## 4 Chitosan as Catalytic Support
### Chitosan-Containing Composite Biocatalysts
Magnetite NPs were prepared using theco-precipitation method and placed into CS acidic solution. Further addition of NaOH solution allowed for the formation of CS gel with the entrapped Fe\({}_{3}\)O\({}_{4}\) NPs. CS-based magnetic composite was activated with EDAC/NHS for immobilization of inulinase. Hydrolysis of 5% inulin using immobilized inulinase (2.5 U/mg) led to a fructose release of 35.8 g/L at pH 6.0 and a temperature of 60 \({}^{\circ}\)C in 3 h. Obtained fructose was further used to produce hydroxymethylfurfural over TiO\({}_{2}\)magnetic silica spheres. The immobilized inulinase was recycled with up to 10 cycles of inulin hydrolysis with 9.2 g/L of fructose released at the 10 th cycle. Medium-chain, oleic (18:1n-9) and medium-chain fatty acid structured lipids were produced by [PERSON] et al. (2003) via acidosis reaction in solvent-free medium with capric (10:0) and lauric (12:0) free fatty acids and triolein or olive oil using _Yarrowia lipolytica_ LIP as a biocatalyst. This LIP was immobilized by microencapsulation in CS-alginate beads, which were produced by ionotropic gelation of CS with SA. Prior to gelation, SA was solubilized in the enzyme extract. The resulting mixture was dripped into a solution of calcium chloride and CS, resulting in the formation of microcapsules. The immobilized LIP resulted in a similar incorporation efficiency for lauric acid with olive oil triacylglycerol and this reaction could be performed for fivecyles without catalytic activity loss. Co-immobilization of LAC and ABTS was carried out (204) for removal of 2,4-dichlorophenol, bisphenol A, indole and anthracene. LAC was immobilized on magnetic CS NPs modified with amino-functionalized ionic liquid containing ABTS (MACS-NIL) based on Cu ion chelation (MACS-NIL-Cu-LAC). First, CS was modified with NIL, and then the obtained CS-NIL was dissolved in acetic acid and added dropwise to the suspension of Fe\({}_{3}\)O\({}_{4}\) NPs containing ABTS. After that, a solution of NaOH was added, resulting in the formation of MACS-NIL. The MACS-NIL NPs were activated with Cu(II) and then the LAC was immobilized (Figure 30). Figure 30: Simplified representation of MACS–NIL–Cu–LAC synthesis (based on Ref. (204)). MACS-NIL-Cu-LAC revealed enhanced activity (1.7-fold more than of free LAC), improved pH and temperature adaptability, and increased thermal and storage stability. The MACS-NIL-Cu-LAC had good removal efficiency with 100% for 2,4-dichlorophenol in water at 25 \({}^{\circ}\)C, even when the concentration reached 50 mg/L. The reusability study showed that after six catalytic runs, the removal efficiency of 2,4-dichlorophenol using MACS-NIL-Cu-LAC could still reach 93.2%. Moreover, the MACS-NIL-Cu-LAC exhibited higher catalytic efficiencies with 100%, 70.5% and 93.3% for bisphenol A, indole and anthracene, respectively [204]. [PERSON] et al. [205] carried out a one-pot immobilization of \(\beta\)-galactosidase using the silica sol-gel encapsulation process combined with metal chelation using CS and Ca(II), Zn(II) or Cu(II). Briefly, sodium silicate and CS solutions in acetic acid were mixed (pH = 6), salt solution (500 mM CaCl\({}_{2}\), ZnCl\({}_{2}\) or CuCl\({}_{2}\)) was added and then the enzyme was added (100 IU/mL). No additional crosslinking agents were used. Metal chelation was proposed to play an important role in the encapsulation process. It was found that the presence of cations does not affect encapsulation efficiency (81%) and has positive effects on the maximum catalytic potential. However, depending on the metal type, at 60 \({}^{\circ}\)C, different thermal behavior was observed: Ca(II)--hyperactivating (1.6 times higher activity compared to the soluble enzyme)/stabilizing; Zn(II)--hyperactivating/non-stabilizing; and Cu(II)--non-hyperactivating/non-stabilizing effects. As a result, hydrolytic and transgalactoyslation activities of the enzyme (33.6-57.4% total lactose conversion) were higher than thosereported with analogue biocatalysts. [PERSON] et al.