Nylon-6 cylinders and a sponge-like derivative as supports for immobilizing trypsin

1. Two types of nylon-6 supports (small cylinders and a sponge-like derivative) were prepared for immobilizing enzymes. Nylon-6 beads were solubilized by immersion in 80 formic acid and then reprecipitated using two different types of non-solvent solutions (distilled water or a 1:1 acetone:water solution) giving rise to a sponge-like derivative and to a colloidal suspension, respectively. The latter was molded into a thin thread which was cut into small cylinders. 2. Trypsin (EC 3.4.21.4) was covalently bound to glutaraldehyde-activated nylon-6 cylinders as well as to the sponge-like derivative. The maximum (100) apparent initial enzymatic activity was found for the trypsin bound to small cylinders, while the initial activity of trypsin bound to the sponge-like material was 61 in comparison with that of trypsin-small cylinders, under the same conditions of enzyme immobilization reaction (1 g of nylon support and 5 ml of 1.3 mg/ml trypsin in 0.1 M sodium phosphate buffer, pH 8.5, at 10 degrees C for 18 h) and of enzymatic reaction (1 g of trypsin-nylon in a batch reactor, 2 ml of 0.7 w/v azocasein solution in 50 mM borate buffer, pH 8.5, at 37 degrees C, with shaking, for 1 h). However, the decrease of activity after enzyme immobilization was more conspicuous for the trypsin-small cylinders than for the trypsin-sponge. The former retained approximately 25 of its initial activity, while the latter retained approximately 67 of its initial activity, after seven cycles of utilization for 1 h, pH 8.5, at 37 degrees C and 8 days of storage, pH 8.5, at 4 degrees C in the presence of azocasein. 3. Scanning electron microscopy was performed to visualize the surface of the support after each step of the immobilization process. The electron micrographs show that the two types of nylon supports had a rough surface, which became rougher and full of craters after treatment with 5 N HCl. On the other hand, the partially hydrolyzed nylon surface acquired the appearance of Swiss cheese after treatment with 2.5 glutaraldehyde. After reaction with the enzyme molecules the surface became rougher again.

Periplasmic trehalase from Escherichia coli: characterization and immobilization on spherisorb

1. Trehalase was partially purified from Escherichia coli and characterized. The Km for trehalose was 0.78 mM, the pH optimum 5.5 and the temperature optimum 30 degrees C. 2. Trehalase represented approximately 50 per cent of the total protein released by osmotic shock. The preparation was free of nonspecific carbohydrate hydrolases, which act on sucrose, galactose and maltose, permitting trehalose determination in biological samples, such as insect hemolymph and free cell extracts among others. 3. The enzyme was stable in 50 mM maleate buffer, pH 6.2, at -8 degrees C for at least 6 months and could be used to determine trehalose in the range of 6 to 30 nmol. 4. Immobilization of the enzyme was achieved by covalent linkage to spherisorb-5NH2 (spherical silica gel). Retention of total catalytic activity averaged 32 per cent . 5. The reactor, stored for one month at -5 degrees C, retained 98 per cent of its initial immobilized activity. 6. This immobilized form of the enzyme could be used routinely for specific determinations of trehalose