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1.
Future Microbiol ; : 1-14, 2024 Jul 16.
Artículo en Inglés | MEDLINE | ID: mdl-39011856

RESUMEN

Aim: This work aims to standardize the three-dimensional hydroxyethyl-alginate-gelatin (HAG) scaffold as a model to evaluate Aspergillus fumigatus biofilm and antifungal treatments. Methods: The scaffold was characterized by physical, rheological and microscopic analyses; the antibiofilm action was evaluated by determination of cfu and metabolic activity. Results: The scaffold was non-toxic showing stability in aqueous media, swelling capacity, elasticity and had homogeneously distributed pores averaging 190 µm. The A. fumigatus biofilm established itself very well on the scaffold and treatment with amphotericin B and voriconazole reduced viable cells and metabolic activity. Conclusion: The HAG scaffold proved to be a model to mimic lung parenchyma, suitable for establishing a 3D biofilm culture of A. fumigatus and evaluating the efficacy of antifungals.


[Box: see text].

2.
Mol Biol Rep ; 51(1): 767, 2024 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-38878205

RESUMEN

BACKGROUND: Esterases (EC 3.1.1.X) are enzymes that catalyze the hydrolysis ester bonds. These enzymes have large potential for diverse applications in fine industries, particularly in pharmaceuticals, cosmetics, and bioethanol production. METHODS AND RESULTS: In this study, a gene encoding an esterase from Thermobifida fusca YX (TfEst) was successfully cloned, and its product was overexpressed in Escherichia coli and purified using affinity chromatography. The TfEst kinetic assay revealed catalytic efficiencies of 0.58 s-1 mM-1, 1.09 s-1 mM-1, and 0.062 s-1 mM-1 against p-Nitrophenyl acetate, p-Nitrophenyl butyrate, and 1-naphthyl acetate substrates, respectively. Furthermore, TfEst also exhibited activity in a pH range from 6.0 to 10.0, with maximum activity at pH 8.0. The enzyme demonstrated a half-life of 20 min at 70 °C. Notably, TfEst displayed acetyl xylan esterase activity as evidenced by the acetylated xylan assay. The structural prediction of TfEst using AlphaFold indicated that has an α/ß-hydrolase fold, which is consistent with other esterases. CONCLUSIONS: The enzyme stability over a broad pH range and its activity at elevated temperatures make it an appealing candidate for industrial processes. Overall, TfEst emerges as a promising enzymatic tool with significant implications for the advancement of biotechnology and biofuels industries.


Asunto(s)
Acetilesterasa , Esterasas , Thermobifida , Acetilesterasa/metabolismo , Acetilesterasa/genética , Acetilesterasa/química , Concentración de Iones de Hidrógeno , Cinética , Especificidad por Sustrato , Thermobifida/enzimología , Thermobifida/genética , Esterasas/metabolismo , Esterasas/genética , Esterasas/química , Estabilidad de Enzimas , Temperatura , Escherichia coli/genética , Escherichia coli/metabolismo , Clonación Molecular/métodos , Hidrólisis , Xilanos/metabolismo , Butiratos/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Nitrofenoles
3.
Vaccines (Basel) ; 11(11)2023 Nov 20.
Artículo en Inglés | MEDLINE | ID: mdl-38006064

RESUMEN

Mucosal vaccination appears to be suitable to protect against SARS-CoV-2 infection. In this study, we tested an intranasal mucosal vaccine candidate for COVID-19 that consisted of a cationic liposome containing a trimeric SARS-CoV-2 spike protein and CpG-ODNs, a Toll-like receptor 9 agonist, as an adjuvant. In vitro and in vivo experiments indicated the absence of toxicity following the intranasal administration of this vaccine formulation. First, we found that subcutaneous or intranasal vaccination protected hACE-2 transgenic mice from infection with the wild-type (Wuhan) SARS-CoV-2 strain, as shown by weight loss and mortality indicators. However, when compared with subcutaneous administration, the intranasal route was more effective in the pulmonary clearance of the virus and induced higher neutralizing antibodies and anti-S IgA titers. In addition, the intranasal vaccination afforded protection against gamma, delta, and omicron virus variants of concern. Furthermore, the intranasal vaccine formulation was superior to intramuscular vaccination with a recombinant, replication-deficient chimpanzee adenovirus vector encoding the SARS-CoV-2 spike glycoprotein (Oxford/AstraZeneca) in terms of virus lung clearance and production of neutralizing antibodies in serum and bronchial alveolar lavage (BAL). Finally, the intranasal liposomal formulation boosted heterologous immunity induced by previous intramuscular vaccination with the Oxford/AstraZeneca vaccine, which was more robust than homologous immunity.

4.
Int J Mol Sci ; 24(7)2023 Mar 23.
Artículo en Inglés | MEDLINE | ID: mdl-37047028

RESUMEN

Cultivated meat (CM) technology has the potential to disrupt the food industry-indeed, it is already an inevitable reality. This new technology is an alternative to solve the environmental, health and ethical issues associated with the demand for meat products. The global market longs for biotechnological improvements for the CM production chain. CM, also known as cultured, cell-based, lab-grown, in vitro or clean meat, is obtained through cellular agriculture, which is based on applying tissue engineering principles. In practice, it is first necessary to choose the best cell source and type, and then to furnish the necessary nutrients, growth factors and signalling molecules via cultivation media. This procedure occurs in a controlled environment that provides the surfaces necessary for anchor-dependent cells and offers microcarriers and scaffolds that favour the three-dimensional (3D) organisation of multiple cell types. In this review, we discuss relevant information to CM production, including the cultivation process, cell sources, medium requirements, the main obstacles to CM production (consumer acceptance, scalability, safety and reproducibility), the technological aspects of 3D models (biomaterials, microcarriers and scaffolds) and assembly methods (cell layering, spinning and 3D bioprinting). We also provide an outlook on the global CM market. Our review brings a broad overview of the CM field, providing an update for everyone interested in the topic, which is especially important because CM is a multidisciplinary technology.


Asunto(s)
Productos de la Carne , Ingeniería de Tejidos , Ingeniería de Tejidos/métodos , Reproducibilidad de los Resultados , Carne , Biotecnología , Andamios del Tejido
5.
Int J Biol Macromol ; 167: 93-100, 2021 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-33259843

RESUMEN

Glutathione peroxidases (GPx) are a family of enzymes with the ability to reduce organic and inorganic hydroperoxides to the corresponding alcohols using glutathione or thioredoxin as an electron donor. Here, we report the functional and structural characterization of a GPx identified in Trichoderma reesei (TrGPx). TrGPx was recombinantly expressed in a bacterial host and purified using affinity. Using a thioredoxin coupled assay, TrGPx exhibited activity of 28 U and 12.5 U in the presence of the substrates H2O2 and t-BOOH, respectively, and no activity was observed when glutathione was used. These results indicated that TrGPx is a thioredoxin peroxidase and hydrolyses H2O2 better than t-BOOH. TrGPx kinetic parameters using a pyrogallol assay resulted at Kmapp = 11.7 mM, Vmaxapp = 10.9 IU/µg TrGPx, kcat = 19 s-1 and a catalytic efficiency of 1.6 mM-1 s-1 to H2O2 as substrate. Besides that, TrGPx demonstrated an optimum pH ranging from 9.0-12.0 and a half-life of 36 min at 80 °C. TrGPx 3D-structure was obtained in a reduced state and non-catalytic conformation. The overall fold is similar to the other phospholipid-hydroperoxide glutathione peroxidases. These data contribute to understand the antioxidant mechanism in fungi and provide information for using antioxidant enzymes in biotechnological applications.


Asunto(s)
Hypocreales/enzimología , Peroxirredoxinas/química , Peroxirredoxinas/metabolismo , Secuencia de Aminoácidos , Antioxidantes/química , Antioxidantes/farmacología , Fraccionamiento Químico , Clonación Molecular , Activación Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expresión Génica , Glutatión Peroxidasa/química , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Concentración de Iones de Hidrógeno , Hypocreales/genética , Modelos Moleculares , Peroxirredoxinas/genética , Peroxirredoxinas/aislamiento & purificación , Conformación Proteica , Relación Estructura-Actividad , Temperatura
6.
Int J Biol Macromol, v. 167, p. 93-100, jan. 2021
Artículo en Inglés | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-3507

RESUMEN

Glutathione peroxidases (GPx) are a family of enzymes with the ability to reduce organic and inorganic hydroperoxides to the corresponding alcohols using glutathione or thioredoxin as an electron donor. Here, we report the functional and structural characterization of a GPx identified in Trichoderma reesei (TrGPx). TrGPx was recombinantly expressed in a bacterial host and purified using affinity. Using a thioredoxin coupled assay, TrGPx exhibited activity of 28 U and 12.5 U in the presence of the substrates H2O2 and t-BOOH, respectively, and no activity was observed when glutathione was used. These results indicated that TrGPx is a thioredoxin peroxidase and hydrolyses H2O2 better than t-BOOH. TrGPx kinetic parameters using a pyrogallol assay resulted at Kmapp = 11.7 mM, Vmaxapp = 10.9 IU/μg TrGPx, kcat = 19 s−1 and a catalytic efficiency of 1.6 mM−1 s−1 to H2O2 as substrate. Besides that, TrGPx demonstrated an optimum pH ranging from 9.0–12.0 and a half-life of 36 min at 80 °C. TrGPx 3D-structure was obtained in a reduced state and non-catalytic conformation. The overall fold is similar to the other phospholipid-hydroperoxide glutathione peroxidases. These data contribute to understand the antioxidant mechanism in fungi and provide information for using antioxidant enzymes in biotechnological applications.

7.
Int J Biol Macromol ; 129: 653-658, 2019 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-30771398

RESUMEN

Epoxide hydrolases (EHs) are enzymes involved in the metabolism of endogenous and exogenous epoxides, and the development of EH inhibitors has important applications in the medicine. In humans, EH inhibitors are being tested in the treatment of cardiovascular diseases and show potent anti-inflammatory effects. EH inhibitors are also considerate promising molecules against infectious diseases. EHs are functionally very well studied, but only a few members have its three-dimensional structures characterized. Recently, a new EH from the filamentous fungi Trichoderma reseei (TrEH) was reported, and a series of urea or amide-based inhibitors were identified. In this study, we describe the crystallographic structures of TrEH in complex with five different urea or amide-based inhibitors with resolutions ranging from 2.6 to 1.7 Å. The analysis of these structures reveals the molecular basis of the inhibition of these compounds. We could also observe that these inhibitors occupy the whole extension of the active site groove and only a few conformational changes are involved. Understanding the structural basis EH interactions with different inhibitors might substantially contribute for the study of fungal metabolism and in the development of novel and more efficient antifungal drugs against pathogenic Trichoderma species.


Asunto(s)
Amidas/química , Amidas/farmacología , Epóxido Hidrolasas/antagonistas & inhibidores , Trichoderma/enzimología , Urea/química , Urea/farmacología , Amidas/metabolismo , Dominio Catalítico/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Epóxido Hidrolasas/química , Epóxido Hidrolasas/metabolismo , Concentración 50 Inhibidora , Modelos Moleculares , Urea/metabolismo
8.
Mol Biol Rep ; 46(1): 371-379, 2019 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-30426381

RESUMEN

Epoxide hydrolases (EHs) are present in all living organisms and catalyze the hydrolysis of epoxides to the corresponding vicinal diols. EH are involved in the metabolism of endogenous and exogenous epoxides, and thus have application in pharmacology and biotechnology. In this work, we describe the substrates and inhibitors selectivity of an epoxide hydrolase recently cloned from the filamentous fungus Trichoderma reesei QM9414 (TrEH). We also studied the TrEH urea-based inhibitors effects in the fungal growth. TrEH showed high activity on radioative and fluorescent surrogate and natural substrates, especially epoxides from docosahexaenoic acid. Using a fluorescent surrogate substrate, potent inhibitors of TrEH were identified. Interestingly, one of the best compounds inhibit up to 60% of T. reesei growth, indicating an endogenous role for TrEH. These data make TrEH very attractive for future studies about fungal metabolism of fatty acids and possible development of novel drugs for human diseases.


Asunto(s)
Epóxido Hidrolasas/fisiología , Trichoderma/metabolismo , Catálisis , Epóxido Hidrolasas/antagonistas & inhibidores , Epóxido Hidrolasas/metabolismo , Compuestos Epoxi/metabolismo , Ácidos Grasos/fisiología , Hidrólisis , Trichoderma/fisiología
9.
Appl Environ Microbiol ; 85(4)2019 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-30504211

RESUMEN

The hypersaline Kebrit Deep brine pool in the Red Sea is characterized by high levels of toxic heavy metals. Here, we describe two structurally related mercuric reductases (MerAs) from this site which were expressed in Escherichia coli Sequence similarities suggest that both genes are derived from proteobacteria, most likely the Betaproteobacteria or Gammaproteobacteria We show that one of the enzymes (K35NH) is strongly inhibited by NaCl, while the other (K09H) is activated in a NaCl-dependent manner. We infer from this difference that the two forms might support the detoxification of mercury in bacterial microorganisms that employ the compatible solutes and salt-in strategies, respectively. Three-dimensional structure modeling shows that all amino acid substitutions unique to each type are located outside the domain responsible for formation of the active MerA homodimer, and the vast majority of these are found on the surface of the molecule. Moreover, K09H exhibits the predominance of acidic over hydrophobic side chains that is typical of halophilic salt-dependent proteins. These findings enhance our understanding of how selection pressures imposed by two environmental stressors have endowed MerA enzymes with catalytic properties that can potentially function in microorganisms that utilize distinct mechanisms for osmotic balance in hypersaline environments.IMPORTANCE Analysis of two structurally homologous but catalytically distinct mercuric reductases from the Kebrit Deep brine in the Red Sea sheds light on the adaptations that enable microorganisms to cope simultaneously with extreme salinity and toxic mercury compounds. One is strongly inhibited by high NaCl concentrations, while the other exhibits NaCl-dependent activation. Their different activity profiles imply that they may derive from bacterial microorganisms that utilize compatible solutes and salt-in strategies, respectively, to maintain osmotic balance. Three-dimensional modeling reveals that regions not involved in formation of the active homodimer are conserved between the two. However, in the NaCl-dependent form, distinct amino acid substitutions are found in areas that are critical for stability in high salt. The work provides insights into how two environmental stressors have shaped the structure of orthologous enzymes through selection and adaptation, enabling them to retain their catalytic function in what may be very different cellular contexts.


Asunto(s)
Adaptación Fisiológica/fisiología , Bacterias/enzimología , Mercurio/metabolismo , Oxidorreductasas/química , Oxidorreductasas/genética , Adaptación Fisiológica/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Bacterias/genética , Regulación Bacteriana de la Expresión Génica , Océano Índico , Modelos Moleculares , Oxidorreductasas/efectos de los fármacos , Oxidorreductasas/metabolismo , Filogenia , Conformación Proteica , Salinidad , Sales (Química) , Agua de Mar/microbiología , Alineación de Secuencia , Análisis de Secuencia , Cloruro de Sodio/farmacología , Microbiología del Agua
10.
Biochim Biophys Acta Proteins Proteom ; 1865(8): 1039-1045, 2017 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-28502798

RESUMEN

Epoxide hydrolases (EHs) are enzymes that have high biotechnological interest for the fine and transformation industry. Several of these enzymes have enantioselectivity, which allows their application in the separation of enantiomeric mixtures of epoxide substrates. Although two different families of EHs have been described, those that have the α/ß-hidrolase fold are the most explored for biotechnological purpose. These enzymes are functionally very well studied, but only few members have three-dimensional structures characterised. Recently, a new EH from the filamentous fungi Trichoderma reseei (TrEH) has been discovered and functionally studied. This enzyme does not have high homology to any other EH structure and have an enatiopreference for (S)-(-) isomers. Herein we described the crystallographic structure of TrEH at 1.7Å resolution, which reveals features of its tertiary structure and active site. TrEH has a similar fold to the other soluble epoxide hydrolases and has the two characteristic hydrolase and cap domains. The enzyme is predominantly monomeric in solution and has also been crystallised as a monomer in the asymmetric unit. Although the catalytic residues are conserved, several other residues of the catalytic groove are not, and might be involved in the specificity for substrates and in the enantioselectivy of this enzyme. In addition, the determination of the crystallographic structure of TrEH might contribute to the rational site direct mutagenesis to generate an even more stable enzyme with higher efficiency to be used in biotechnological purposes.


Asunto(s)
Epóxido Hidrolasas/química , Epóxido Hidrolasas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Trichoderma/metabolismo , Dominio Catalítico/fisiología , Cristalografía por Rayos X/métodos , Modelos Moleculares , Mutagénesis Sitio-Dirigida/métodos
11.
Data Brief ; 8: 436-40, 2016 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-27366781

RESUMEN

The data presented in this article are related to the research article entitled "Epoxide hydrolase of Trichoderma reesei: Biochemical properties and conformational characterization" [1]. Epoxide hydrolases (EHs) are enzymes that catalyze the hydrolysis of epoxides to the corresponding vicinal diols. This article describes the optimal parameters for the colorimetric red assay to determine the enzymatic activity, with an emphasis on the characterization of the kinetic parameters, pH optimum and thermal stability of this enzyme. The effects of reagents that are not resistant to oxidation by sodium periodate on the reactions can generate false positives and interfere with the final results of the red assay.

12.
Int J Biol Macromol ; 89: 569-74, 2016 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-27177457

RESUMEN

Epoxide hydrolases (EHs) are enzymes that are present in all living organisms and catalyze the hydrolysis of epoxides to the corresponding vicinal diols. EHs have biotechnological potential in chiral chemistry. We report the cloning, purification, enzymatic activity, and conformational analysis of the TrEH gene from Trichoderma reesei strain QM9414 using circular dichroism spectroscopy. The EH gene has an open reading frame encoding a protein of 343 amino acid residues, resulting in a molecular mass of 38.2kDa. The enzyme presents an optimum pH of 7.2, and it is highly active at temperatures ranging from 23 to 50°C and thermally inactivated at 70°C (t1/2=7.4min). The Michaelis constants (Km) were 4.6mM for racemic substrate, 21.7mM for (R)-(+)-styrene oxide and 3.0mM for (S)-(-)-styrene oxide. The kcat/Km analysis indicated that TrEH is enantioselective and preferentially hydrolyzes (S)-(-)-styrene oxide. The conformational stability studies suggested that, despite the extreme conditions (high temperatures and extremely acid and basic pHs), TrEH is able to maintain a considerable part of its regular structures, including the preservation of the native cores in some cases. The recombinant protein showed enantioselectivity that was distinct from other fungus EHs, making this protein a potential biotechnological tool.


Asunto(s)
Epóxido Hidrolasas/química , Proteínas Recombinantes/química , Trichoderma/enzimología , Secuencia de Aminoácidos/genética , Clonación Molecular , Epóxido Hidrolasas/genética , Epóxido Hidrolasas/aislamiento & purificación , Compuestos Epoxi/química , Escherichia coli/genética , Hidrólisis , Proteínas Recombinantes/genética , Especificidad por Sustrato
13.
Appl Microbiol Biotechnol ; 100(6): 2567-77, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26810078

RESUMEN

Fungal habitats include soil, water, and extreme environments. With around 100,000 fungus species already described, it is estimated that 5.1 million fungus species exist on our planet, making fungi one of the largest and most diverse kingdoms of eukaryotes. Fungi show remarkable metabolic features due to a sophisticated genomic network and are important for the production of biotechnological compounds that greatly impact our society in many ways. In this review, we present the current state of knowledge on fungal biodiversity, with special emphasis on filamentous fungi and the most recent discoveries in the field of identification and production of biotechnological compounds. More than 250 fungus species have been studied to produce these biotechnological compounds. This review focuses on three of the branches generally accepted in biotechnological applications, which have been identified by a color code: red, green, and white for pharmaceutical, agricultural, and industrial biotechnology, respectively. We also discuss future prospects for the use of filamentous fungi in biotechnology application.


Asunto(s)
Biodiversidad , Biotecnología/métodos , Hongos/clasificación , Hongos/metabolismo
14.
PLoS One ; 9(6): e97338, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24921648

RESUMEN

Metagenomics-based functional profiling analysis is an effective means of gaining deeper insight into the composition of marine microbial populations and developing a better understanding of the interplay between the functional genome content of microbial communities and abiotic factors. Here we present a comprehensive analysis of 24 datasets covering surface and depth-related environments at 11 sites around the world's oceans. The complete datasets comprises approximately 12 million sequences, totaling 5,358 Mb. Based on profiling patterns of Clusters of Orthologous Groups (COGs) of proteins, a core set of reference photic and aphotic depth-related COGs, and a collection of COGs that are associated with extreme oxygen limitation were defined. Their inferred functions were utilized as indicators to characterize the distribution of light- and oxygen-related biological activities in marine environments. The results reveal that, while light level in the water column is a major determinant of phenotypic adaptation in marine microorganisms, oxygen concentration in the aphotic zone has a significant impact only in extremely hypoxic waters. Phylogenetic profiling of the reference photic/aphotic gene sets revealed a greater variety of source organisms in the aphotic zone, although the majority of individual photic and aphotic depth-related COGs are assigned to the same taxa across the different sites. This increase in phylogenetic and functional diversity of the core aphotic related COGs most probably reflects selection for the utilization of a broad range of alternate energy sources in the absence of light.


Asunto(s)
Metagenoma , Microbiota/genética , Agua de Mar/microbiología , Adaptación Fisiológica , Luz , Microbiota/fisiología , Familia de Multigenes , Filogenia
15.
J Biol Chem ; 289(3): 1675-87, 2014 Jan 17.
Artículo en Inglés | MEDLINE | ID: mdl-24280218

RESUMEN

A unique combination of physicochemical conditions prevails in the lower convective layer (LCL) of the brine pool at Atlantis II (ATII) Deep in the Red Sea. With a maximum depth of over 2000 m, the pool is characterized by acidic pH (5.3), high temperature (68 °C), salinity (26%), low light levels, anoxia, and high concentrations of heavy metals. We have established a metagenomic dataset derived from the microbial community in the LCL, and here we describe a gene for a novel mercuric reductase, a key component of the bacterial detoxification system for mercuric and organomercurial species. The metagenome-derived gene and an ortholog from an uncultured soil bacterium were synthesized and expressed in Escherichia coli. The properties of their products show that, in contrast to the soil enzyme, the ATII-LCL mercuric reductase is functional in high salt, stable at high temperatures, resistant to high concentrations of Hg(2+), and efficiently detoxifies Hg(2+) in vivo. Interestingly, despite the marked functional differences between the orthologs, their amino acid sequences differ by less than 10%. Site-directed mutagenesis and kinetic analysis of the mutant enzymes, in conjunction with three-dimensional modeling, have identified distinct structural features that contribute to extreme halophilicity, thermostability, and high detoxification capacity, suggesting that these were acquired independently during the evolution of this enzyme. Thus, our work provides fundamental structural insights into a novel protein that has undergone multiple biochemical and biophysical adaptations to promote the survival of microorganisms that reside in the extremely demanding environment of the ATII-LCL.


Asunto(s)
Mercurio/química , Metagenoma , Océanos y Mares , Oxidorreductasas/química , Agua de Mar/microbiología , Microbiología del Agua , Secuencia de Bases , Concentración de Iones de Hidrógeno , Cinética , Mercurio/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oxidorreductasas/biosíntesis , Oxidorreductasas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
16.
Fungal Genet Biol ; 60: 9-18, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23872076

RESUMEN

Brazil houses over 10% of the total number of known species on Earth, with a great diversity of plants and fungi. The collection, isolation, identification and conservation of filamentous fungi with relevance to agriculture, pharmaceutical, food and biotechnological industries in Biological Resource Centers (CRBs) is very important to the development of a nation's scientific and technological infrastructure. In Brazil, 36 fungal collections are registered in the database of International Collections. Several federal and state programs have encouraged the formation of a researcher's network in order to study natural resources and the nation's biodiversity. In this context, Brazilian researchers have been on the frontiers of knowledge, investigating the enzymatic systems from native filamentous fungi with potential for biomass degradation and biotechnological application. In this review, we address recent progress in Brazilian fungal research, focusing on the identification and study of fungi and enzymes with potential for biomass degradation and application in bioenergy.


Asunto(s)
Metabolismo Energético , Hongos/clasificación , Hongos/enzimología , Biodiversidad , Biocombustibles , Biomasa , Biotecnología/métodos , Brasil , Celulasas/metabolismo , Oxigenasas/metabolismo
17.
Int J Biol Macromol ; 50(1): 19-24, 2012 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-21986542

RESUMEN

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°C for 1h at pH from 8 to 11.5, while maintaining its biological activity.


Asunto(s)
Proteínas Recombinantes/química , Superóxido Dismutasa/química , Trichoderma/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Dicroismo Circular , Clonación Molecular , Escherichia coli/metabolismo , Calor , Concentración de Iones de Hidrógeno , Sustancias Macromoleculares/química , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Conformación Proteica , Estructura Secundaria de Proteína , Especies Reactivas de Oxígeno , Temperatura
19.
Biochemistry ; 45(26): 8184-92, 2006 Jul 04.
Artículo en Inglés | MEDLINE | ID: mdl-16800643

RESUMEN

The filamentous fungus Trichoderma reesei is adapted to nutrient-poor environments, in which it uses extracellular cellulases to obtain glucose from the available cellulose biomass. We have isolated and characterized Trhxt1, a putative glucose transporter gene, as judged by the glucose accumulation phenotype of a DeltaTrhxt1 mutant. This gene is repressed at high glucose concentrations and expressed at micromolar levels and in the absence of glucose. The gene is also induced during the growth of T. reesei on cellulose when the glucose concentration generated from the hydrolysis of cellulose present in the culture medium is in the micromolar range. We also show that oxygen availability controls the expression of the Trxht1 gene. In this regard, the gene is down-regulated by hypoxia and also by the inhibition of the flow of electrons through the respiratory chain using antimycin A. Intriguingly, anoxia but not hypoxia strongly induces the expression of the gene in the presence of an otherwise repressive concentration of glucose. These results indicate that although the absence of repressing concentrations of glucose and an active respiratory chain are required for Trhxt1 expression under normoxic conditions these physiological processes have no effect on the expression of this gene under an anoxic state. Thus, our results highlight the presence of a novel coordinated interaction between oxygen and the regulatory circuit for glucose repression under anoxic conditions.


Asunto(s)
Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Trichoderma/metabolismo , Algoritmos , Animales , Secuencia de Bases , Celulasa/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glucosa/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Humanos , Cinética , Ratones , Datos de Secuencia Molecular , Oxígeno , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico
20.
Biochemistry ; 45(12): 3912-24, 2006 Mar 28.
Artículo en Inglés | MEDLINE | ID: mdl-16548518

RESUMEN

Oxygen is essential for the survival of obligatorily aerobic eukaryotic microorganisms, such as the multicellular fungus Trichoderma reesei. However, the molecular basis for the inability of such cells to survive for extended periods under anoxic conditions is not fully understood. Using cDNA microarray analysis, we show that changes in oxygen availability have a drastic effect on gene expression in T. reesei. The expression levels of 392 (19.6%) out of 2000 genes examined changed significantly in response to hypoxia, transient anoxia, and reoxygenation. In addition to modulating many genes with no previously assigned function, cells respond to hypoxia by readjusting the balance of expression between genes required for energy production and consumption, and altering the expression of genes involved in protective mechanisms and signaling pathways. Moreover, we show that transient anoxia strongly represses genes for enzymes that are critical for glycolysis, and are essential for energy production under anaerobic conditions. Our study thus reveals crucial differences between the facultative anaerobe Saccharomyces cerevisiae and T. reesei with regard to the oxygen-dependent transcriptional control of the glycolytic pathway, which can account for the differential survival of the two species in the absence of oxygen.


Asunto(s)
Oxígeno/metabolismo , Transcripción Genética/fisiología , Trichoderma/fisiología , Metabolismo Energético , Perfilación de la Expresión Génica , Genes Fúngicos , Glucosa/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Trichoderma/genética
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