2011 Elsevier B.V. All rights reserved.

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.