Produção heteróloga de endoglucanase de Moniliophthora perniciosa em E. coli: otimização da expressão

Abstract

The increase in agro-industrial production has intensified the generation of lignocellulosic residues, making it necessary to seek sustainable alternatives for their utilization. These materials are mainly composed of cellulose, hemicellulose, and lignin, recalcitrant structures that require efficient treatments for their degradation. In this context, cellulases stand out due to their ability to hydrolyze cellulose, generating fermentable sugars with potential biotechnological applications. Among these enzymes, endoglucanases act by cleaving internal β-1,4-glycosidic bonds in amorphous cellulose. Phytopathogenic fungi, such as Moniliophthora perniciosa, represent promising sources of these enzymes, including the endoglucanase EG45, which is the focus of this study. The aim of this work was to evaluate the heterologous expression of EG45 in Escherichia coli in order to identify favorable conditions for its recombinant production. For this purpose, different host strains (BL21 Star, C41, and C43), IPTG concentrations (0.1, 0.5, 1.0, and 2.0 mM), and cultivation temperatures (16, 20, 25, and 30 °C) were evaluated. Protein expression was analyzed by SDS-PAGE, allowing the selection of the most promising conditions for subsequent solubility assessment through cell lysis by sonication and analysis of the soluble and insoluble fractions. The results demonstrated EG45 expression under most of the tested conditions, with the highest levels observed in the BL21 Star and C41 strains. Furthermore, a saturation pattern of expression was identified from 0.5 mM IPTG onward, with no significant gains observed at higher inducer concentrations. Regarding temperature, the highest expression intensities were detected at 25 °C and 30 °C. Solubility analysis revealed a predominance of the protein in the insoluble fraction, indicating the formation of inclusion bodies. Although the BL21 Star and C43 strains showed lower accumulation of insoluble protein, suggesting the possibility of an undetected soluble fraction, the C41 strain was selected for continuation of the bioprocess because it combined high expression yield with greater metabolic stability associated with the T7 expression system. Thus, an IPTG concentration of 0.5 mM, a cultivation temperature of 25 °C, and the C41 strain were defined as the most promising conditions for future optimization and scale-up ofrecombinant endoglucanase EG45 production.