## 4 Chitosan as Catalytic Support
### Chitosan-Containing Composite Biocatalysts
CS-GA-Gly-TLL was \(\sim\)four times more active than CS-GA-TLL in both olive oil emulsion hydrolysis and alkyl palmitate synthesis via esterification. Isoamyl palmit synthesis in isooctane at 50 \({}^{\circ}\)C using CS-GA-Gly-TLL provided maximum conversion of 85% after 90 min of reaction. After nine consecutive runs, the biocatalyst retained 34% of its initial activity. [PERSON] et al. [181] immobilized trypsin on the surface of GA-activated ZnO/CS nanocomposite through covalent attachment via Schiffbase linkages. GA-ZnO/CS nanocomposite was synthesized using are relatively simple procedure including the following steps:(i) synthesis of ZnO NPs; (ii) addition of ZnO NPs to CS solution; (iii) addition of NaOH solution to suspension of ZnO NPs to form ZnO/CS nanocomposite; (iv) activation of ZnO/CS with GA. Optimal immobilization conditions (incubation time (16 h), enzyme concentration (1.8 mg/mL) and pH (7.8)) were investigated to obtain the maximum activity of the immobilized trypsin. The immobilized trypsin exhibited optimum catalytic activity at pH 8.5 and 60 \({}^{\circ}\)C. Immobilized trypsin preserved 67% of initial activity at 50 \({}^{\circ}\)C during 2 h of incubation and sustained nearly 50% of catalytic activity until the 9 th repeated cycle of utilization. Moreover, the immobilized trypsin retained 50% of its enzymatic activity after 90 days of storage at 4 \({}^{\circ}\)C.  
[PERSON] et al. [182] studied the immobilization of dextransucrase produced by an indigenously isolated strain of _Leuconstoc mesenteroides_ AA1 on CS beads, which were crosslinked and activated with GA. Immobilized dextransucrase exhibited higher catalytic performance in a broader range of pH than the soluble form. The immobilized version worked better at lower pH values of 4.0 and 4.5 and exhibited approximately 75.0% and 82.0% of relative activity compared to that of the soluble form, respectively. [PERSON] et al. [183] developed a magnetically separable biocatalyst based on amamo LIP immobilized on Fe\({}_{3}\)O\({}_{4}\) NPs (nanomagnetite (nM)) either modified with 3-aminopropylriteoxysilane (APTES) or stabilized with oleic acid (OA) (Figure 27) and then modified with CS (nM-OA-CS). For the modification of nM-OA with CS, nM-OA particles were dispersed in acidic solution of CS and then dripped in NaOH solution. The nM-APTES and nM-OA samples disclosed crystal sizes of 8.07 and 8.69 nm, respectively. The nM-OA-CS displayed an average particles size of 918.6 nm. Lip was immobilized by covalent binding with GA on both nM-APTES-GA and nM-OA-CS-GA supports with different surface/volume ratios. The catalytic activity of nM-APTES-GA-LIP (85%) was higher than that of nM-OA-CS-GA-LIP (46%), which reflected the correlation between enzyme loading efficiency on the magnetic nanosupports and their relative activity, essential to potential use in a magnetically stabilized fluidized bed reactor. A two-step approach was proposed by [PERSON] et al. [184] for the synthesis of a novel type of bio-friendly composite membrane of poly(methyl acrylate-co-acrylic acid) and CS (PMA-co-PAA@CS), which served as a support for the immobilization of horseradish peroxididases (HRP). A composite membrane was obtained by the following steps:  
1. The PMA-co-PAA membrane was obtained using the electrospinning method;
2. Carboxyl groups of PMA-co-PAA were activated with EDAC/NHS;
3. The activated membrane was immersed in CS solution in 0.2 M acetic acid. Figure 27: Synthesis of nM-OA or nM–APTS T NPs (based on Ref. [183]). Then, PMA-co-PAA@CS was treated with GA and HRP was immobilized. Immobilized HRP (about 110 mg/g) revealed noticeably higher thermal (25-60 \({}^{\circ}\)C), pH (4-10) and storage stabilities compared to native HRP. Moreover, the PMA-co-PAA@CS-HRP was demonstrated to be an active and recyclable biocatalyst for the catalytic oxidation of 4-NP over 10 cycles. The activity of PMA-co-PAA@CS-HRP reached maximum at a concentration of 4-NP of 100 mg/L, allowing 86% removal efficiency.