## 4 Chitosan as Catalytic Support
### Chitosan-Containing Composite Biocatalysts
[206] developed a promising strategy for LIP immobilization based on the magnetic microspheres functionalized with hyaluronic acid (HA) and CS (Fe\({}_{3}\)O\({}_{4}\)@SiO\({}_{2}\)@[CS/HA]\({}_{3}\)). First, Fe\({}_{3}\)O\({}_{4}\) magnetic microspheres and the core-shell were synthesized using hydrothermal reaction and the sol-gel method, respectively. Then, a layer-by-layer assembly of HA and CS on the surface of the Fe\({}_{3}\)O\({}_{4}\)@SiO\({}_{2}\) microspheres was carried out. LIP was covalently bonded on the surface of the resulting microspheres using EDAC/NHS to produce Fe\({}_{3}\)O\({}_{4}\)@SiO\({}_{2}\)@[CS/HA]\({}_{3}\)@LIP. Fe\({}_{3}\)O\({}_{4}\)@SiO\({}_{2}\)@[CS/HA]\({}_{3}\)@LIP showed good thermal and long-term stability (after storage for 30 days at 4 \({}^{\circ}\)C Fe\({}_{3}\)O\({}_{4}\)@SiO\({}_{2}\)@[CS/HA]\({}_{3}\)@LIP retained 78% of the initial activity), reusability (85% of initial activity was retained after nine consecutive cycles) and catalytic activity for the synthesis of the structured lipid of 1,3-diolely-2-palmitoylglycerol--healthy component of food, oil and pharmaceutical intermediates. [PERSON] et al. [36] synthesized CS modified with 4-acetylsulfanilchloride (see Figure 2) for the immobilization of proteases (ficin, papain and bromelin). Enzyme immobilization was carried out by complexation via the addition of modified CS to an enzyme solution having pH = 9 or 10. No additional shaping or crosslinking was applied. It was found that the hydroxy, thionyl and amino groups of 2-(4-acetamido-2-sulfanilamide)CS were involved in the complexation process. The immobilized papain (protein content was about 12 mg/gsupport) was more active compared to other immobilized proteases. Moreover, the total and specific proteolytic activities of immobilized papain were higher (122% and 102%, respectively) compared to the native enzyme due to hyperactivation. Moreover, [PERSON] et al. [207] and [PERSON] et al. [208] synthesized a series of nano- and microparticles of CS with the addition of ascorbic acid for further immobilization of papain and bromelin. CS particles were prepared by dissolving CS in acetic acid solution with the subsequent addition of NaOH under constant stirring. Ascorbic acid was added to the CS solution before gelation. The immobilized papain exhibited 40% of its catalytic activity after incubation for 168 h in Tris-HCl buffer (0.05 M, pH 7.5) at 37 \({}^{\circ}\)C, while the free enzyme retained only 15% of its activity [207]. The free bromelin retained 13% of its activity after incubation for 168 h, while the immobilized enzyme exhibited between 33% and 39% of its activity depending on the molecular weight of the CS and its particle sizes [208]. A novel organic-inorganic hybrid nanoflower was obtained by [PERSON] et al. [209] via the CS-regulated biomimetic mineralization of calcium phosphate (CaP) to immobilize sucrose phosphorylase (SPase) via self-assembly (CS-CaP@SPase). CS was dissolved in acetic acid with a subsequent addition of TPP aqueous solution duringstirring. Then, CaP and SPase were added. The obtained CS-CaP@SPase exhibited excellent pH, byproduct and organic solvent (acetonitrile) tolerance, and storage stability. CS-CaP@SPase maintained about 70% of relative activity, while free SPase retained only 5% at 60 \({}^{\circ}\)C. The CS-CaP@SPase activity reached its maximum at pH = 8; at pH = 7 it retained 90% of relative activity, while the free SPase retained 65% of relative activity. It was proposed that the active groups on the CS-CaP surface could interact with H+, which could reduce the effects of H+ on the activity of SPase. Moreover, the relative activity of CS-CaP@SPase was still retained at about 80% after 10 cycles and maintained 75% after 15 days. [PERSON] et al. [210] developed bienzyme biocatalysts by co-immobilization of HRP and GOx on DA-modified cellulose-CS composite beads via covalent binding (cellulose-CS@DA/HRP-GOx) (Figure 31).