Clonagem, expressão heteróloga e caracterização funcional de uma endoglicanase de Penicillium echinulatum

AUTOR(ES)
DATA DE PUBLICAÇÃO

2009

RESUMO

This work describes the pioneering molecular biology studies with the fungus Penicillium echinulatum lineage 9A02S1 (DSM 18942), which resulted in the first isolation and characterization of a cDNA corresponding to an egl1 gene, encoding a endoglucanase 1 (EGL1). The egl1 cDNA was obtained from a library constructed under induction conditions of the cellulolytic system of this fungus. The cDNA sequence is 1,161 base pairs long, showing high identity scores with genes of fungal endoglicanases family 5A. It encodes a predicted protein of 387 amino acid residues, with molecular mass of 41.1 kDa, theoretical isoelectric point of 4.99 and a potential site of N-glycosylation. The predicted protein has three different domains: a catalytic domain, a highly conserved carbohydrate binding domain (CBD), and a region linking the two domains (hinge). From the predicted amino acid sequence of EGL1 it was possible to perform a three-dimensional modeli, using protein sequences deposited in data bank (PDB). The solved structure revealed that this protein displays structural properties similar to those observed in family 5A endoglucanases. The egl1 cDNA was cloned in a Pichia pastoris expression vector, pPIC9. Following transformation, recombinant clones with higher enzymatic activity were selected, using microfermentations in Deep Well plates. After selecting the best producer clone, we evaluated the biochemical properties of recombinant enzyme, as well as the optimization of culture conditions and subsequent characterization of the enzyme kinetics. The recombinant EGL1 secreted by the recombinant P. pastoris revealed characteristics of particular interest for industrial applications: an optimal activity over a broad range pH (5.0 9.0) and an optimal temperature of 60C. The recombinant EGL1 showed high thermostability at 70C after 1 h of pre-incubation, and the activity of recombinant EGL1 was strongly stimulated by the addition of calcium ion in the reaction.

ASSUNTO(S)

biologia molecular biologia molecular enzimas de fungos clonagem molecular fungos

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