## 4 Chitosan as Catalytic Support
### Chitosan-Containing Composite Biocatalysts
Almost no decrease in catalytic activity was found for this system due to the active conformation assumed by the covalently immobilized enzyme and to the contribution to the activity of the CS-coated support [188]. A magnetically separable, CS-based material was applied for immobilization of LIP from _Candida antarctica_ by [PERSON] et al. [189]. For the synthesis of composite support, maghemite NPs (2-3 nm) were placed into CS solution in 1.5% acetic acid. Then, the excess CS was removed by washing the solution with deionized water and separated with an external magnet. The resulting material was dispersed into solution of sodium TPP to form a maghemite/CS/TPP composite. For the immobilization of LIP, GA was applied. Optimization of the conditions for LIP immobilization allowed for the selection of the process variables: 9 h incubation time, 55 \({}^{\circ}\)C incubation temperature and 12% (\(v/v\)) GA content. The optimized immobilized LIP activity was 1.8 U for the \(p\)-nitrophenyl palmitatehydrolysisreaction. [PERSON] et al. [190] immobilized \(\beta\)-glucosidase from _Humicola insolens_ (Bglhi) and endoglucanase from _Scytalidium thermophilum_ (Egst) on Fe\({}_{3}\)O\({}_{4}\) NPs derivatized with CS/GA/N-(5-amino-1-carboxy-pentyl) imidioacetic acid (AB-NTA) and functionalized with NiCl\({}_{2}\) using the procedure developed by [PERSON] et al. [191]. Briefly, CS solution in acetic acid was added to asuspension of Fe\({}_{3}\)O\({}_{4}\) NPs and then CS-covered magnetic NPs were treated with crosslinking agent (GA). After removing the excess of GA, composite NPs were treated with AB-NTA and NiCl\({}_{2}\). Immobilization of Bglhi and Egst proceeded withthe coordination of the imidazole group of a 6-histidine tag added to either the N-terminal (in the case of Bglhi) or C-terminal (in the case of Egst) of the protein with the Ni\({}^{2+}\) coordinated with NTA group of NPs. The immobilization yields were about 20% for Bglhi and Egst with efficiencies of 132% and 115%, respectively. The two enzymes were also co-immobilized with a yield ofabout 49%. The optimal temperatures of the immobilized enzymes were 70 \({}^{\circ}\)C and 55 \({}^{\circ}\)C for Egst and Bglhi, respectively. The individually immobilized enzymes, when combined, showed a synergistic effect on the substrates tested (\(p\)-nitrophenyl-\(\beta\)-D-glucopyranoside for Bglhi and carboxymethylcellulose for Egst) and similar action when compared to the co-immobilized enzymes. Core-shell CS beads (CSBs) and dried CS beads were synthesized by [PERSON] et al. [192]. DBs were obtained using thetypical procedure with slight modifications: CS was dissolved in a mixed acid solution containing acetic acid, lactic acid and citric acid and then dripped into NaOH solution, washed and dried at 60 \({}^{\circ}\)C. For CSB preparation, dried CS beadswere soaked in a 0.5% acetic acid solution for 60 s and then neutralized by the addition of an equimolar amount of NaOH. CSBs were found to be an effective support for immobilization of recombinant mulosucarse (INU) from _Lactobacillus reuteri_ 121 (GA was used as an activating agent). INU-CSBs were used for the synthesis of inulin-type fructoligosaccharide and revealed a higher optimum temperature (60 \({}^{\circ}\)C) compared to free INU (50 \({}^{\circ}\)C). Moreover, the pH stability and thermostability of the INU-CSBs improved significantly. After being reused for 12 cycles, the INU-CSBs retained about 45% of their initial activity. The inulin-type fructoligosaccharide was also continuously synthesized in a fixed-bed bioreactor for at least 30 h using INU-CSBs. Crosslinked enzyme aggregates (CLEAs) are an alternative to carrier-bound enzymes, allowing for the immobilization of enzymes in their own macromolecular matrix. Several enzymes were crosslinked using a bifunctional reagent (e.g., GA) and exhibited 10 to 1000 times higher specific activity (IU/g catalyst) than their carrier-bound enzyme counterparts [193].