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Heterologous expression of novel endoglucanases from Myceliophthora heterothallica in Pichia pastoris and Escherichia coli

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Author(s):
Carolina Bezerra Bussoli
Total Authors: 1
Document type: Doctoral Thesis
Press: São José do Rio Preto. 2016-06-30.
Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências Letras e Ciências Exatas. São José do Rio Preto
Defense date:
Advisor: Eleni Gomes
Abstract

Thermophilic microorganisms have gain attention in the last years because of the biotechnological interest. Essentially these organisms secrete thermostable proteins displaying suitable features, such as biochemical and structural, for industrial platforms. The thermophilic fungus Myceliophthora heterothallica F.2.1.4, isolated from poultry litter, showed high activity for β-1,4- endoglucanase (31 U/ml). A new sequence enconding for glycoside hydrolase was identified and expressed in Pichia pastoris and Escherichia coli. Sequence analysis allowed to classify this new β-1,4-endoglucanase as a member of the glycoside hydrolase 5 (GH5). The enzyme, named Mh_GH5, has N-terminal family 1 carbohydrate binding module (CBM1). The recombinant enzymes expressed in Pichia pastoris and Escherichia coli were purified and subjected to biochemical characterization. Recombinant endoglucanases showed optimal temperature of 55°C (P. pastoris) and 60°C (E. coli) and optimal pH of 4,0 for both heterologous systems. The thermostability of these new enzymes were evaluated and demonstrated that post-translational modifications, such as glycosylation, found on Pichia pastoris heterologous enzymes, contributed to higher thermostability. Structural analysis of the catalytic domain of the new GH5 from M. heterothallica expressed in E. coli was solved at 1.1 Å resolution and shows a common (β/α)8 TIM-barrel fold found in members of GH5 family. Based on the biochemical and structural information we have obtained for Mh_GH5 it is possible to suggest potential features involved in the thermostability of proteins from thermophilic microorganisms and might contribute for the development of new efficient biocatalysts required for biomass conversion to bioethanol and/or chemicals. (AU)

FAPESP's process: 12/00506-9 - Heterologous expression of cellulases from Myceliophthora heterotalica F.2.1.4 in Pichia pastoris with characterization and purification of the enzymes produced
Grantee:Carolina Bezerra Bussoli
Support type: Scholarships in Brazil - Doctorate