## 4 Chitosan as Catalytic Support
### Chitosan-Containing Composite Biocatalysts
To produce cellulose-CS@DA/HRP-GOx, cellulose and CS were dissolved in the ionic liquid ([Emim][OAc]); then, the resulting solution was added dropwise into ultrapure water to obtain cellulose-CS composite beads. The cellulose-CS composite beads and a certain amount of DA were placed in Tris-HCl buffer solution (pH 8.5) and left to react. Then, cellulose-CS@DA beads were used as a support for the co-immobilization of HRP and GOx at different ratios (3:1, 2:1, 1:1, 1:2 and 1:3). Cellulose-CS@DA/HRP-GOx beads showed good ability to catalytically degrade acridine in wastewater. The highest degradation rate of acridine (99.5%, 8 h) was found at anHRP-to-GOx ratio of 2:1. For comparison, the degradation rate of acridine in the case of cellulose-CS@DA/HRP and cellulose-CS@DA/GOx was 93.8% and 15.8%, respectively,  
Figure 31: Simplified representation of synthesis of cellulose–CS@DA/HRP–GOx composite beads (based on Ref. [210]). for 8 h. Additionally, the developed bienzyme biocatalyst maintained a degradation rate of 61.2% after six cycles of acridine degradation [210]. HRP was immobilized with 90% efficiency on the agarose-CS hydrogel (ACS-HRP) using NHS as a mild chemical crosslinker [211] and used for degradation of reactive blue 19. CS was dissolved in 1% acetic acid and then added to agarose aqueous solution under continuous mixing. After that, freshly prepared NHS solution was added to the agarose-CS solution. This mixture was used to incorporate HRP; the resulting suspension was poured in sterile glass plates and placed at 4 \({}^{\circ}\)C for 1 h to solidify the gel matrix completely. ACS-HRP preserved higher activity under acidic environments with a pH of 4.0 (38 vs. 5.9%), and well stabilized under alkaline conditions, retaining 3.9-fold greater activity than free HRP at pH 10. Using ACS-HRP, 1.6- and 4-fold greater activity was achieved at 50 and 70 \({}^{\circ}\)C, respectively, compared to the free enzyme. After 5 and 10 cycles of use, the ACS-HRP retained above 90% and 60% of original activity, respectively. [PERSON] et al. [212] reported the microencapsulation of LIP from _Yarrowia lipolytica_ IMUFRJ 50,682 with ionotropic gelation of CS with SA. Briefly, SA was dissolved in LIP enzymatic extract, and then microcapsules were formed by dripping in calcium chloride and CS solution (CS was preliminarily dissolved in acetic acid). Microapsules remained in the CaCl\({}_{2}\) solution for a determined time at pH = 7 and were then lyophilized. The catalytic activity of the immobilized LIP was estimated using p-nitrophenyl laurate as a substrate. The optimum parameters of immobilization were found (3.1% (\(w/v\)) SA, 0.2% (\(w/v\)) CS, 0.14 M CaCl\({}_{2}\) and 1 min complexation time) allowing for high immobilization yield (99.8%) and 150.7 U/g of LIP activity. Microapsules containing LIP from _Y. lipolytica_ were more stable at 37 \({}^{\circ}\)C than the free enzyme and retained 50% of activity after the second reaction cycle and could be used for five reactions with 20% of initial activity. A biocatalyst for the continuous production of gluten-reduced beer was developed by [PERSON] et al. [213] based on immobilized prolyl endopeptidase from _Aspergillus niger_ (AN-PEP). AN-PEP was immobilized on CS beads, which we obtained by common dripping CS acidic solution into NaOH solution and then functionalized with a polydialdehyde-hyde starch crosslinker. The maximum specific activity of the immobilized enzyme was 0.016 U/mgBSAeq. (BSA (boxing serum albumin) was used as standard protein). The immobilization increased the thermal stability of AN-PEP, which showed similar catalytic properties in synthetic beer (toward the synthetic substrate Z-Gly-Pro-pNA) when it was applied at 20 \({}^{\circ}\)C or at 50 \({}^{\circ}\)C. The continuous treatment in a fluidized bed reactorallowed for the reductionof the initial gluten content (65 mg/kg) in commercial beer from barley malt, reaching 19 mg/kg after 9 h and 15 mg/kg after 10 h of treatment. [PERSON] et al.