A poly(vinylpyrrolidone) end-functionalized with a carboxylic acid group (PVP-CO2H) was synthesized by RAFT/MADIX polymerization mediated by 4-(O-ethylxanthyl)methyl benzoic acid. The molecular weight of the as-synthesized PVP-CO2H was estimated through UV Vis spectroscopy (Mn(UV-Vis) = 7322 g/mol), Gel Permeation Chromatography (GPC) (Mn(GPC) = 8670 g/mol), and 1H-NMR, (Mn(NMR) = 8207 g/mol). The values obtained were close with the theoretical molecular weight (Mn(th) = 7925 g/mol). Subsequently, the preformed PVP-CO2H was activated to produce N-succinimidyl poly(vinylpyrrolidone) (PVP-NHS). This precursor was covalently coupled to papain to produce bioconjugate PVP-papain. The functional group modifications in the PVP chain-end were observed by the variations in the chemical shift values by 1H and 13C-NMR, and FT-IR analysis at each step of the synthesis. The molecular weight of the PVP-papain was obtained by SEC-HPLC and suggests that, on average, four or five chains of PVP-CO2H were attached to one papain molecule. Compared to papain, the PVP-papain exhibited significantly improved catalytic activity, pH, and thermal stability, also, the storage studies shown that the catalytic activity of PVP-papain was about 79% versus the native enzyme (29%) and was kept it even when it was stored for 25 days.
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