New recombinant β-1,4-endoglucanase from the thermophilic Myceliophthora heterothallica expressed in Pichia pastoris: partial characterization and structural analysis of thermostability

The thermophilic filamentous fungus Myceliophthora heterothallica constitutes a powerful cellulolytic microorganism. A new sequence encoding for glycoside hydrolase was identified and expressed in Pichia pastoris X-33. 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). Two derivatives of this enzyme were constructed, Mh_GH5 attached to CBM1 (Mh_GH5-CBM1) and a second one comprising only the catalytic domain (CD) of the enzyme (Mh_GH5-CD) in order to evaluate the interactions of the CBM, CD and cellulosic substrate. The enzymes were successfully expressed as active in P. pastoris. The recombinant enzymes were purified and subjected to partial biochemical characterization. Recombinant endoglucanases showed optimal temperature of 55°C and optimal pH of 4.0 for both constructs. Additionally, Mh_GH5-CD was expressed in E. coli, purified and submitted to structural analysis of the catalytic domain of this new GH5. The structure was solved at 1.1 Å resolution, determined by molecular-replacement method, shows a common (β/α)₈ TIM-barrel fold found in members of GH5 family. Mh_GH5-CD exhibited short loops, high percentage of tryptophan residues, 50.3% of aminoacid residues contributing for secondary  structure formation and two potential glycosylation sites. Based on the structural information we have obtained for Mh_GH5 it is possible to suggest features involved in the thermostability of proteins from thermophilic microorganisms and might contribute for the development of new efficient biocatalysts for biomass conversion.