Caracterização estrutural in silico e produção recombinante de uma lignina peroxidase de Trametes villosa

Abstract

Lignin peroxidase (LiP) (EC 1.11.1.14) is an enzyme produced by white decay fungi and is of great biotechnological interest due to its ability to degrade lignin and its potential use in industrial and environmental applications, especially in biofuel production. Despite numerous studies with fungal extracts of LiP, production still faces challenges such as low yield, instability, and difficult purification. Alternatives that can be explored to overcome these limitations include recombinant production in Escherichia coli. Therefore, the objective of this work was to select, characterize in silico, and recombinantly produce, in E. coli, a consensus sequence for lignin peroxidase from the genome of the fungus Trametes villosa, for future application in bioethanol production. The results demonstrated that it was possible to select a consensus sequence for LiP from the genome of T. villosa (LiP1820), and the model generated from the sequence showed high reliability. In molecular dynamics studies, RMSD, ΔRMSF, and GR data indicate greater flexibility, better conformation, and compaction at pH 3, indicating a possible facilitator for substrate entry. rSurf presented a larger contact surface at pH 3, favoring a more adjusted conformation. However, when we evaluated MM-PBSA and FEL, we observed that at neutral pH the protein was more stable, indicating that this high stability may be associated with high rigidity at neutral pH, hindering substrate access, which is not observed at acidic pH. The T. villosa LiP1820 enzyme was expressed in E. coli with pET28a and pET32a vectors and strains BL21 (DE3), pLySs, and Rosetta™ (DE3). Only the pET32a construct in Rosetta™ (DE3), grown in TB medium, resulted in an active enzyme after refolding, with maximum activity at pH 4.0 and 6.0 at 50°C. Given this scenario of obtaining this enzyme in its active form and its biotechnological value, advances in recombinant production pave the way for industrial application, making its production viable and strategicfor the development of clean technologies. This is the first report of recombinant production of LiP from T. villosa in E. coli.