RESUMEN
BACKGROUND: Factor C (FC) is widely used as a standard material for endotoxin testing. It functions as a zymogenic serine protease and serve as a biosensor that detects lipopolysaccharides. Prior investigations involving molecular docking and molecular dynamics simulations of FC demonstrated an interaction between the C-type lectin domain (CLECT) and the ligand lipopolysaccharide (lipid A). In this study, our aim was to assess the stability of the interaction between fragment FC and the lipid A ligand using protein modeling approaches, molecular docking, molecular dynamics simulation, and gene construction into the pPIC9K expression vector. METHODS AND RESULTS: The FC structure was modelled by online tools. In this case, both molecular docking and MD simulations were applied to identify the interaction between protein and ligand (lipid A) including its complex stability. The FC structure model using three modeling websites has varied values, according to a Ramachandran plot study. When compared to other models, AlphaFold server modeling produced the best Ramachandran findings, with residues in the most advantageous area at 88.3%, followed by ERRAT values at 89.83% and 3D Verify at 71.93%. From the docking simulation of FC fragments with three ligands including diphosphoryl lipid A, FC-Core lipid A, and Kdo2 lipid A can be an activator of FC protein by binding to receptor regions to form ligand-receptor complexes. MD simulations were performed on all three complexes to assess their stability in water solvents showing that all complexes were stable during the simulation. The optimization of recombinant protein expression in Pichia pastoris was conducted by assessing the OD value and protease activity. Induction was carried out using 1% (v/v) methanol in BMMY media at 30°C for 72 h. CONCLUSIONS: Protein fragments of Factor C has been proven to detect endotoxins and serve as a potential biomarker. Molecular docking simulation and MD simulation were employed to study the complex formation of protein fragments FC with ligands. The expression of FC fragments was successfully achieved through heterologous expression. We propose optimizing the expression of FC fragments by inducing them with 1% methanol at 30°C and incubating them for 72 h. These optimized conditions are well-suited for upscaling the production of recombinant FC fragments using a bioreactor.
RESUMEN
Microplastics are proven as heavy metals vector, but the adsorption mechanism still unclear. This study investigated the adsorption of Pb and Cu in microplastics in the Musi River and the environment effect. This study was conducted in 10 stations along the Musi River to the estuary. The polymers of microplastics were dominated by PP and followed by PE, PES, PVC, and nylon. The average concentration of Pb (0.0347 mg L-1 for water and 0.470 mg kg-1 for microplastics) was higher than Cu (0.0138 mg L-1 for water and 0.091 mg kg-1 for microplastics). The highest concentration of both metals in water and microplastics were found in the estuary. Environment parameters have different effects on the adsorption. The present study found that the adsorption processes were following the Freundlich model. The interaction metal-microplastic was physisorption. Pb and Cu will attach through weak bonds and easy to release into the aquatic ecosystem.
