Produção e caracterização bioquímica de uma endoglucanase recombinante de Moniliophthora perniciosa (STAHEL) Aime & Phillips-Mora a partir de células de Escherichia coli

Abstract

Cellulases are hydrolytic enzymes that play a crucial role in the degradation of plant cell walls by cleaving β-1,4-glycosidic bonds in cellulose, making them essential for converting lignocellulosic biomass into fermentable sugars. Among the various microorganisms capable of producing cellulases, filamentous fungi stand out due to their high enzymatic capacity and extracellular secretion. One such fungus is Moniliophthora perniciosa, a basidiomycete phytopathogen responsible for witches' broom disease in cacao (Theobroma cacao L.), which employs thermostable cellulases as part of its infection strategy. Despite its biological relevance, recombinant production of enzymes from this fungus remains underexplored. This study aimed to both review recent advances in the recombinant production of fungal cellulases and to report, for the first time, the heterologous expression, purification, and functional characterization of the endoglucanase EG45 from M. perniciosa in Escherichia coli. The literature review highlights multiple successful cases of expressing cellulolytic genes from filamentous fungi in heterologous systems, particularly E. coli, due to its advantages including ease of genetic manipulation, fast growth rate, high cell density, and a wide range of expression vectors. Recombinant DNA technology has enabled large-scale production of optimized enzymatic cocktails with significant gains in yield, stability, and cost-effectiveness, especially for biorefinery applications. Experimentally, the EG45 gene with codon optimization was cloned into both pET28a and pET32a vectors. The expression of the fusion protein pET32a-EG45 was achieved in E. coli Rosetta (DE3), following induction with IPTG (1 mM) at 37 °C for 4 hours. A protein band of approximately 38 kDa, matching the predicted molecular weight, was detected by SDS-PAGE in both soluble and insoluble fractions. Purification from the insoluble fraction using urea solubilization and refolding yielded high purity but insufficient concentration for enzymatic assays. In contrast, purification from the soluble fraction provided a functional enzyme in adequate quantity and quality. The recombinant enzyme exhibited optimal activity at 50 °C and pH 6.0, demonstrating thermal stability and industrially relevant functionality. In conclusion, the successful recombinant production of EG45 from Moniliophthora perniciosa, reported here for the first time, represents a significant advancement in understanding the biology of this phytopathogen and exploring its biotechnological potential. The proven activity of the enzyme supports the feasibility of using E. coli as a heterologous host for the efficient production of active fungal enzymes, with promising applications in agro-industrial waste valorization and sustainable biofuel production.