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Previous issue date: 2014-12-08 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / 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. / A obten c~ao da estrutura tridimensional de uma enzima pode ser utilizada como
passo inicial em projetos de engenharia gen etica e engenharia enzim atica com intuito
de optimiza c~ao da atividade catal tica e/ou a produ c~ao em escala industrial de enzimas
alvo. Atualmente, a cristalogra a e o m etodo mais empregado para a determina c~ao de
estruturas tridimensionais de macromol eculas.
A biomassa vegetal, na forma de celulose, hemicelulose e lignina, apresenta grande
potencial para aplica c~oes biotecnol ogicas. Com a crescente demanda por fontes renov aveis
de energia, tem-se proposto sua utiliza c~ao para a obten c~ao de energia, os ditos biocombust
veis. Para esse m, a principal abordagem que se tem procurado e a degrada c~ao da
biomassa via hidr olise enzim atica. Neste contexto, o estudo de micro-organismos capazes
de realizar a degrada c~ao da biomassa e o estudo das enzimas envolvidas no processo
apresentam papel chave.
Particularmente, o fungo term o lo Humicola grisea var. thermoidea apresenta
produ c~ao signi cativa de enzimas lignocelulol ticas ativas a alta temperatura e tem sido
considerado um forte candidato para aplica c~oes industriais. Contudo, a literatura cient
ca ainda carece de informa c~oes estruturais sobre as enzimas do fungo envolvidas na
hidr olise da lignocelulose. Em trabalhos anteriores, a enzima celulol tica celobiohidrolase
(CBH1.2) de H. grisea foi identi cada e clonada em Pichia pastoris bem como uma das
endoxilanases (HXYN2) deste mesmo organismo.
Neste projeto de mestrado, foram expressadas as enzimas CBH1.2 e HXYN2 utilizando
sistema heter ologo de express~ao, obtendo rendimento satisfat orio para ensaios in
vitro. Foram estabelecidos protocolos de puri ca c~ao via precipita c~ao por sulfato de am^onio
e realizados experimentos iniciais de atividade enzim atica via o m etodo dos a c ucares redutores
para as duas enzimas.
XVIII
Foi encontrada condi c~ao de cristaliza c~ao para a enzima CBH1.2r onde foram obtidos
pequenos cristais em forma de agulha nos ensaios de cristaliza c~ao. Al em deste,
tamb em foi realizada a clonagem da enzima CBH1.2 de Humicola grisea no vetor de
express~ao pHIL-D2 (para express~ao extracelular). Este vetor foi utilizado para transformar
as linhagens SMD1168 e GS115 da levedura P. pastoris ambas com o gen otipo
his4-. Foram detectados transformantes capazes de secretar a prote na ativa em ambas as linhagens.
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.bc.ufg.br:tede/4254 |
| Date | 08 December 2014 |
| Creators | Malaspina, Lorraine Andrade |
| Contributors | Marques, Ivo de Almeida, Faria, Fabrícia Paula de, Marques, Ivo de Almeida, Faria, Fabrícia Paula de, Ulhoa, Cirano José, Valadares, Napoleão Fonseca |
| Publisher | Universidade Federal de Goiás, Programa de Pós-graduação em Fisica (IF), UFG, Brasil, Instituto de Física - IF (RG) |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da UFG, instname:Universidade Federal de Goiás, instacron:UFG |
| Rights | http://creativecommons.org/licenses/by-nc-nd/4.0/, info:eu-repo/semantics/openAccess |
| Relation | 3162138865744262028, 600, 600, 600, 600, -4029658853652049306, -8327146296503745929, 2075167498588264571 |
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