Nonconducting Cellulosic Textiles 921

Pepsin and Trypsin Immobilization on Cotton

Cotton, which is used for the manufacture of clothes is not expensive and is highly accessible, has been used with success in the immobilization of enzymes and microorganisms, and in the fermentation process (Albayrak and Yang 2002). Cotton covered with polyethyleneimine has recently been used as a support for the immobilization of glucose oxidase (Kumar et al. 1997), urease (Kamath et al. 1988), and invertase (Yamazaki et al. 1984) by ionic adsorption followed by reticulation with glutaraldehyde. Cotton fibersthathaveaskeletonofcellulose(Fig. 1) canbeactivatedwith avariety of reagents such as cyanogen bromide (CNBr), sulfonyl chlorides, and periodates to form covalent bonds with the enzyme (Confort et al. 1989). The activation of cellulose by CNBr produces a slightly reactive cyclic imidocarbonate with a labile bond that provokes the release of the coupled catalyst (Confort et al. 1988). However, CNBr is poisonous, and its use implies some serious health risks during the activation because of the

Fig. 1. Basic structure of cellulose molecules ii-2

Cellulose

Fig. 1. Basic structure of cellulose molecules possible presence of cyanate residues during food ingredient production (Giacomini et al. 1998).

Chloride p-toluenesulfonyl (tosyl chloride) is one of the least expensive reagents that can be used to activate hydroxyl groups under moderate conditions (Albayrak and Yang 2002). While 2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride) is more reactive than tosyl chloride, it is extremely expensive and too volatile to be used conveniently, unlike tosyl (Albayrak and Yang 2002). Tosylated materials are stable in a dilute acidic environment (e.g., pepsin in an acidic environment) and the covalent bond formed after the displacement of the tosyl should be very strong and stable.

Among the proteolytic enzymes, immobilized proteases have shown better properties regarding activity and stability. They catalyze the hydrolysis of certain peptide bonds in protein molecules. The general reaction is illustrated in Fig. 2.

Fig. 2. Hydrolysis of peptide bonds in protein molecules (a reaction that is catalyzed by immobilized proteases)

Fig. 2. Hydrolysis of peptide bonds in protein molecules (a reaction that is catalyzed by immobilized proteases)

Proteases attack proteins in two ways, yielding different products. Endo-proteases attack peptide bonds in the interior of the peptide chain, yielding smaller polypeptides and peptides. Exoproteases act as cleaving agents of single amino acids from either end of the peptide chain. The pancreatic enzyme trypsin, which facilitates the digestion of proteins, breaks a peptide bond adjacent to the basic amino acid residues of lysine and arginine, whilst the enzyme chymotrypsin, another digestive enzyme, hydrolyzes the peptide bond adjacent to aromatic amino acid residues (phenylalanine, tryptophan, and tyrosine). These two enzymes have serine at the active site and are referred to as serine proteases. Pepsin effects a direct interaction between the peptide bonds and two aspartate radicals, which play alternatively the role of acid and basic catalysts. It is supposed that one of the carboxylic radicals of aspartate is dissociated whilst the other is protonated.

Cotton was chosen for its highly porous fibrous structure and its high mechanical strength. Hence, it allows high flow rates and efficient mass transfer through the matrix, properties that are advantageous not only during treatments for chemical activation and enzyme immobilization, but also for the applications of immobilized enzymes. For this reason, in the present work we are interested in the establishment of cotton catalytic materials upon which the proteases pepsin and trypsin will be immobilized in an irreversible manner using covalent bonding via tosyl chloride activation.

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