%0 Journal Article
%T Continuous and autonomous-flow separation of laccase enzyme utilizing functionalized aqueous two-phase system with computer vision control.
%A Sharma V
%A Joo JU
%A Mottafegh A
%A Kim DP
%J Bioresour Technol
%V 403
%N 0
%D 2024 Jul 22
%M 38788804
%F 11.889
%R 10.1016/j.biortech.2024.130888
%X Downstream processing of biomolecules, particularly therapeutic proteins and enzymes, presents a formidable challenge due to intricate unit operations and high costs. This study introduces a novel cysteine (cys) functionalized aqueous two-phase system (ATPS) utilizing polyethylene glycol (PEG) and potassium phosphate, referred as PEG-K3PO4/cys, for selective extraction of laccase from complex protein mixtures. A 3D-baffle micro-mixer and phase separator was meticulously designed and equipped with computer vision controller, to enable precise mixing and continuous phase separation under automated-flow. Microfluidic-assisted ATPS exhibits substantial increase in partition coefficient (Kflow = 16.3) and extraction efficiency (EEflow = 88 %) for laccase compared to conventional batch process. Integrated and continuous-flow process efficiently partitioned laccase, even in low concentrations and complex crude extracts. Circular dichroism spectra of laccase confirm structural stability of enzyme throughout the purification process. Eventually, continuous-flow microfluidic bioseparation is highly useful for seamless downstream processing of target biopharmaceuticals in integrated and autonomous manner.