The cotton used was obtained locally and had a density of 1.27 g/cm3. The value of specific surface for the cotton fibers was about 55 x 103 m2/kg (Kaewprasit et al. 1998). The immobilization procedure comprised four main steps: mercerization of the cotton with NaOH, pretreatment with pyridine, tosyl activation of the cotton, and enzyme coupling to the cotton fibers. For mercerization, 0.1 g of cotton was soaked in 10 ml of 3 N NaOH solution for several hours (24 h, but about 4 h can be sufficient; Albayrak and Yang 2002). The cotton was then rinsed thoroughly with distilled water to remove the excess NaOH. The wet cotton was blotted between paper towels to remove as much water as possible. Then blotted cotton was then further rinsed with dry acetone to remove water. A sample of 10 ml dry pyridine was then added to the cotton and incubated for (pyridine pretreatment) 18 h.
For tosylation of the cotton, 10 ml of a 5-M tosyl solution (in excess) in dry acetone was added to 0.1 g of the pyridine-pretreated cotton, without removing the pyridine, for 48 h. When tosylation was completed, the cotton was removed from the reaction mixture and washed first with acetone and then with excess amounts of a 10 mM HCl solution to remove tosyl and
pyridine residues from the cotton. Tosyl-activated cotton was kept in 10 mM HCl solution and stored at 4 °C until used for enzyme immobilization.
Enzyme immobilization was carried out by immersing 0.1 g of cotton for 2 h in 4 ml of a 10-mM HCl solution in the case of pepsin, and Tris-HCl, CaCl2 (pH 7.5) in the case of trypsin, containing 2 mg of enzyme. After immobilization, the cotton was rinsed with copious amounts of the same acid or buffer solution and then kept in it. Enzyme activity was determined immediately and with time. All reactions during tosyl activation and enzyme immobilization were carried out in 20-ml flasks at room temperature and shaken at 200 rpm. The flasks were tightly sealed with rubber stoppers to prevent evaporation of the reactants and solvents.
The proposed mechanism for cotton tosylation followed by enzyme immobilization is described in Fig. 3.
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