Estudos bioquímicos e estruturais das enzimas celobiohidrolase e endoxilanase do fungo Humicola grisea var. thermoidea
Carregando...
Data
2014-12-08
Autores
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Federal de Goiás
Resumo
Obtaining 3-dimensional structure of an enzyme can be used as an initial step in
projects of genetic engineering and enzymatic engineering when aiming for optimization of
catalytic activity and/or production of target enzymes on an industrial scale. Currently,
crystallography is the most widely used method for the determination of three-dimensional
structures of macromolecules.
The plant biomass in the form of cellulose, hemicellulose and lignin, have great potential
for biotechnological applications. With the growing demand for renewable energy
sources, its been proposed it's use to obtain energy, called biofuels. For such purpose,
the main approach is the search of the degradation of biomass via enzymatic hydrolysis.
In this context, the study of microorganisms capable of carrying out the degradation of
biomass and the study of the enzymes involved in this process play a key role.
Particularly, the thermophilic fungus Humicola grisea var. thermoidea presents
signi cant production of active lignocellulolytic enzymes at high temperature and has
been considered a strong candidate for industrial applications. However, the scienti c
literature still lacks of structural information on fungal enzymes involved in the hydrolysis
of lignocellulose. In previous work, the cellulolytic enzyme cellobiohydrolase (CBH1.2)
from Humicola grisea has been identi ed and cloned into Pichia pastoris as well as one
of the endoxylanases (HXYN2) from this same organism.
In this master's project, the enzymes CBH1.2 and HXYN2 were expressed using
heterologous expression system obtaining satisfactory yield for in vitro assays. Puri -
cation protocols were established via precipitation by ammonium sulfate and initial experiments
of enzyme activity were performed via the reducing sugars method for both
enzymes.
XX
Crystallization conditions were found for the enzyme CBH1.2r, where small needleshaped
crystals were obtained in crystallization trials. In addition to this, the cloning of
the enzyme CBH1.2 from Humicola grisea with the pHIL-D2 expression vector (for extracellular
expression) was performed. This vector was used to transform GS115 and
SMD1168 strains of the yeast P. pastoris both with the genotype his4-. Transformants
that were able to secrete active protein were detected in both strains.
Descrição
Palavras-chave
Citação
MALASPINA, L. A. Estudos bioquímicos e estruturais das enzimas celobiohidrolase e endoxilanase do fungo Humicola grisea var. thermoidea. 2014. 121 f. Dissertação (Mestrado em Física)–Universidade Federal de Goiás, Goiânia, 2014.