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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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The strong catalytic performance, eco-friendly reaction systems, and selectivity of enzyme-based biocatalysts are extremely interesting. Immobilization has been shown to be a good way to improve enzyme stability and recyclability. Chitosan-incorporated metal oxides, among other support matrices, are an intriguing class of support matrices for the immobilization of various enzymes. Herein, the cross-linked chitosan/zinc oxide nanocomposite (CS/ZnO) was synthesized and further improved by adding iron oxide (Fe2O3) nanoparticles. The final cross-linked CS/ZnO/Fe2O3 nanocomposite was used as an immobilized support for catalase and is characterized by SEM, EDS, and FTIR. The nanocomposite CS/ZnO/Fe2O3 enhanced the biocompatibility and immobilized system properties. CS/ZnO/Fe2O3 achieved a higher immobilization yield (84.32%) than CS/ZnO (37%). After 10 repeated cycles, the remaining immobilized catalase activity of CS/ZnO and CS/ZnO/Fe2O3 was 14% and 45%, respectively. After 60 days of storage at 4 °C, the remaining activity of immobilized enzyme onto CS/ZnO and CS/ZnO/Fe2O3 was found to be 32% and 47% of its initial activity. The optimum temperature was noticed to be broad at 25–30 °C for the immobilized enzyme and 25 °C for the free enzyme. Compared with the free enzyme optimum pH (7.0), the optimum pH for the immobilized enzyme was 7.5. The Km and Vmax values for the free and immobilized enzyme on CS/ZnO, and the immobilized enzyme on CS/ZnO/Fe2O3, were found to be 91.28, 225.17, and 221.59 mM, and 10.45, 15.87, and 19.92 µmole ml−1, respectively. Catalase immobilization on CS/ZnO and CS/ZnO/Fe2O3 offers better stability than free catalase due to the enzyme's half-life. The half-life of immobilized catalase on CS/ZnO/Fe2O3 was between 31.5 and 693.2 min. [ABSTRACT FROM AUTHOR]