Continuous and autonomous-flow separation of laccase enzyme utilizing functionalized aqueous two-phase system with...

Area of Science:

• Biochemical Engineering
• Separation Science
• Biotechnology

Background:

• Downstream processing of biomolecules, especially therapeutic proteins and enzymes, is costly and complex.
• Current methods often involve intricate unit operations, limiting efficiency.

Purpose of the Study:

• To develop a novel cysteine (cys) functionalized aqueous two-phase system (ATPS) for selective laccase extraction.
• To integrate this system into a continuous-flow microfluidic device for automated bioseparation.

Main Methods:

• A polyethylene glycol (PEG) and potassium phosphate (K3PO4) based ATPS was functionalized with cysteine.
• A 3D-baffle micro-mixer and phase separator with computer vision control was designed for automated, continuous-flow operation.
• Laccase extraction efficiency and partition coefficient were compared to conventional batch processes.

Main Results:

• The microfluidic-assisted ATPS (PEG-K3PO4/cys) achieved a partition coefficient (Kflow) of 16.3 and extraction efficiency (EEflow) of 88% for laccase.
• The continuous-flow process demonstrated effective laccase partitioning from complex mixtures, even at low concentrations.
• Circular dichroism confirmed the structural stability of laccase during the process.

Conclusions:

• Continuous-flow microfluidic bioseparation using functionalized ATPS offers a highly efficient and automated solution for downstream processing.
• This approach is valuable for seamless purification of target biopharmaceuticals.
• The developed system enables integrated and autonomous bioseparation processes.