Avaliação da atividade fungicida de óleos essenciais e suas substâncias ativas no controlo de fungos de armazenamento

Mestrado em Engenharia Agronómica - Proteção da Plantas - Instituto Superior de Agronomia - UL

Enzyme Optimization for Lignocellulose Hydrolysis using Mechanistic Modeling

A novel mechanistic model for the saccharification of cellulose and hemicellulose is utilized to predict the products of hydrolysis over a range of enzyme loadings and times. The mechanistic model considers the morphology of the substrate and the kinetics of enzymes to optimize enzyme concentrations for the enzymatic hydrolysis of cellulose and hemicellulose simultaneously. Substrates are modeled based on their fraction of accessible sites, glucan content, xylan content, and degree of polymerizations. This enzyme optimization model takes into account the kinetics of six core enzymes for lignocellulose hydrolysis: endoglucanase I (EG1), cellobiohydrolase I (CBH1), cellobiohydrolase II (CBH2), and endo-xylanase (EX) from Trichoderma reesei; β-glucosidase (BG), and β-xylosidase (BX) from Aspergillus niger. The model employs the synergistic action of these enzymes to predict optimum enzyme concentrations for hydrolysis of Avicel and ammonia fiber explosion (AFEX) pretreated corn stover. Glucan, glucan + xylan, glucose and glucose + xylose conversion predictions are given over a range of mass fractions of enzymes, and a range of enzyme loadings. Simulation results are compared with optimizations using statistically designed experiments. BG and BX are modeled in solution at later time points to predict the effect on glucose conversion and xylose conversion.

DETECTION OF SPORES AND HYPHAE OF ARTWORKS’ BIODETERIOGENIC FILAMENTOUS FUNGI BY RNA-FISH

Filamentous fungi are a threat to the conservation of Cultural Heritage. Thus, detection and identification of viable filamentous fungi are crucial for applying adequate Safeguard measures. RNA-FISH protocols have been previously applied with this aim in Cultural Heritage samples. However, only hyphae detection was reported in the literature, even if spores and conidia are not only a potential risk to Cultural Heritage but can also be harmful for the health of visitors, curators and restorers. Thus, the aim of this work was to evaluate various permeabilizing strategies for their application in the detection of spores/conidia and hyphae of artworks’ biodeteriogenic filamentous fungi by RNA-FISH. Besides of this, the influence of cell aging on the success of the technique and on the development of fungal autofluorescence (that could hamper the RNA-FISH signal detection) were also investigated. Five common biodeteriogenic filamentous fungi species isolated from biodegradated artworks were used as biological model: Aspergillus niger, Cladosporium sp, Fusarium sp, Penicillium sp. and Exophialia sp. Fungal autofluorescence was only detected in cells harvested from Fusarium sp, and Exophialia sp. old cultures, being aging-dependent. However, it was weak enough to allow autofluorescence/RNA-FISH signals distinction. Thus, autofluorescence was not a limitation for the application of RNA-FISH for detection of the taxa investigated. All the permeabilization strategies tested allowed to detect fungal cells from young cultures by RNA-FISH. However, only the combination of paraformaldehyde with Triton X-100 allowed the detection of conidia/spores and hyphae of old filamentous fungi. All the permeabilization strategies failed in the Aspergillus niger conidia/spores staining, which are known to be particularly difficult to permeabilize. But, even in spite of this, the application of this permeabilization method increased the analytical potential of RNA FISH in Cultural Heritage biodeterioration. Whereas much work is required to validate this RNA-FISH approach for its application in real samples from Cultural Heritage it could represent an important advance for the detection, not only of hyphae but also of spores and conidia of various filamentous fungi taxa by RNA-FISH.

Drought, pod yield, pre-harvest Aspergillus infection and aflatoxin contamination on peanut in Niger

Soil moisture and soil temperature affect pre-harvest infection with Aspergillus flavus and production of aflatoxin. The objectives of our field research in Niger, West Africa, were to: (i) examine the effects of sowing date and irrigation treatments on pod yield, infection with A. flavus and aflatoxin concentration; and (ii) to quantify relations between infection, aflatoxin concentration and soil moisture stress. Seed of an aflatoxin susceptible peanut cv. JL24 was sown at two to four different sowing dates under four irrigation treatments (rainfed and irrigation at 7, 14 and 21 days intervals) between 1991 and 1994, giving 40 different 'environments'. Average air and soil temperatures of 28-34 degrees C were favourable for aflatoxin contamination. CROPGRO-peanut model was used to simulate the occurrence of moisture stress. The model was able to simulate yields of peanut well over the 40 environments (r(2) = 0.67). In general, early sowing produced greater pod yields, as well as less infection and lower aflatoxin concentration. There were negative linear relations between infection (r(2) = 0.62) and the average simulated fraction of extractable soil water (FESW) between flowering and harvest, and between aflatoxin concentration (r(2) = 0.54) and FESW in the last 25 days of pod-filling. This field study confirms that infection and aflatoxin concentration in peanut can be related to the occurrence of soil moisture stress during pod-filling when soil temperatures are near optimal for A. flavus. These relations could form the basis of a decision-support system to predict the risk of aflatoxin contamination in peanuts in similar environments. (c) 2005 Elsevier B.V. All rights reserved.

Benzodiazepine Alkaloids from Marine-Derived Endophytic Fungus Aspergillus ochraceus

A new fungus-derived benzodiazepine analogue, 2-hydroxycircumdatin C (1), and a compound which has been isolated from a natural resource for the first time, but has been previously synthesized, namely (11aS)-2,3-dihydro-7-methoxy-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione (2), along with five structurally related known alkaloids (3-7), were isolated from Aspergillus ochraceus, an endophytic fungus derived from the marine brown alga Sargassum kjellmanianum. Their structures were established on the basis of spectroscopic methods. The absolute configuration of I was determined through CD evidence. Compound 1 displayed significant DPPH radical-scavenging activity with an IC50 value of 9.9 mu M, which is 8.9-fold more potent than that of butylated hydroxytoluene (BHT), a well-known synthetic positive control.

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.

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.

Characterization of a thermostable extracellular tannase produced under submerged fermentation by Aspergillus ochraceus

Background: Tannases are enzymes that may be used in different industrial sectors as, for example, food and pharmaceutical. They are obtained mainly from microorganisms, as filamentous fungi. However, the diversity of fungi stays poorly explored for tannase production. In this article, Aspergillus ochraceus is presented as a new source of tannase with interesting features for biotechnological applications. Results: Extracellular tannase production was induced when the fungus was cultured in Khanna medium with tannic acid as carbon source. The extracellular tannase was purified 9-fold with 2% recovery and a single band corresponding to 85 kDa was observed in SDS-PAGE. The native apparent molecular mass was estimated as 112 kDa. Optima of temperature and pH were 40 degrees C and 5.0, respectively. The enzyme was fully stable from 40 degrees C to 60 degrees C during 1 hr. The activity was enhanced by Mn2+ (33-39%) and NH4+ (15%). The purified tannase hydrolyzed tannic acid and methyl gallate with Km of 0.76 mM and 0.72 mM, respectively, and Vmax of 0.92 U/mg protein and 0.68 U/mg protein, respectively. The analysis of a partial sequence of the tannase encoding gene showed an open read frame of 567 bp and a sequence of 199 amino acids were predicted. TLC analysis revealed the presence of gallic acid as a tannic acid hydrolysis product. Conclusion: The extracellular tannase produced by A. ochraceus showed distinctive characteristics such as monomeric structure and activation by Mn2+, suggesting a new kind of fungal tannases with biotechnological potential. Further, it was the first time that a partial gene sequence for A. ochraceus tannase was described.

Functional characterization and oligomerization of a recombinant xyloglucan-specific endo-beta-1,4-glucanase (GH12) from Aspergillus niveus

Xyloglucan is a major structural polysaccharide of the primary (growing) cell wall of higher plants. It consists of a cellulosic backbone (beta-1,4-linked glucosyl residues) that is frequently substituted with side chains. This report describes Aspergillus nidulans strain A773 recombinant secretion of a dimeric xyloglucan-specific endo-beta-1,4-glucanohydrolase (XegA) cloned from Aspergillus niveus. The ORF of the A. niveus xegA gene is comprised of 714 nucleotides, and encodes a 238 amino acid protein with a calculated molecular weight of 23.5 kDa and isoelectric point of 4.38. The optimal pH and temperature were 6.0 and 60 degrees C, respectively. XegA generated a xyloglucan-oligosaccharides (XGOs) pattern similar to that observed for cellulases from family GH12, i.e., demonstrating that its mode of action includes hydrolysis of the glycosidic linkages between glucosyl residues that are not branched with xylose. In contrast to commercial lichenase, mixed linkage beta-glucan (lichenan) was not digested by XegA, indicating that the enzyme did not cleave glucan beta-1,3 or beta-1,6 bonds. The far-UV CD spectrum of the purified enzyme indicated a protein rich in beta-sheet structures as expected for GH12 xyloglucanases. Thermal unfolding studies displayed two transitions with mid-point temperatures of 51.3 degrees C and 81.3 degrees C respectively, and dynamic light scattering studies indicated that the first transition involves a change in oligomeric state from a dimeric to a monomeric form. Since the enzyme is a predominantly a monomer at 60 degrees C. the enzymatic assays demonstrated that XegA is more active in its monomeric state. (c) 2012 Elsevier B.V. All rights reserved.

Inactivation of Aspergillus flavus spores by curcumin-mediated photosensitization

Minimizing fungal infection is essential to the control of mycotoxin contamination of foods and feeds but many potential control methods are not without their own safety concerns for the consumers. Photodynamic inactivation is a novel light-based approach which offers a promising alternative to conventional methods for the control of mycotoxigenic fungi. This study describes the use of curcumin to inactivate spores of Aspergillus flavus, one of the major aflatoxin producing fungi in foods and feeds. Curcumin is a natural polyphenolic compound from the spice turmeric (Curcuma longa). In this study the plant has shown to be an effective photosensitiser when combined with visible light (420 nm). The experiment was conducted in in vitro and in vivo where A. flavus spores were treated with different photosensitiser concentration and light dose both in buffer solution and on maize kernels. Comparison of fungal load from treated and untreated samples was determined, and reductions of fungal spore counts of up to 3 log CFU ml−1 in suspension and 2 log CFU g−1 in maize kernels were obtained using optimal dye concentrations and light dose combinations. The results in this study indicate that curcumin-mediated photosensitization is a potentially effective method to decontaminate A. flavus spores in foods and feeds.

Characterisation, cloning and production of industrially interesting enzymes: gluconolactone oxidase of Penicillium cyaneo-fulvum and gluconate 5-dehydrogenase of Gluconobacter suboxydans

The work covered in this thesis is focused on the development of technology for bioconversion of glucose into D-erythorbic acid (D-EA) and 5-ketogluconic acid (5-KGA). The task was to show on proof-of-concept level the functionality of the enzymatic conversion or one-step bioconversion of glucose to these acids. The feasibility of both studies to be further developed for production processes was also evaluated. The glucose - D-EA bioconversion study was based on the use of a cloned gene encoding a D-EA forming soluble flavoprotein, D-gluconolactone oxidase (GLO). GLO was purified from Penicillium cyaneo-fulvum and partially sequenced. The peptide sequences obtained were used to isolate a cDNA clone encoding the enzyme. The cloned gene (GenBank accession no. AY576053) is homologous to the other known eukaryotic lactone oxidases and also to some putative prokaryotic lactone oxidases. Analysis of the deduced protein sequence of GLO indicated the presence of a typical secretion signal sequence at the N-terminus of the enzyme. No other targeting/anchoring signals were found, suggesting that GLO is the first known lactone oxidase that is secreted rather than targeted to the membranes of the endoplasmic reticulum or mitochondria. Experimental evidence supports this analysis, as near complete secretion of GLO was observed in two different yeast expression systems. Highest expression levels of GLO were obtained using Pichia pastoris as an expression host. Recombinant GLO was characterised and the suitability of purified GLO for the production of D-EA was studied. Immobilised GLO was found to be rapidly inactivated during D-EA production. The feasibility of in vivo glucose - D-EA conversion using a P. pastoris strain co-expressing the genes of GLO and glucose oxidase (GOD, E.C. 1.1.3.4) of A. niger was demonstrated. The glucose - 5-KGA bioconversion study followed a similar strategy to that used in the D-EA production research. The rationale was based on the use of a cloned gene encoding a membrane-bound pyrroloquinoline quinone (PQQ)-dependent gluconate 5-dehydrogenase (GA 5-DH). GA 5-DH was purified to homogeneity from the only source of this enzyme known in literature, Gluconobacter suboxydans, and partially sequenced. Using the amino acid sequence information, the GA 5-DH gene was cloned from a genomic library of G. suboxydans. The cloned gene was sequenced (GenBank accession no. AJ577472) and found to be an operon of two adjacent genes encoding two subunits of GA 5-DH. It turned out that GA 5-DH is a rather close homologue of a sorbitol dehydrogenase from another G. suboxydans strain. It was also found that GA 5-DH has significant polyol dehydrogenase activity. The G. suboxydans GA 5-DH gene was poorly expressed in E. coli. Under optimised conditions maximum expression levels of GA 5-DH did not exceed the levels found in wild-type G. suboxydans. Attempts to increase expression levels resulted in repression of growth and extensive cell lysis. However, the expression levels were sufficient to demonstrate the possibility of bioconversion of glucose and gluconate into 5-KGA using recombinant strains of E. coli. An uncharacterised homologue of GA 5-DH was identified in Xanthomonas campestris using in silico screening. This enzyme encoded by chromosomal locus NP_636946 was found by a sequencing project of X. campestris and named as a hypothetical glucose dehydrogenase. The gene encoding this uncharacterised enzyme was cloned, expressed in E. coli and found to encode a gluconate/polyol dehydrogenase without glucose dehydrogenase activity. Moreover, the X. campestris GA 5-DH gene was expressed in E. coli at nearly 30 times higher levels than the G. suboxydans GA 5-DH gene. Good expressability of the X. campestris GA-5DH gene makes it a valuable tool not only for 5-KGA production in the tartaric acid (TA) bioprocess, but possibly also for other bioprocesses (e.g. oxidation of sorbitol into L-sorbose). In addition to glucose - 5-KGA bioconversion, a preliminary study of the feasibility of enzymatic conversion of 5-KGA into TA was carried out. Here, the efficacy of the first step of a prospective two-step conversion route including a transketolase and a dehydrogenase was confirmed. It was found that transketolase convert 5-KGA into TA semialdehyde. A candidate for the second step was suggested to be succinic dehydrogenase, but this was not tested. The analysis of the two subprojects indicated that bioconversion of glucose to TA using X. campestris GA 5-DH should be prioritised first and the process development efforts in future should be focused on development of more efficient GA 5-DH production strains by screening a more suitable production host and by protein engineering.

Estimating specific yield and transmissivity with magnetic resonance sounding in an unconfined sandstone aquifer (Niger)

The unconfined aquifer of the Continental Terminal in Niger was investigated by magnetic resonance sounding (MRS) and by 14 pumping tests in order to improve calibration of MRS outputs at field scale. The reliability of the standard relationship used for estimating aquifer transmissivity by MRS was checked; it was found that the parametric factor can be estimated with an uncertainty a parts per thousand currency sign150% by a single point of calibration. The MRS water content (theta (MRS)) was shown to be positively correlated with the specific yield (Sy), and theta (MRS) always displayed higher values than Sy. A conceptual model was subsequently developed, based on estimated changes of the total porosity, Sy, and the specific retention Sr as a function of the median grain size. The resulting relationship between theta (MRS) and Sy showed a reasonably good fit with the experimental dataset, considering the inherent heterogeneity of the aquifer matrix (residual error is similar to 60%). Interpreted in terms of aquifer parameters, MRS data suggest a log-normal distribution of the permeability and a one-sided Gaussian distribution of Sy. These results demonstrate the efficiency of the MRS method for fast and low-cost prospection of hydraulic parameters for large unconfined aquifers.

Contribution of geophysical surveys to groundwater modelling of a porous aquifer in semiarid Niger: An overview

Subsurface geophysical surveys were carried out using a large range of methods in an unconfined sandstone aquifer in semiarid south-western Niger for improving both the conceptual model of water flow through the unsaturated zone and the parameterization of numerical a groundwater model of the aquifer. Methods included: electromagnetic mapping, electrical resistivity tomography (ERT), resistivity logging, time domain electromagnetic sounding (TDEM), and magnetic resonance sounding (MRS). Analyses of electrical conductivities, complemented by geochemical measurements, allowed us to identify preferential pathways for infiltration and drainage beneath gullies and alluvial fans. The mean water content estimated by MRS (13%) was used for computing the regional groundwater recharge from long-term change in the water table. The ranges in permeability and water content obtained with MRS allowed a reduction of the degree of freedom of aquifer parameters used in groundwater modelling.

Immobilization of Aspergillus oryzae alpha-galactosidase in gelatin and its application in removal of flatulence-inducing sugars in soymilk

Raffinose oligosaccharides (RO) are the major factors responsible for flatulence following ingestion of soybean-derived products. Removal of RO from seeds or soymilk would then have a positive impact on the acceptance of soy-based foods. In this study, alpha-galactosidase from Aspergillus oryzae was entrapped in gelatin using formaldehyde as the hardener. The immobilization yield was 64.3% under the optimum conditions of immobilization. The immobilized alpha-galactosidase showed a shift in optimum pH from 4.8 to 5.4 in acetate buffer. The optimum temperature also shifted from 50 degrees C to 57 degrees C compared with soluble enzyme. Immobilized alpha-galactosidase was used in batch, repeated batch and continuous mode to degrade RO present in soymilk. In the repeated batch, 45% reduction of RO was obtained in the fourth cycle. The performance of immobilized alpha-galactosidase was tested in a fluidized bed reactor at different flow rates and 86% reduction of RO in soymilk was obtained at 25 ml h(-1) flow rate. The study revealed that immobilized alpha-galactosidase in continuous mode is efficient in reduction of RO present in soymilk.