## 4 Chitosan as Catalytic Support
### Chitosan-Containing Composite Biocatalysts  
CS is widely used for the immobilization of enzymes as a part of support for covalent binding or encapsulation [167; 168; 169; 170; 171; 172]. The ability of CS to shape beads/microspheres, microcapsules, fibers, membranes, coatings, sponges and gels has expanded its applicability as a support for the immobilization of a wide range of biomolecules and enzymes [173]. However, despite the advantages of using CS as a support for enzyme immobilization, CS beads and fibers are quite fragile and mechanically unstable [174; 175]. As previously mentioned, the mechanical properties of CS-based materials can be increased via covalent crosslinking (i.e., with GA or ECH). However, the cytotoxicity of the aforementioned crosslinking agents may hinder the use of CS hydrogels for pharmaceutical and biomedical applications. Thus, in contrast to CS-based adsorbents and metal-containing catalysts, toxic GA is replaced with the safer TPP at the crosslinking stage. For example, [PERSON] et al. [176] used CM prepared by dripping of CS acid solution in the solution of sodium TPP (crosslinking agent) for further co-immobilization of 7\(\alpha\)- and 7\(\beta\) hydroxysteroid dehydrogenases (HSDH). It is noteworthy that the immobilization of these enzymes was carried out by preliminary activation of CM with GA that is common for enzyme deposition on heterogeneous supports. CM-immobilized HSDHs were used for the conversion of taurochenodeoxycholic acid (a component of chicken bile). The catalytic reaction reached equilibrium within 4 h. The yield of reaction product (tauoursodeoxycholic acid) was about 62% after equilibrium and the content of tauoursodeoxycholic acidin the reaction product was as high as 33.16% [176]. A similar procedure was applied by [PERSON] et al. [177] for the immobilization of extracellular inulinase from _Kluyveromyces marxianus_ on CS beads. Under the optimized conditions, a maximum yield 65.5% of immobilized inulinase was obtained. The maximum hydrolysis of inulin--84.5%--was observed at 125 rpm after 4 h using an immobilized enzyme. The developed immobilized biocatalyst was successfully used for the hydrolysis of inulin for 14 batches. An unusual approach was proposed by [PERSON] et al. [178], who synthesized ahybrid support consisting of CS and chitin nanowhiskers (CNWs). CNWs were introduced into CS solution in 2% acetic acid containing tannic acid as a crosslinking agent, and then the obtained mixture was dripped into NaOH solution. The resulting CS/CNWs beads were treated with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDAC) hydrochloride and N-hydroxysuccinimide (NHS) for further immobilization of _Rhizomucor miehei_ lipase (LIP). The LIP loading of 2 mg/g was achieved, corresponding to the specific and residual activity of 537 U/g and 137%, respectively. Immobilized LIP was used for synthesis of eugenyl benzoate with the highest yield of 56.3% achieved for 5 h using 3 mg/mL of LIP-CS/CNWs with a molar ratio of eugenolbenzoic acid of 3:1. [PERSON] et al. [179] immobilized \(\beta\)-galactosidase produced by the yeast _Kluyveromyces lactis_ NRRL Y1564 and delivered an enzymatic activity of 4418.37 U/g\({}_{\text{cell}}\) after 12 h of processing on the CS particles without any crosslinker (CS was dissolved in 2% acetic acid, and then coagulating agent (KOH) was added during stirring). After coagulation, CS particles were activated with GA, ECH or glycidol. GA was chosen as an optimal activating agent. The biocatalyst obtained by immobilization in CS-GA allowed for the effective hydrolyzation lactose of milk (42.8% of conversion) and produced lactose (17.32 g/L). [PERSON] et al. [180] prepared GA-activated CS hydrogel (anacidic solution of CS was dripped into NaOH solution), which was further treated with glycine solution (CS-GA-Gly). The obtained CS-GA-Gly was used for the immobilization of the LIP from _Thermomyces lanuginos_ (TLL).