Innovative separation of melittin from bee venom using micro-free-flow electrophoresis: An experimental and theoretical study.

Background: Bee venom consists of more than 50 % melittin (MLT), which has anti-cancer, anti-inflammatory, and antimicrobial properties. Bee venom also contains toxic components such as phospholipase A2 (PLA2) and hyaluronidase (HYA), which cause allergic reactions, so the toxic components must be removed to use MLT. In previous studies, analytical methods were used to separate MLT. This study used micro-free flow electrophoresis (μFFE) for MLT separation. This separation was simulated using computational fluid dynamics (CFD) and molecular dynamics simulation (MD). The glass chip was fabricated using the wet etched method, and MLT separation was investigated experimentally.

Results: The CFD results demonstrated that MLT was separated from PLA2 and HYA in less than 3 min, and MLT and toxic components took different paths in the channel. The operating conditions, such as the sample flow rate, buffer flow rate, electric field strength, and channel dimensions, were optimized during the simulation. The MD results showed that MLT's conformation does not change the separation process. MD simulation with an atomistic resolution could give us more accurate results of molecular interactions. Also, MLT separation was performed on a glass chip, adding to the precision of the process. The channel outputs were analyzed using high-performance liquid chromatography (HPLC). MLT separation using μFFE was performed for the first time. The results indicated that melittin was purified 90 % in this separation, and PLA2 was rejected by 90 %.

Significance: The results indicated that, for the first time, the separation of MLT from bee venom using μFFE could be achieved with a high recovery rate, thereby demonstrating its potential for biological applications. This separation in less than 3 min has higher yield and purity than analytical methods such as HPLC.