## 4 Chitosan as Catalytic Support
### Chitosan-Containing Composite Biocatalysts
Co-immobilization of different enzymes (combi-CLEAs) allows cascade reactions to be carried out; however, the small and variable particle size of combi-CLEAs (5-50 \(\mu\)m) remains as a problem. Thus, [PERSON] et al. [193] entrapped combi-CLEAs of \(\beta\)-D-glucosidase (\(\beta\)G) and \(\alpha\)-L-arabinofuranosidase (ARA) in CS beads. The beads were obtained by dripping the mixture of solutions of combi-CLEAs and CS into sodium TPP solution. The resulting biocatalyst beads (average diameter 1.24 mm) retained full activity after 91 days of incubation under winemaking conditions, having specific activities of 0.91 and 0.88 IU/g \(\beta\)G and ARA, respectively. Such characteristics make them suitable for aroma enhancement in wines. Some researchers proposed the use of genipin (GP) extracted from the fruits _Genipa americana_ and _Gardenia jasminoides Ellis_ as a crosslinking reagent (Figure 28) [194; 195; 196; 197]. GP is 5000-10,000 times less cytotoxic than GA. [PERSON] et al. [198] suggested a new anodic catalyst consisting of CNTs, 4-nitrobenzoic acid (4-NBA), CS, GP and glucose oxidase (GOx) to promote glucose oxidation and the performance of an enzymatic biofuel cell. The new CNT/4-NBA/[CS/GOx/GP] catalyst contained a large amount of GOx (17.8 mg/mL) and produced a high anodic current (331 \(\upmu\)A/cm\({}^{2}\) at 0.3 V vs. Ag/AgCl) with low onset potential (0.05 V vs. Ag/AgCl). [PERSON] et al. [199] used GP for the activation of CS beads prepared using typical dripping of CS acidic solution into NaOH solution and immobilization of LAC (Figure 29) from white rot fungus _Trantes pubescens_ for removal of different dyes including CR, MB, etc. Maximum immobilization was obtained with 0.10% (\(v/v\)) GP and 4.0 U mL\({}^{-1}\) of enzyme solution after activation and contact for 14 h and 6 h, respectively. Improved pH, thermal and storage stabilities of the immobilized LAC were obtained when compared with the free counterpart. The CS-LAC system exhibited a residual activity of >55% after 11 cycles, demonstrating better durability than the free LAC. The activity loss of CS-LAC after 30 days of storage at 4 \({}^{\circ}\)C was 42.86%. [PERSON] et al. [200] immobilized \(\beta\)-D-galactosidase from _Aspergillus oryzae_ on CS particles, which were obtained by dissolution of CS in acidic medium with subsequent dripping into NaOH solution. After the enzyme adsorption, GP was applied as crosslinking agent. \(\beta\)-D-galactosidase-CS-GP particles were applied for the synthesis of galactooligosaccharides at a different variation of the initial lactose concentration, pH and temperature, and yields of 30% were achieved. \(\beta\)-D-galactosidase immobilized using GP had higher activity than  
Figure 28: Crosslinking of CS with GP [198]. Figure 29: Activation of CS with GP and further immobilization of LAC [199]. the biocatalyst prepared using GA. The immobilized enzyme maintained 100% of its initial activity after 25 batches of lactose hydrolysis. The biotechnological route of utilizing _Rhizomucor mielei_ LIP conjugated to a magnetite support was proposed by [PERSON] et al. (2010) to produce pentyl valerate--a fuel additive. Magnetic Fe\({}_{3}\)O\({}_{4}\) NPs were added to a suspension of chitin in CS solution in the presence of mineral oil and Tween 80. This mixture was treated with GA and then the pH was adjusted to 10 with NaOH solution. The resulting magnetically separable composite material was separated using an external magnetic field, washed and dried, and, after activation with EDAC/NHS, used for immobilization of LIP. The highest yield of pentyl valerate (96%) was reached under an optimized condition (50 \({}^{\circ}\)C, using a valeric acid/pentanol molar ratio of 1:2, with a LIP loading of 1.5 mg/mL and incubation time of 3 h). The immobilized LIP retained 90% activity after 40 days of storage and leached an initial 3.5% of the LIP, likely due to insufficient rinsing during preparation. [PERSON] et al. (2020) immobilized inulinase from _Aspergillus niger_ on CS-coated magnetic NPs.