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Estudio de lacasas desplegadas en superficie de saccharomyces cerevisiae en la oxidación de compuestos fenólicos presentes en hidrolizados lignocelulósicos
No full textRESUMEN
Uno de los mayores retos de la actualidad es el desarrollo y producción de energías
renovables y sustentables, que presenten bajo impacto ambiental y que logren contender
con la alta demanda energética. Este interés se ha ido intensificando debido al uso excesivo
de los combustibles fósiles, así como al incremento en los gases de efecto invernadero
(GEI). En los últimos años, se han adoptado estrategias que permitan sustituir a los
combustibles fósiles, en las que se incluyen la energía solar, la eólica y el uso de
biocombustibles.
El bioproceso consolidado es una de las estrategias con mayor potencial en la producción
de bioetanol y su desarrollo puede ser abordado utilizando diferentes enfoques. En este
trabajo se propone el uso de la tecnología de despliegue en superficie de proteínas para
integrar etapas del proceso de bioetanol, lo que permitiría acercarse al bioproceso
consolidado.
La integración de las etapas de detoxificación y fermentación se puede lograr empleando
un sistema levadura/lacasa. La lacasa lleva a cabo la oxidación de los compuestos fenólicos
presentes en los hidrolizados lignocelulósicos, reduciendo su toxicidad lo que favorece el
crecimiento de la levadura y la conversión de azúcares a etanol. No obstante, al desarrollar
esta idea surgieron diferentes preguntas sobre los factores que podrían afectar el proceso
final. Cada una de estas preguntas fueron planteadas a lo largo de los capítulos en los que
se dividió esta tesis, los cuales son descritos a continuación:
Capítulo I
En este capítulo se seleccionó y caracterizó el material lignocelulósico. De los diferentes
materiales disponibles, la cascarilla de arroz es uno de los materiales más interesantes, por
su abundancia, por ser un residuo agrícola sin valor en el mercado de la alimentación
humana y por ser un cultivo importante en el estado de Morelos. La composición química
de este material fue determinada, encontrando 28% de carbohidratos, 12% de lignina y 22%
de cenizas. Este tipo de materiales deben ser pretratados para liberar el mayor porcentaje
de azúcares, por lo que se decidió utilizar el método de ácido diluido. Las condiciones a las
cuales se realiza el pretratamiento favorece la generación de compuestos inhibidores, como
derivados de furano, ácidos alifáticos y compuestos fenólicos. Ya que el interés principal
Resumen
xiii
era la remoción de los compuestos fenólicos, se determinó que factores favorecen la
liberación de dichos compuestos y algunos fueron identificados y cuantificados en el
hidrolizado, por ejemplo, el ácido ferúlico, ácido vanílico y ácido p-cumárico.
Capítulo II
Al conocer qué compuestos estaban presentes en el hidrolizado, se continuó por evaluar el
efecto sobre el crecimiento de levadura del hidrolizado lignocelulósico, observando un
incremento en la fase lag, así como una disminución en el rendimiento de bioetanol
comparado con el cultivo sin hidrolizado. El efecto negativo sobre el crecimiento y
producción de etanol del cultivo en presencia del hidrolizado fue evidente, no obstante,
debido a la complejidad del hidrolizado no fue posible determinar cuál era el compuesto
más tóxico o si todos afectaban de igual manera. Así que, en este capítulo se decidió
evaluar individualmente el efecto de los compuestos fenólicos y determinar su efecto
inhibidor sobre la levadura.
Para ello fueron utilizadas 2 cepas de S. cerevisiae, una levadura comercial para la
producción de cerveza y la levadura utilizada para el despliegue en superficie. Se observó
que el ácido ferúlico afectaba negativamente ambas cepas, y fue el compuesto con mayor
efecto inhibidor de los 8 compuestos probados. Además, se evaluó el efecto biológico de
los compuestos fenólicos sobre modelos de membrana. Encontrando un efecto diferencial
sobre los liposomas dependiente del compuesto probado, esto para probar si los
compuestos fenólicos afectan la membrana celular de la levadura.
Una vez determinado el efecto sobre la célula, se decidió realizar el tratamiento de los
compuestos fenólicos y determinar si los compuestos al ser oxidados reducían o
incrementaban su efecto negativo sobre la levadura. Se evaluó la capacidad oxidativa de 2
lacasas comerciales sobre compuestos fenólicos, observando que el tratamiento con
lacasas del ácido ferúlico y la vainillina favorecía el crecimiento y la producción de etanol.
Mientras que, el tratamiento enzimático de compuestos como el ácido siríngico y el ácido
p-cumárico mostraban un mayor efecto inhibidor al ser tratados con lacasas. Por lo que se
llegó a la conclusión que la detoxificación con lacasas es apropiada para hidrolizados en
los que se encuentren como compuestos mayoritarios el ácido ferúlico o la vainillina.
Resumen
xiv
Capítulo III
Los resultados obtenidos con las enzimas comerciales demostraron la capacidad de las
lacasas de oxidar compuestos fenólicos de diferente naturaleza. El siguiente paso fue
evaluar la lacasa desplegada en superficie de S. cerevisiae en el tratamiento del hidrolizado
de cascarilla de arroz, los porcentajes de oxidación obtenidos fueron entre 1.7-7%. Para
solucionar la baja oxidación, se decidió probar otras lacasas desplegadas en superficie, así
como incrementar la expresión de las mismas. La evolución dirigida de péptidos señal ha
permitido incrementar la expresión de diferentes proteínas de interés, tomando en cuenta
este enfoque se decidió realizar mutaciones sobre el gen Aga2, el cual funciona como
péptido señal y ancla en el sistema de despliegue en superficie. Las variantes obtenidas
del ancla Aga2 incrementaron hasta 8 veces la producción de lacasas desplegadas
comparadas con el ancla silvestre. Estas variantes fueron probadas en la oxidación de
compuestos fenólicos individuales mostrando porcentajes de oxidación entre 20 y 60%.
Hasta el momento no existían reportes en los que se hubieran realizado mutaciones en el
ancla del sistema de despliegue. Con este trabajo se logró mostrar la flexibilidad del ancla
para ser evolucionada y de esta manera mejorar la expresión de proteínas, sin perder su
capacidad de anclaje. Además, se respondieron ciertas interrogantes y se solucionaron
inconvenientes del sistema de despliegue que ahora permitirían continuar con el siguiente
paso que es evaluar el sistema lacasa/levadura en condiciones reales del hidrolizado.SUMMARY
Nowadays, the development and production of renewable and sustainable energies are the
main challenges in the energy field. These energies have to present low environmental
impact and should be enough to the high energy demand. This interest has been intensifying
due to the overuse of fossil fuels and the increase of greenhouse gases. In recent years,
strategies have been adopted to replace fossil fuels, including solar energy, wind energy
and the use of biofuels.
Consolidated bioprocessing is one of the strategies with high potential for bioethanol
production and its development can be approached using different technologies. This work
proposed the use of protein surface display technology to integrate stages of the bioethanol
process, which would allow us to approach consolidated bioprocessing.
Integration of the detoxification and fermentation steps can be accomplished using a
yeast/laccase system. Laccase carries out the oxidation of phenolic compounds present in
lignocellulosic hydrolysates, reducing their toxicity, which promotes yeast growth and the
conversion of sugars to ethanol. However, in developing this idea, different questions
appeared related to the factors that could affect the final process. Each question was posed
throughout the chapters of this thesis, which are described below:
Chapter I
In this chapter, the lignocellulosic material was selected and characterized. Rice husk was
selected based on its abundancy, to be an agricultural residue without human feeding value
and to be an important crop in Morelos state. The chemical composition of this material was
determined, founding 28% carbohydrates, 12% lignin and 22% ashes. This kind of material
has to be pretreated to release the high carbohydrate content possible. It was decided to
use acid-diluted pretreatment. Operational conditions used in the pretreatment step promote
the generation of inhibitory compounds, such as furans derivatives, aliphatic acids and
phenolic compounds. Since the main interest was the removal of phenolic compounds, it
was determined which factors release these compounds and some of them were identified
and quantified in the hydrolysate, for example, ferulic acid, vanillic acid and p-coumaric acid.
Summary
xvi
Chapter II
Once the phenolic compounds were identified in hydrolysate, the effect of these compounds
on yeast growth was evaluated. An increase in lag phase and reduction of ethanol yield was
observed in cultures with the addition of hydrolysate compared to control cultures. The
negative effect on yeast growth and ethanol production of hydrolysate compounds was
evident. However, is not possible to determine which compound shows the greatest toxicity
or if all the compounds affect in the same way, mainly due to the complexity of hydrolysate.
Thus, in this chapter, the biological effect of phenolic compounds was evaluated individually.
Two yeast strains were used, one of them a beer commercial strain and the other the strain
used for yeast surface display. Ferulic acid negatively affects both strains and it was the
most toxic compound of the 8 compounds tested. In order to reduce the negative effects of
these compounds, two commercial laccases were used. Besides, the biological effect of
these compounds on the yeast membrane model was evaluated. Differential effects were
observed on liposomes that are dependent on the phenolic compound tested, this assay
was to probe the effect of phenolic compounds on yeast membrane.
Once the effect on yeast cells was established, laccase treatment of phenolic compounds
was performed to determine if enzymatic treatment reduces or increases the inhibitory
effects of phenolic compounds on yeast. Laccase treatment of ferulic acid and vanillin
reduces their negative effects increasing yeast growth and ethanol yield. While laccase
treatment of syringic acid and p-coumaric acid showed a higher inhibitory effect than the
untreated ones. For that reason, laccase detoxification is an appropriate strategy for
hydrolysates that contain ferulic acid or vanillin as major compounds.
Chapter III
The results obtained in chapter II demonstrated that laccases can oxidize different types of
phenolic compounds. Subsequently, laccase treatment of the rice husk hydrolysate was
performed using a laccase displayed on S. cerevisiae surface, obtaining oxidation
percentages between 1.7-7%. To solve the low oxidation, it was decided to test other
laccases, as well as to increase the expression through directed evolution. Directed
evolution of signal peptides allows to increase the expression of diverse proteins of interest,
thus, based on this approach, directed evolution was performed on the Aga2 gene, which
Summary
xvii
acts as signal peptide and anchor of yeast surface display system. Aga2 mutants increased
up to 8 times the laccase production compared with the Aga2 wild type. Phenolic compounds
were oxidized between 20-60% using these mutants.
Until now, no reports were found in which mutations had been made in the anchor Aga2 to
increase the expression of the protein fused to it. With this work, it was possible to show the
flexibility of the anchor to be evolved and in this way improve the expression of proteins,
without losing its anchoring capacity. Besides, some questions and drawbacks were solved
to continue with the next step that is to evaluate the yeast/laccase system in real hydrolysate
conditions
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
Author Under Sail The Imagination of Jack London, 1893-1902
No full textIn Author Under Sail, Jay Williams offers the first complete literary biography of Jack London as a professional writer engaged in the labor of writing. It examines the authorial imagination in London's work, the use of imagination in both his fiction and nonfiction, and the ways he defined imagination in the creative process in his business dealings with his publishers, editors, and agents. In this first volume of a two-volume biography, Williams traverses the years 1893 to 1902, from London's "Story of a Typhoon" to The People of the Abyss. The Jack London who emerges in the pages of Author Under Sail is a writer whose partnership with publishers, most notably his productive alliance with George Brett of Macmillan, was one of the most formative in American literary history. London pioneered many author models during the heyday of realism and naturalism, blurring the boundaries of these popular genres by focusing on absorption and theatricality and the representation of the seen and unseen. London created an impassioned, sincere, and extremely personal realism unlike that of other American writers of the time. Author Under Sail is a literary tour de force that reveals the full range of London as writer, creative citizen, and entrepreneur at the same time it sheds light on the maverick side of machine-age literature.Intro -- Title Page -- Copyright Page -- Dedication -- Contents -- Acknowledgments -- Introduction -- 1. Spirit Truth -- 2. From Absorption to Theatricality and Back Again -- 3. "I Will Build a New Present" -- 4. Sons as Authors -- 5. Fathers as Publishers -- 6. The Daughter as Author -- 7. Lovers as Authors -- 8. At Sea with the Family -- 9. Yellow News, Yellow Stories -- 10. The Return Home -- Notes -- Bibliography -- Index -- About Jay WilliamsIn Author Under Sail, Jay Williams offers the first complete literary biography of Jack London as a professional writer engaged in the labor of writing. It examines the authorial imagination in London's work, the use of imagination in both his fiction and nonfiction, and the ways he defined imagination in the creative process in his business dealings with his publishers, editors, and agents. In this first volume of a two-volume biography, Williams traverses the years 1893 to 1902, from London's "Story of a Typhoon" to The People of the Abyss. The Jack London who emerges in the pages of Author Under Sail is a writer whose partnership with publishers, most notably his productive alliance with George Brett of Macmillan, was one of the most formative in American literary history. London pioneered many author models during the heyday of realism and naturalism, blurring the boundaries of these popular genres by focusing on absorption and theatricality and the representation of the seen and unseen. London created an impassioned, sincere, and extremely personal realism unlike that of other American writers of the time. Author Under Sail is a literary tour de force that reveals the full range of London as writer, creative citizen, and entrepreneur at the same time it sheds light on the maverick side of machine-age literature.Description based on publisher supplied metadata and other sources.Electronic reproduction. Ann Arbor, Michigan : ProQuest Ebook Central, YYYY. Available via World Wide Web. Access may be limited to ProQuest Ebook Central affiliated libraries
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