## 4 Chitosan as Catalytic Support
### Chitosan-Containing Composite Biocatalysts
The improved performance of the immobilized HRP was attributed to CS, which provided not only a hydrophilic environment but also multiple binding sites for the HRP [184]. [PERSON] et al. immobilized LIP of _Cercospora kikuchi_[185] on CS beads, which were prepared using a generally accepted method and were activated with sodium meteoriodate, ECH or GA. Among the chosen activating agents, GA allowed the best immobilized enzyme for all the analyzed parameters, mainly LIP activity retention, which represented an improvement of 46.5% on the free LIP. It is noteworthy that CS beads underwent fluidized bed drying that provided excellent features (low moisture content (3.84% \(w/w\)) and water activity (\(\sim\)0.10)) for LIP application in organic media for the esterification/transesterification process. The obtained immobilized LIP system was used [185] to catalyze the synthesis of butyl butyrate with high yield (98.96%). A further improvement of the biocatalyst preparation procedure was the application of the magnetic separation principle and spray drying method [186]. Fe\({}_{3}\)O\({}_{4}\)-CS-LIP microparticles were prepared by mixing magnetic NPs (Fe\({}_{3}\)O\({}_{4}\)/CS ratio was 1/4 \(w/w\)) and enzyme solution (LIP of _Candida rugosa_) with CS solution in acetic acid. Optionally, activating agents (e.g., GA) were added. The resulting mixture was either dripped to the solution of sodium TPP or, alternatively, underwent spray drying. It was found that all the immobilized derivatives retained between 73.6% and 84.6% of initial enzyme activity in the reaction of isoamyl caprylate synthesis. Long-term storage (6 months) at 5 \({}^{\circ}\)C revealed that the immobilized derivatives retained more than 70% of their activity. For comparison, free LIP retained only 18.3% of its activity. The immobilization process also made LIP reuse possible: composite materials retained between 40% and about 60% of their initial activity after ten reuse cycles. [PERSON] et al. studied the immobilization of lacase (LAC) from _Trametes versicolor_ on CS-based supports [187; 188]. CS beads were prepared and crosslinked with GA or ECH and used for LAC immobilization (CS-GA-LAC or CS-ECH-LAC). Moreover, for the immobilization of LAC, for the first time, the direct conjugation of the enzyme was applied through the interaction between the carbonyl group, formed after the periodate oxidation of the LAC carbohydrate portion, and the amino groups of CS (CS-LAC). All the three methods were reliable for the immobilization of LAC. However, the CS-LAC system revealed higher storage stability: it retained initial activity for 30 days in the oxidation reaction of 2,2\({}^{\prime}\)-azino-bis-(3-ethyl-benzothazoline-6-sulfonic acid) (ABTS) to its cation radical ABTS\({}^{+}\). On the contrary, CS-GA-LAC and CS-ECH-LAC showed a decrease inactivity of 50 and 40%, respectively [187]. Another catalytic system used for LAC immobilization was based on manganese iron oxide (MnFe\({}_{2}\)O\({}_{4}\)) magnetic NPs coated with CS using two methods: (i) after the formation of magnetic NPs; (ii) during the synthesis of magnetic NPs (in situ). Both methods, in general, followed the well-known procedures described in previous sections of this review. LAC was covalently bonded to the resulting composites with GA. It was found that both CS-based supports possessed more specific activity against ABTS than the naked magnetic NPs, which exhibited not-Fenton-type intrinsic activity, attributed to the Fe\({}^{3+}\) surface ions. The potential oxidant activity of the developed biocatalytic systems was tested for the degradation of diclofenac at room temperature in the presence of ABTS as a mediator. It was shown that in contrast to the free LAC, the enzyme immobilized on magnetic supports possessed both good resistance to temperature denaturation and storage stability for 30 days. The most promising system, based on the in situ-formed magnetic NPs covered with CS, showed adiclofenac removal efficiency of 78%, higher than that of the free enzyme (70%).