Immobilization of papain on cold-plasma functionalized polyethylene and glass surfaces

Polyethylene and glass surfaces were functionalized under dichlorosilane-RF-cold-plasma environments and were employed as substrates for further in situ derivatization reactions and immobilization of papain. Surface functionality changes of RF-plasma-exposed surfaces were monitored under 40 kHz continuous discharge environments. The nature and morphology of derivatized substrates and the substrates bearing the immobilized enzyme were analyzed using survey and high resolution ESCA, ATR-FTIR, and fluorescence of chemical derivatization techniques. Spacer molecules intercalated between the substrates and the enzyme significantly increased the enzyme activity (comparable with the that of the free enzyme). Computer-aided conformational modeling of the substrate-spacer systems corroborated with experimental data indicated that an optimal distance might exist between the enzyme and the substrate. The activity of free and immobilized papain was monitored using benzoyl arginine ethyl ester assay. The pH data were recorded every 0.3 s over 25 min. The Michaelis-Menten kinetic constants were evaluated for immobilized enzymes. It was shown, that the immobilized papain retains most of its activity after several washing/assay cycles.