Synthesis of hemicyanine-based chitosan ligands in dye-affinity chromatography for the purification of chewing stick peroxidase

Abstract

A novel built-in approach for the in situ formed hemicyanine dyes in the chitosan matrix is presented. Chitosan was reacted with salicylaldehyde to afford the corresponding Schiff base in good yield. This derivative was then converted to two heterocyclic quaternary ammonium salts, namely chitosan benzothiazolium and chitosan picolinium salts, which upon reaction with p-dimethylaminobenzaldehyde by Knoevenagel condensation, the corresponding built-in hemicyanine dyes were obtained. Characterization was made by NMR, FTIR, SEM, and UV-visible. The two dyed samples (benzothiazolium and picolinium) were used for the purification of chewing stick peroxidase by affinity chromatography method. 1.0 M ammonium sulfate was used as eluent to check whether the purification of enzyme proceeds by hydrophobic interactions and the result indicated that the purification proceeds by rather ionic interactions, and therefore 1 M NaCl was used instead. The overall result indicates that benzothiazolum column showed better affinity with a high specific activity of the separated enzymes compared with those obtained with the picolinium column. This novel dye ligand built-in approach onto a biopolymeric substrate is promising and would pave the way for more future work ahead in the field of the purification of proteins and other biological macromolecules.

Keywords: Affinity chromatography; Chemical modification; Chewing stick peroxidase; Chitosan; Enzyme purification; Fixation; Hemicyanine.