Chitosan beads (CBs) were produced by the dropwise addition of chitosan gel in an alkaline coagulating mixture (H2O:methanol:NaOH = 4:5:1, w/w/w) [20]. This gel was prepared by dissolving 6.5 g of chitosan in 210 mL of 8% acetic acid, and then the solution was degassed. The collected wet CBs were cleaned with deionized water until the water became neutral and were subsequently dried at 60 °C for 12 h.
The crosslinking was conducted by adding CBs to a glass vessel containing GA, where the molar ratio of the CHO groups of GA and the amino groups of chitosan was adjusted to 1:2, which was specified elsewhere [20]. Basically, 70 g of wet CBs was placed in 250 mL of a GA solution (25 wt%) at room temperature for 6 h. The GA-CBs were washed three times with deionized water to eliminate the residual GA. Then, they were dried at 65 °C for 18 h. The resulting GA-CBs were weighed and used in the following biosorption experiments and for enzyme immobilization.