Biochemical ans structural studies of cellobiohydrolase and endoxylanase enzymes of the fungus Humicola grisea var. thermoidea

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.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