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Previous issue date: 2014-08-22 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / Os microrganismos apresentam uma imensa diversidade gen?tica e est?o presentes em toda biosfera, no entanto cerca de 1% das esp?cies pode ser cultivada por t?cnicas laboratoriais padr?o. A metagen?mica tornou poss?vel o acesso direto ao genoma microbiano derivado de amostras ambientais, utilizando t?cnicas independentes de cultivo. A metodologia permite obter informa??o funcional de prote?nas, assim como a identifica??o de potenciais produtos com interesse biotecnol?gico e de novos recursos biol?gicos industrialmente explor?veis, a exemplo novas solu??es para impactos ambientais. ?reas contaminadas com petr?leo s?o caracterizadas por um grande ac?mulo de hidrocarbonetos e surfactantes s?o utilizados para sua biorremedia??o. Sendo assim, a abordagem metagen?nima foi utilizada para selecionar genes envolvidos no processo de degrada??o e biossurfacta??o de hidrocarbonetos. Em um trabalho anterior, o DNA ambiental (eDNA) foi extra?do de amostras de solo coletadas em duas diferentes ?reas (Caatinga e rio salino) do Rio Grande do Norte (Brasil), as bibliotecas metagen?micas foram constru?das e analisadas funcionalmente. Os clones capazes de degradar o ?leo foram avaliados quanto ? capacidade de sintetizar biossurfactante. O clone foi sequenciado e an?lise da sequencia revelou uma ORF com 897 pb, 298 amino?cidos e prote?na de peso molecular pr?ximo a 34 kDa. A busca por homologia no GenBank revelou similaridade com a sequ?ncia que codifica uma prote?na hipot?tica de representantes da fam?lia Halobacteriaceae, que foram mostradas recentemente como produtoras de biossurfactantes. A presen?a da sequ?ncia codificante inserida e do fen?tipo adquirido foram confirmadas. Primers foram desenhados e suas ORFs amplificadas por PCR. Em seguida, foram subclonadas em vetor de express?o pETDuet-1, contendo uma cauda de histidina, para express?o e posterior purifica??o da prote?na de interesse. Os testes foram realizados para confirma??o da atividade de biossurfactante e degrada??o de hidrocarbonetos e apresentaram resultados positivos. O ensaio de imunodetec??o (western blot) com a utiliza??o do anticorpo monoclonal Anti-His? confirmou a presen?a da prote?na ambiental. Esse estudo foi o primeiro a relatar uma poss?vel prote?na com atividade biossurfactante obtida a partir de uma abordagem metagen?mica. / The microorganisms have a vast genetic diversity and they are present
throughout the biosphere, however, only about 1% of the species can be
cultivated by traditional cultivation techniques. Within this diversity there is a
huge pool genetic and biological being explored. The metagenomics has
enabled direct access to microbial genome derived from environmental samples
using independent methods of cultivation. The methodology enables to obtain
functional information about the proteins, as well as identify potential products
with biotechnological interest and new industrially exploitable biological
resources, such as new solutions to environmental impacts. Oil-contaminated
areas are characterized by a large accumulation of hydrocarbons and
surfactants may be used for bioremediation. Thus, the metagenomic approach
was used in this study in order to select genes involved in the degradation and
hydrocarbon emulsification. In a previous work, the environmental DNA (eDNA)
was extracted from soil samples collected from two different areas (Caatinga
and Saline River) of Rio Grande do Norte (Brazil), the metagenomic libraries
were constructed and functionally analyzed. The clone able to degrade the oil
was evaluated for the ability to synthesize biosurfactants. The sequence
analysis revealed an ORF with 897 bp, 298 amino acids and a protein with
around 34 kDa. The search for homology in GenBank revealed sequence
similarity with a hypothetical protein of representatives Halobacteriaceae family,
who were recently shown as strains producing biosurfactants. The presence of
the inserted coding sequence and the acquired phenotype was confirmed.
Primers were designed and the ORF amplified by PCR. The ORF was
subcloned into pETDuet-1 expression vector for subsequent purification of the
protein of interest containing a histidine tail. The tests performed to confirm the
biosurfactant activity and the ability of hydrocarbon degradation showed positive
results. The immunodetection test (western blot) using the monoclonal AntiHis?
confirmed the presence of the environmental protein. This study was the
first to report a possible protein with biosurfactant activity obtained from a
metagenomic approach
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.ufrn.br:123456789/19512 |
| Date | 22 August 2014 |
| Creators | Ara?jo, Sinara Carla da Silva |
| Contributors | 00297997750, http://lattes.cnpq.br/1083882171718362, Lanza, Daniel Carlos Ferreira, 05372896663, http://lattes.cnpq.br/6851351991421755, Duarte, F?bio Teixeira, 03063680486, http://lattes.cnpq.br/4371235140013662, Agnez, Lucymara Fassarella |
| Publisher | Universidade Federal do Rio Grande do Norte, PROGRAMA DE P?S-GRADUA??O EM BIOQU?MICA, UFRN, Brasil |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Source | reponame:Repositório Institucional da UFRN, instname:Universidade Federal do Rio Grande do Norte, instacron:UFRN |
| Rights | info:eu-repo/semantics/openAccess |
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