Identifica????o de genes envolvidos na degrada????o de xilana por meio de abordagens gen??mica e metagen??mica

Autor: Schroeder, Lu??s Felipe
Jazyk: portugalština
Rok vydání: 2014
Předmět:
Zdroj: Biblioteca Digital de Teses e Dissertações da UCBUniversidade Católica de BrasíliaUCB.
Druh dokumentu: masterThesis
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There are different process being developed for cellulosic ethanol production, with possible different pretreatments with varying temperatures and pH, in addition to several biomasses can be used as the source of fermentable sugars. Among the important enzymes for deconstruction of plant biomass, stand out xylanases. These enzymes are responsible for deconstruction of the hemicellulose present in the structure of the plant cell walls. There are several ways to accomplish the identification of these enzymes: purification from an isolated microorganism is one. In this study, genomic and metagenomic approaches were used to carry out the prospection of the genes responsible for coding these enzymes. Clones from two libraries were used for detection and evaluation of activity on solid medium, supplemented with xylan and acid pretreated sugarcane bagasse. Nineteen clones of a goat rumen metagenomic library and five clones from an AB60 bacterium genomic library, with 15,000 clones constructed in this study, were selected initially. Fourteen clones from the metagenomic library were completely sequenced and their ORFs were analyzed. Four clones from the genomic library were partially sequenced and one clone had its sequence completely determined and 104 ORFs were obtained for all clones completely or partially sequenced ORFs were analyzed. Eleven ORFs showed some similarity to genes of importance for the degradation of complex polysaccharides. Among the most important ORFs and most likely to be related to the detected activity, are genes coding for ??-glucosidase, ??-xylosidase and ??-glucuronidase. Furthermore, also other ORFs with lower probability of relation with the activity or necessity to full sequencing of the clones for a few more conclusive analysis were identified. About 40% of the ORFs present in the rumen clones and 37.8% of the ORFs present in Acidobacteria clones showed similarity with hypothetical or uncharacterized proteins, which could be important in the activity detected. A ??-glucuronidase gene detected in a clone from the goat rumen metagenomic library was synthesized, its sequence was optimized for expression in Escherichia coli. However, in the present work, it was not possible to sub-cloning, made expression and purification of this enzyme. Some ORFs detected can be used for future studies of expression and characterization in order to improve knowledge about biotechnological potential present in the rumen and acidobacteria AB60, besides the ecological role of these microorganisms in their environment.
Existem diversos processos em desenvolvimento para a produ????o de etanol celul??sico, havendo diferentes poss??veis pr??-tratamentos, com temperaturas e pH variados, al??m das diversas biomassas que podem ser utilizadas como fonte de a????cares ferment??veis. Dentre as enzimas importantes para a desconstru????o de biomassa vegetal, destacam-se as xilanases. Estas enzimas s??o respons??veis pela desconstru????o da estrutura hemicelul??sica presente na parede celular das plantas. H?? diversas maneiras para alcan??ar a identifica????o destas enzimas: purifica????o a partir de micro-organismo isolado sendo uma delas. No presente trabalho, foram utilizadas abordagens gen??mica e metagen??mica a fim de realizar a prospec????o dos genes respons??veis pela codifica????o para estas enzimas. Foram utilizados clones oriundos de duas bibliotecas para a detec????o e avalia????o da atividade em meio s??lido suplementado com xilana e baga??o de cana-de-a????car pr??-tratado com ??cido. Dezenove clones de uma biblioteca metagen??mica de r??men de caprinos e cinco clones de uma biblioteca gen??mica da bact??ria AB60, com 15.000 clones constru??da no presente trabalho, foram selecionados inicialmente. Quatorze clones da biblioteca metagen??mica foram sequenciados completamente e tiveram as suas ORFs analisadas. Quatro clones da biblioteca gen??mica foram parcialmente sequenciados e um clone teve a sua sequ??ncia completa determinada e as ORFs analisadas. Das 104 ORFs obtidas de todos os clones completamente ou parcialmente sequenciados, onze ORFs apresentaram alguma similaridade com genes de import??ncia para a degrada????o de polissacar??deos complexos. Dentre as ORFs de maior import??ncia e com maior probabilidade de estarem relacionadas com a atividade detectada, est??o genes codificantes para ??-glicosidase, ??-xilosidase e ??-glicuronidase. Al??m disso, tamb??m foram identificadas outras ORFs com menor probabilidade de rela????o com a atividade ou necessidade de sequenciamento completo de alguns dos clones para uma an??lise mais conclusiva. Cerca de 40% das ORFs presentes nos clones selecionados de r??men e 37,8% das ORFs presentes nos clones selecionados de Acidobacteria apresentaram similaridade com prote??nas hipot??ticas ou prote??nas n??o caracterizadas que podem ter import??ncia na atividade detectada. Um gene de ??-glicuronidase detectado em um clone da biblioteca metagen??mica de r??men de caprino foi sintetizado, otimizando-se sua sequ??ncia para express??o em Escherichia coli. Entretanto, n??o foi poss??vel a sub-clonagem, express??o e purifica????o desta enzima no presente trabalho. Algumas ORFs detectadas podem ser utilizadas para estudos futuros de express??o e caracteriza????o a fim de aprimorar o conhecimento a respeito do potencial biotecnol??gico presente no r??men e na Acidobact??ria AB60, al??m do papel ecol??gico destes micro-organismos em seu ambiente.
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