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Dilute sulfuric acid (DA), sulfur dioxide (SO(2)), liquid hot water (LHW), soaking in aqueous ammonia (SAA), ammonia fiber expansion (AFEX), and lime pretreatments were applied to Alamo, Dacotah, and Shawnee switchgrass. Application of the same analytical methods and material balance approaches facilitated meaningful comparisons of glucose and xylose yields from combined pretreatment and enzymatic hydrolysis. Use of a common supply of cellulase, beta-glucosidase, and xylanase also eased comparisons. All pretreatments enhanced sugar recovery from pretreatment and subsequent enzymatic hydrolysis substantially compared to untreated switchgrass. Adding beta-glucosidase was effective early in enzymatic hydrolysis while cellobiose levels were high but had limited effect on longer term yields at the enzyme loadings applied. Adding xylanase improved yields most for higher pH pretreatments where more xylan was left in the solids. Harvest time had more impact on performance than switchgrass variety, and microscopy showed changes in different features could impact performance by different pretreatments.
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Biotecnología/métodos , Carbohidratos/biosíntesis , Enzimas/metabolismo , Panicum/metabolismo , Amoníaco/metabolismo , Carbohidratos/análisis , Carbohidratos/aislamiento & purificación , Celulasa/metabolismo , Ecotipo , Endo-1,4-beta Xilanasas/metabolismo , Glucosa/análisis , Hidrólisis , Panicum/citología , Panicum/ultraestructura , Estaciones del Año , Xilosa/análisis , beta-Glucosidasa/metabolismoRESUMEN
This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.
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Grass species represent a major source of food, feed, and fiber crops and potential feedstocks for biofuel production. Most of the biomass is contributed by cell walls that are distinct in composition from all other flowering plants. Identifying cell wall-related genes and their functions underpins a fundamental understanding of growth and development in these species. Toward this goal, we are building a knowledge base of the maize (Zea mays) genes involved in cell wall biology, their expression profiles, and the phenotypic consequences of mutation. Over 750 maize genes were annotated and assembled into gene families predicted to function in cell wall biogenesis. Comparative genomics of maize, rice (Oryza sativa), and Arabidopsis (Arabidopsis thaliana) sequences reveal differences in gene family structure between grass species and a reference eudicot species. Analysis of transcript profile data for cell wall genes in developing maize ovaries revealed that expression within families differed by up to 100-fold. When transcriptional analyses of developing ovaries before pollination from Arabidopsis, rice, and maize were contrasted, distinct sets of cell wall genes were expressed in grasses. These differences in gene family structure and expression between Arabidopsis and the grasses underscore the requirement for a grass-specific genetic model for functional analyses. A UniformMu population proved to be an important resource in both forward- and reverse-genetics approaches to identify hundreds of mutants in cell wall genes. A forward screen of field-grown lines by near-infrared spectroscopic screen of mature leaves yielded several dozen lines with heritable spectroscopic phenotypes. Pyrolysis-molecular beam mass spectrometry confirmed that several nir mutants had altered carbohydrate-lignin compositions.
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Pared Celular/genética , Pared Celular/fisiología , Zea mays/genética , Arabidopsis/genética , Metabolismo de los Hidratos de Carbono/genética , Carbohidratos/biosíntesis , Elementos Transponibles de ADN/genética , Flores/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Redes y Vías Metabólicas/genética , Datos de Secuencia Molecular , Familia de Multigenes/genética , Mutagénesis Insercional/genética , Mutación/genética , Nucleótidos/metabolismo , Oryza/genética , Fenotipo , Propanoles/metabolismo , Especificidad por Sustrato/genética , Zea mays/citologíaRESUMEN
Oil prices and government mandates have catalyzed rapid growth of nonfossil transportation fuels in recent years, with a large focus on ethanol from energy crops, but the food crops used as first-generation energy crops today are not optimized for this purpose. We show that the theoretical efficiency of conversion of whole spectrum solar energy into biomass is 4.6-6%, depending on plant type, and the best year-long efficiencies realized are about 3%. The average leaf is as effective as the best PV solar cells in transducing solar energy to charge separation (ca. 37%). In photosynthesis, most of the energy that is lost is dissipated as heat during synthesis of biomass. Unlike photovoltaic (PV) cells this energetic cost supports the construction, maintenance, and replacement of the system, which is achieved autonomously as the plant grows and re-grows. Advances in plant genomics are being applied to plant breeding, thereby enabling rapid development of next-generation energy crops that capitalize on theoretical efficiencies while maintaining environmental and economic integrity.
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Productos Agrícolas/metabolismo , Fuentes Generadoras de Energía , Fuentes de Energía Bioeléctrica/tendencias , Biomasa , Biotecnología , Cruzamiento , Productos Agrícolas/genética , Productos Agrícolas/crecimiento & desarrollo , Productos Agrícolas/efectos de la radiación , Lignina/metabolismo , Modelos Biológicos , Fotosíntesis , Energía SolarRESUMEN
Endoglucanase E1 from Acidothermus cellulolyticus was expressed cytosolically under control of the cauliflower mosaic virus 35S promoter in transgenic duckweed, Lemna minor 8627 without any obvious observable phenotypic effects on morphology or rate of growth. The recombinant enzyme co-migrated with the purified catalytic domain fraction of the native E1 protein on western blot analysis, revealing that the cellulose-binding domain was cleaved near or in the linker region. The duckweed-expressed enzyme was biologically active and the expression level was up to 0.24% of total soluble protein. The endoglucanase activity with carboxymethylcellulose averaged 0.2 units mg protein(-1) extracted from fresh duckweed. The optimal temperature and pH for E1 enzyme activity were about 80 degrees C and pH 5, respectively. While extraction with HEPES (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) buffer (pH 8) resulted in the highest recovery of total soluble proteins and E1 enzyme, extraction with citrate buffer (pH 4.8) at 65 degrees C enriched relative amounts of E1 enzyme in the extract. This study demonstrates that duckweed may offer new options for the expression of cellulolytic enzymes in transgenic plants.
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Celulasa/metabolismo , Bacterias Grampositivas/enzimología , Plantas Modificadas Genéticamente , Tampones (Química) , Celulasa/genética , Celulasa/aislamiento & purificación , Concentración de Iones de Hidrógeno , TemperaturaRESUMEN
An attempt was made to obtain a high-level production of intact Acidothermus cellulolyticus endoglucanase (E1) in transgenic tobacco plants. The E1 expression was examined under the control of the constitutive and strong Mac promoter or light-inducible tomato Rubisco small sub-unit (RbcS-3C) promoter with its original or Alfalfa Mosaic Virus (AMV) RNA4 5'-untranslated leader (UTL) and targeted to different sub-cellular compartments via transit peptides. The transit peptides included native E1, endoplasmic reticulum, vacuole, apoplast, and chloroplast. E1 expression and its stability in transgenic plants were determined via E1 activity, protein immunoblotting, and RNA gel-blotting analyses. Effects of sub-cellular compartments on E1 production and its stability were determined in transgenic tobacco plants carrying one of six transgene expression vectors, where the E1 was under the control of Mac promoter, mannopine synthase transcription terminator, and one of the five transit peptides. Transgenic tobacco plants with an apoplastic transit peptide had the highest average E1 activity and protein accumulation, which was about 0.25% of total leaf soluble proteins estimated via E1 specific activity and protein gel blots. Intercellular fluid analyses confirmed that E1 signal peptide functioned properly in tobacco cells to secret E1 protein into the apoplast. By replacing RbcS-3C UTL with AMV RNA4 UTL E1 production was enhanced more than twofold, while it was less effective than the mannopine synthase UTL. It was observed that RbcS-3C promoter was more favorable for E1 expression in transgenic plants than the Mac promoter. E1 activity in dried tobacco seeds stored one year at room temperature was 45% higher than that observed immediately after harvesting, suggesting that E1 protein can be stored at room temperature for a long period. E1 stability in different sub-cellular compartments and the optimal combination of promoter, 5'-UTL, and sub-cellular compartmentation for heterologous protein production in transgenic plants are discussed.
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Actinomycetales/enzimología , Celulasas/biosíntesis , Nicotiana/enzimología , Nicotiana/genética , Regiones no Traducidas 5' , Actinomycetales/genética , Virus del Mosaico de la Alfalfa/genética , Secuencia de Bases , Celulasas/genética , ADN Recombinante/genética , Estabilidad de Enzimas , Expresión Génica , Genes Bacterianos , Vectores Genéticos , Datos de Secuencia Molecular , Orgánulos/enzimología , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
Screening of the Merck compound collection identified 6 as an unusually simple, low molecular weight hit with moderate affinity for GABAA receptors. The structural novelty of 6, compared to our advanced series of GABAA alpha5 inverse agonists, made it an attractive molecule for further exploration. This paper will describe the evolution of 6 into a new series of ligands with nanomolar affinity and functional selectivity for GABAA alpha5 receptor subtypes.
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Piridazinas/síntesis química , Receptores de GABA-A/efectos de los fármacos , Animales , Línea Celular , Humanos , Técnicas In Vitro , Ligandos , Masculino , Ratones , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Técnicas de Placa-Clamp , Subunidades de Proteína/fisiología , Piridazinas/farmacocinética , Piridazinas/farmacología , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/fisiología , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
The binding affinities at the human NK-1 receptor of two spirocyclic compounds were found to be similar despite being epimeric at a key stereocentre. This unexpected result prompted a thorough investigation of the solution conformations of the two compounds. This revealed that a conformational switch in the tetrahydrofuran ring enabled the C-3-aryl group to be equatorial in both cases, leading to a similar juxtaposition of the aryl rings.
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Espectroscopía de Resonancia Magnética/métodos , Antagonistas del Receptor de Neuroquinina-1 , Compuestos de Espiro/química , Cristalografía por Rayos X , Espectroscopía de Resonancia Magnética/normas , Modelos Moleculares , Conformación Molecular , Protones , Compuestos de Espiro/farmacología , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Techniques such as mass spectrometry and NMR spectroscopy form an essential part of the medicinal chemist's toolbox for characterizing and assessing the purity of new molecules. Empowering medicinal chemists to gain early insight into their reaction products has a direct impact on productivity. Devolution of cutting-edge techniques from the specialist to the bench chemist also frees the specialist to concentrate on solving the more demanding of analytical problems. For open-access techniques to be taken up, they must be robust and be able to handle differing sample concentrations and varying sample complexities. This paper details the implementation of high-resolution liquid chromatography/mass spectrometry in open access to aid the medicinal chemist in characterizing desired products and identifying unexpected rearrangements, by-products and complete unknowns.
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Química Farmacéutica/métodos , Evaluación Preclínica de Medicamentos/métodos , Preparaciones Farmacéuticas/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Química Farmacéutica/instrumentación , Cromatografía Líquida de Alta Presión/métodos , Humanos , Preparaciones Farmacéuticas/síntesis química , Espectrometría de Masa por Ionización de Electrospray/instrumentaciónRESUMEN
New, rapid, and inexpensive methods that monitor the chemical composition of corn stover and corn stover-derived samples are a key element to enabling the commercialization of processes that convert stover to fuels and chemicals. These new techniques combine near infrared (NIR) spectroscopy and projection to latent structures (PLS) multivariate analysis to allow the compositional analysis of hundreds of samples in 1 d at a cost of about $10 each. The new NIR/PLS rapid analysis methods can also be used to support a variety of research projects that would have been too costly to pursue by traditional methods.
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Alimentación Animal/análisis , Biomasa , Etanol/metabolismo , Zea mays/química , Biotecnología/métodosRESUMEN
Recent developments in the technologies and approaches to identify metabolites in a drug discovery environment are reviewed. Samples may be generated using either in vitro systems--typically, but not exclusively, liver subcellular fractions, such as microsomes, or whole cells, such as hepatocytes. Alternatively, metabolites are generated in vivo using excreta obtained following dosing in preclinical species. Recombinant drug metabolizing enzymes or microorganisms may offer alternate vectors. New techniques, such as the use of solid-phase microextraction, have found application in the isolation of metabolites from biological matrices. However, this is still dominated by the use of preparative chromatography, which has advanced through the use of mass-directed detection. Detection and structural elucidation by mass spectrometry have improved markedly with increases in sensitivity, allowing lower abundance metabolites to be detected, and increases in selectivity, with the use of high-resolution time-of-flight and quadrupole-time-of-flight instruments. Finally, higher field strength magnets coupled with novel probe designs and increased use of liquid chromatographic hyphenation techniques continue to drive the capabilities of nuclear magnetic resonance spectroscopy as the definitive structural elucidation tool.