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Previous issue date: 2014-11-27 / PAPER 1 Depletion of the xynB2 gene upregulates β-Xylosidase expression in
C. crescentus.
Caulobacter crescentus is able to express several enzymes involved in the utilization of
lignocellulosic biomasses. Five genes, xynB1-5, that encode β-xylosidases are present
in the genome of this bacterium. In this study, the xynB2 gene, which encodes -
xylosidase II (CCNA_02442), was cloned under the control of the PxylX promoter to
generate the O-xynB2 strain, which overexpresses the enzyme in the presence of
xylose. In addition, a null mutant strain, -xynB2, was created by two homologous
recombination events where the chromosomal xynB2 gene was replaced by a copy
that was disrupted by the spectinomycin-resistant cassette. It was demonstrated that C.
crescentus cells lacking -xylosidase II up-regulates the xynB genes inducing β-
xylosidase activity. Transcriptional analysis revealed that xynB1 (RT-PCR analysis)
and xynB2 (lacZ transcription fusion) gene expression was induced in the -xynB2
cells, and high -xylosidase activity was observed in the presence of different agroindustrial
residues in the null mutant strain, a characteristic that can be explored and
applied in biotechnological processes. In contrast, overexpression of the xynB2 gene
caused down-regulation of the expression and activity of the -xylosidase. For
example, the β-xylosidase activity that was obtained in the presence of sugar cane
bagasse was 7-fold and 16-fold higher than the activity measured in the C. crescentus
parental and O-xynB2 cells, respectively. Our results suggest that -xylosidase II may
have a role in controlling the expression of the xynB1 and xynB2 genes in C.crescentus.
PAPER 2 - OPTIMIZATION OF THE PRODUCTION β-XYLOSIDASE: A NEW
Thermomyces lanuginosus ISOLATED FROM ATLANTIC FOREST BIOME.
The successful production of enzymes for the deconstruction of plant biomass depends
not only on the isolation and identification of new microorganism producers of
hemicellulases, but also on the implementation and improvement of experimental
strategies that lead to maximal induction of enzymatic activities. In this work, a new
strain of Thermomyces lanuginosus (T. lanuginosus) was isolated from the Atlantic
Forest biome in Brazil, and its β-xylosidase activity in response to agro-industrial
residues was tested. Using the (CCRD) statistical approach as a strategy for
optimization, the induction of β-xylosidase activity was evaluated in residual corn straw,
which was used as a carbon source, and improved so that the optimum condition
achieved high β-xylosidase activity (1,003 U ml -1; specific activity = 1.683 U mg-1) with
214 U ml -1. The optimal conditions for the crude enzyme extract were pH 5.5 and 60°
C showing better thermostability at 55° C. The saccharification ability of β-xylosidase in
the presence of hemicellulose obtained from corn straw and xylan from beechwood
substrates showed a xylo-oligosaccharide to xylose conversion yield of 80 and 50%,
respectively, at 50° C. These data suggest that β-xylosidase from T. lanuginosus
isolated from the Atlantic Forest can be used for the saccharification of hemicellulose
derived from corn straw, an abundant residue in the American continents, thus
providing an interesting alternative for future tests for energy production that relies on
the conversion of plant biomass. / RESUMO GERAL
As Beta-D-Xilosidases (1,4-β-D-xilano xilohidrolase; EC 3.2.1.37) são glicosídeo
hidrolases que tem papel crucial em catalizar a liberação de unidades de xilose a partir
de xilo-oligossacarídeos derivados da degradação do xilano. A completa degradação
do xilano é um passo chave do ciclo do carbono na natureza e é um processo também
realizado por microrganismos. A bioconversão de materiais lignocelulósicos é
vantajosa não somente do ponto de vista ambiental mais também econômico o que é
recebido nos setores produtivos com um considerável interesse, pois esses materiais
representam vasta fonte de carbono, que podem ser empregados no desenvolvimento
de bioprocessos que resultam em produtos de alto valor agregado; entre os quais
estão os açúcares fermentáveis, combustíveis, fármacos, enzimas e substâncias de
interesse industrial, além de fazer uma gestão integrada do efluente que por não haver
um desenvolvimento biotecnológico adequado é descartado e acumulado na natureza.
Em face disso, o presente trabalho teve por objetivos estudar a regulação gênica do
gene xynB2 da bactéria Caulobacter crescentus que codifica para a Beta-xilosidase II
através de abordagens moleculares e otimizar a produção enzimática de Betaxilosidases
de Thermomyces lanuginosus na presença de diferentes resíduos de
biomassa vegetal por delineamento experimental. No primeiro artigo exploramos a
bactéria aquática Caulobacter crescentus por possuir várias enzimas envolvidas na
utilização de biomassas lignocelulósicas; contendo em seu genoma cinco genes que
codificam β-xilosidases. A partir do gene xynB2, que codifica para enzima -xylosidase
II (CCNA_02442), desenvolvemos duas linhagens mutantes denominadas O-xynB2,
que super-expressa a enzima na presença de xilose e -xynB2 que tem o gene xynB2
interrompido, o que possibilitou avaliar que a ausência da enzima -xylosidase II em
células de C. crescentus regula positivamente os genes xynB, induzindo a atividade
global de β-xilosidases, revelando um papel regulatório para a mesma. No segundo
trabalho um fungo da linhagem Thermomyces lanuginosus isolado de bioma de Mata
Atlântica foi identificado e analisado quanto à capacidade de produzir Beta-xilosidases
na presença de diferentes resíduos vegetais; em decorrência disso foi otimizado a
produção enzimática com delineamento experimental DCCR, o que permitiu alcançar
altos níveis de atividade enzimática beta-xilosidásica na presença de palha de milho.
| Identifer | oai:union.ndltd.org:IBICT/oai:tede.unioeste.br:tede/2659 |
| Date | 27 November 2014 |
| Creators | Corrêa, Juliana Moço |
| Contributors | Simão, Rita de Cássia Garcia, Ernandes, Samara, Kadowaki, Marina Kimiko, Sene, Luciane, Schuster, Ivan |
| Publisher | Universidade Estadual do Oeste do Parana, Programa de Pós-Graduação "Stricto Sensu" em Engenharia Agrícola, UNIOESTE, BR, Engenharia |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações do UNIOESTE, instname:Universidade Estadual do Oeste do Paraná, instacron:UNIOESTE |
| Rights | info:eu-repo/semantics/openAccess |
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