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1.
Appl Microbiol Biotechnol ; 99(22): 9591-604, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26156238

RESUMEN

Trichoderma filamentous fungi have been investigated due to their ability to secrete cellulases which find various biotechnological applications such as biomass hydrolysis and cellulosic ethanol production. Previous studies demonstrated that Trichoderma harzianum IOC-3844 has a high degree of cellulolytic activity and potential for biomass hydrolysis. However, enzymatic, biochemical, and structural studies of cellulases from T. harzianum are scarce. This work reports biochemical characterization of the recombinant endoglucanase I from T. harzianum, ThCel7B, and its catalytic core domain. The constructs display optimum activity at 55 °C and a surprisingly acidic pH optimum of 3.0. The full-length enzyme is able to hydrolyze a variety of substrates, with high specific activity: 75 U/mg for ß-glucan, 46 U/mg toward xyloglucan, 39 U/mg for lichenan, 26 U/mg for carboxymethyl cellulose, 18 U/mg for 4-nitrophenyl ß-D-cellobioside, 16 U/mg for rye arabinoxylan, and 12 U/mg toward xylan. The enzyme also hydrolyzed filter paper, phosphoric acid swollen cellulose, Sigmacell 20, Avicel PH-101, and cellulose, albeit with lower efficiency. The ThCel7B catalytic domain displays similar substrate diversity. Fluorescence-based thermal shift assays showed that thermal stability is highest at pH 5.0. We determined kinetic parameters and analyzed a pattern of oligosaccharide substrates hydrolysis, revealing cellobiose as a final product of C6 degradation. Finally, we visualized effects of ThCel7B on oat spelt using scanning electron microscopy, demonstrating the morphological changes of the substrate during the hydrolysis. The acidic behavior of ThCel7B and its considerable thermostability hold a promise of its industrial applications and other biotechnological uses under extremely acidic conditions.


Asunto(s)
Metabolismo de los Hidratos de Carbono , Celulasas/genética , Celulasas/metabolismo , Celulosa/metabolismo , Trichoderma/enzimología , Celulasas/química , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Temperatura , Trichoderma/genética
2.
Protein Expr Purif ; 99: 35-42, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24680731

RESUMEN

Recent advances in DNA sequencing techniques have led to an explosion in the amount of available genome sequencing data and this provided an inexhaustible source of uncharacterized glycoside hydrolases (GH) to be studied both structurally and enzymatically. Ligation-Independent Cloning (LIC), an interesting alternative to traditional, restriction enzyme-based cloning, and commercial recombinatorial cloning, was adopted and optimized successfully for a high throughput cloning, expression and purification pipeline. Using this platform, 130 genes encoding mainly uncharacterized glycoside hydrolases from 13 different organisms were cloned and submitted to a semi-automated protein expression and solubility screening in Escherichia coli, resulting in 73 soluble targets. The high throughput approach proved to be a powerful tool for production of recombinant glycoside hydrolases for further structural and biochemical characterization and confirmed that thioredoxin fusion tag (TRX) is a better choice to increase solubility of recombinant glycoside hydrolases expressed in E. coli, when compared to His-tag alone.


Asunto(s)
Clonación Molecular/métodos , Glicósido Hidrolasas/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Vectores Genéticos , Glicósido Hidrolasas/biosíntesis , Secuenciación de Nucleótidos de Alto Rendimiento , Proteínas Recombinantes de Fusión/genética , Solubilidad , Tiorredoxinas/química , Tiorredoxinas/genética
3.
Fungal Genet Biol ; 48(8): 806-11, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21396477

RESUMEN

Agrobacterium tumefaciens is widely used for plant DNA transformation and more recently, has also been used to transform yeast, filamentous fungi and even human cells. Using this technique, we developed the first transformation protocol for the saprobic aquatic fungus Blastocladiella emersonii, a Blastocladiomycete localized at the base of fungal phylogenetic tree, which has been shown as a promising and interesting model of study of cellular function and differentiation. We constructed binary T-DNA vectors containing hygromycin phosphotransferase (hph) or enhanced green fluorescent protein (egfp) genes, under the control of Aspergillus nidulans trpC promoter and terminator sequences. 24 h of co-cultivation in induction medium (IM) agar plates, followed by transfer to PYG-agar plates containing cefotaxim to kill Agrobacterium tumefsciens and hygromycin to select transformants, resulted in growth and sporulation of resistant transformants. Genomic DNA from the pool o resistant zoospores were shown to contain T-DNA insertion as evidenced by PCR amplification of hph gene. Using a similar protocol we could also evidence the expression of enhanced green fluorescent protein (EGFP) in zoospores derived from transformed cells. This protocol can also open new perspectives for other non-transformable closely related fungi, like the Chytridiomycete class.


Asunto(s)
Agrobacterium tumefaciens/genética , Blastocladiella/genética , Transformación Genética , Agrobacterium tumefaciens/crecimiento & desarrollo , Blastocladiella/efectos de los fármacos , Blastocladiella/crecimiento & desarrollo , ADN Bacteriano/genética , Resistencia a Medicamentos , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/genética , Higromicina B/farmacología , Microscopía Confocal , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Reacción en Cadena de la Polimerasa , Microbiología del Agua
4.
Eukaryot Cell ; 9(6): 915-25, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20418381

RESUMEN

Global gene expression analysis was carried out with Blastocladiella emersonii cells subjected to oxygen deprivation (hypoxia) using cDNA microarrays. In experiments of gradual hypoxia (gradual decrease in dissolved oxygen) and direct hypoxia (direct decrease in dissolved oxygen), about 650 differentially expressed genes were observed. A total of 534 genes were affected directly or indirectly by oxygen availability, as they showed recovery to normal expression levels or a tendency to recover when cells were reoxygenated. In addition to modulating many genes with no putative assigned function, B. emersonii cells respond to hypoxia by readjusting the expression levels of genes responsible for energy production and consumption. At least transcriptionally, this fungus seems to favor anaerobic metabolism through the upregulation of genes encoding glycolytic enzymes and lactate dehydrogenase and the downregulation of most genes coding for tricarboxylic acid (TCA) cycle enzymes. Furthermore, genes involved in energy-costly processes, like protein synthesis, amino acid biosynthesis, protein folding, and transport, had their expression profiles predominantly downregulated during oxygen deprivation, indicating an energy-saving effort. Data also revealed similarities between the transcriptional profiles of cells under hypoxia and under iron(II) deprivation, suggesting that Fe(2+) ion could have a role in oxygen sensing and/or response to hypoxia in B. emersonii. Additionally, treatment of fungal cells prior to hypoxia with the antibiotic geldanamycin, which negatively affects the stability of mammalian hypoxia transcription factor HIF-1alpha, caused a significant decrease in the levels of certain upregulated hypoxic genes.


Asunto(s)
Blastocladiella/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Blastocladiella/metabolismo , Hipoxia de la Célula , Proteínas Fúngicas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Oxígeno/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación hacia Arriba
5.
Biochimie ; 148: 107-115, 2018 May.
Artículo en Inglés | MEDLINE | ID: mdl-29555372

RESUMEN

Bifidobacterium is an important genus of probiotic bacteria colonizing the human gut. These bacteria can uptake oligosaccharides for the fermentative metabolism of hexoses and pentoses, producing lactate, acetate as well as short-chain fatty acids and propionate. These end-products are known to have important effects on human health. ß-glucosidases (EC 3.2.1.21) are pivotal enzymes for the metabolism and homeostasis of Bifidobacterium, since they hydrolyze small and soluble saccharides, typically producing glucose. Here we describe the cloning, expression, biochemical characterization and the first X-ray structure of a GH3 ß-glucosidase from the probiotic bacteria Bifidobacterium adolescentis (BaBgl3). The purified BaBgl3 showed a maximal activity at 45 °C and pH 6.5. Under the optimum conditions, BaBgl3 is highly active on 4-nitrophenyl-ß-d-glucopyranoside (pNPG) and, at a lesser degree, on 4-nitrophenyl-ß-d-xylopyranoside (pNPX, about 32% of the activity observed for pNPG). The 2.4 Šresolution crystal structure of BaBgl3 revealed a three-domain structure composed of a TIM barrel domain, which together with α/ß sandwich domain accommodate the active site and a third C-terminal fibronectin type III (FnIII) domain with unknown function. Modeling of the substrate in the active site indicates that an aspartate interacts with the hydroxyl group of the C6 present in pNPG but absent in pNPX, which explains the substrate preference. Finally, the enzyme is significantly stabilized by glycerol and galactose, resulting in considerable increase in the enzyme activity and its lifetime. The structural and biochemical studies presented here provide a deeper understanding of the molecular mechanisms of complex carbohydrates degradation utilized by probiotic bacteria as well as for the development of new prebiotic oligosaccharides.


Asunto(s)
Bifidobacterium adolescentis/enzimología , Probióticos , beta-Glucosidasa/química , beta-Glucosidasa/metabolismo , Cristalografía por Rayos X , Modelos Moleculares , Conformación Proteica , Especificidad por Sustrato
6.
Sci Rep ; 8(1): 3678, 2018 02 27.
Artículo en Inglés | MEDLINE | ID: mdl-29487297

RESUMEN

The glycoside hydrolase family 45 (GH45) of carbohydrate modifying enzymes is mostly comprised of ß-1,4-endoglucanases. Significant diversity between the GH45 members has prompted the division of this family into three subfamilies: A, B and C, which may differ in terms of the mechanism, general architecture, substrate binding and cleavage. Here, we use a combination of X-ray crystallography, bioinformatics, enzymatic assays, molecular dynamics simulations and site-directed mutagenesis experiments to characterize the structure, substrate binding and enzymatic specificity of the GH45 subfamily C endoglucanase from Phanerochaete chrysosporium (PcCel45A). We investigated the role played by different residues in the binding of the enzyme to cellulose oligomers of different lengths and examined the structural characteristics and dynamics of PcCel45A that make subfamily C so dissimilar to other members of the GH45 family. Due to the structural similarity shared between PcCel45A and domain I of expansins, comparative analysis of their substrate binding was also carried out. Our bioinformatics sequence analyses revealed that the hydrolysis mechanisms in GH45 subfamily C is not restricted to use of the imidic asparagine as a general base in the "Newton's cradle" catalytic mechanism recently proposed for this subfamily.


Asunto(s)
Celulasa/química , Celulasa/metabolismo , Phanerochaete/enzimología , Catálisis , Biología Computacional , Cristalografía por Rayos X , Pruebas de Enzimas , Simulación de Dinámica Molecular
7.
J Comput Biol ; 23(1): 27-9, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26540331

RESUMEN

Following burgeoning genomic and transcriptomic sequencing data, biochemical and molecular biology groups worldwide are implementing high-throughput cloning and mutagenesis facilities in order to obtain a large number of soluble proteins for structural and functional characterization. Since manual primer design can be a time-consuming and error-generating step, particularly when working with hundreds of targets, the automation of primer design process becomes highly desirable. HTP-OligoDesigner was created to provide the scientific community with a simple and intuitive online primer design tool for both laboratory-scale and high-throughput projects of sequence-independent gene cloning and site-directed mutagenesis and a Tm calculator for quick queries.


Asunto(s)
Clonación Molecular/métodos , Cartilla de ADN/genética , Mutagénesis Sitio-Dirigida/métodos , Programas Informáticos , Algoritmos , Secuencia de Bases
8.
Acta Crystallogr F Struct Biol Commun ; 72(Pt 4): 288-93, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-27050262

RESUMEN

Given the current interest in second-generation biofuels, carbohydrate-active enzymes have become the most important tool to overcome the structural recalcitrance of the plant cell wall. While some glycoside hydrolase families have been exhaustively described, others remain poorly characterized, especially with regard to structural information. The family 43 glycoside hydrolases are a diverse group of inverting enzymes; the available structure information on these enzymes is mainly from xylosidases and arabinofuranosidase. Currently, only one structure of an exo-ß-1,3-galactanase is available. Here, the production, crystallization and structure determination of a putative exo-ß-1,3-galactanase from Bifidobacterium bifidum S17 (BbGal43A) are described. BbGal43A was successfully produced and showed activity towards synthetic galactosides. BbGal43A was subsequently crystallized and data were collected to 1.4 Šresolution. The structure shows a single-domain molecule, differing from known homologues, and crystal contact analysis predicts the formation of a dimer in solution. Further biochemical studies are necessary to elucidate the differences between BbGal43A and its characterized homologues.


Asunto(s)
Bifidobacterium bifidum/enzimología , Enzimas/metabolismo , Galactanos/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Cristalografía por Rayos X , Enzimas/química , Conformación Proteica , Homología de Secuencia de Aminoácido
9.
Sci Rep ; 6: 23473, 2016 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-27032335

RESUMEN

Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials. Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity. Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms. The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite. This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme.


Asunto(s)
Bacillus licheniformis/enzimología , Proteínas Bacterianas/química , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Celulosa/análogos & derivados , Celulosa/metabolismo , Secuencia de Consenso , Cristalografía por Rayos X , Modelos Moleculares , Simulación de Dinámica Molecular , Movimiento (Física) , Mutagénesis Sitio-Dirigida , Filogenia , Conformación Proteica , Dominios Proteicos , Proteínas Recombinantes/química , Dispersión del Ángulo Pequeño , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Tetrosas/metabolismo , Difracción de Rayos X
10.
Genome Announc ; 3(3)2015 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-25977443

RESUMEN

Here, we present the draft genome sequence of Thermus filiformis strain ATCC 43280, a thermophile bacterium capable of producing glycosylated carotenoids acylated with branched fatty acids and enzymes of biotechnological potential.

12.
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
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