PERIPLASMIC TREHALASE FROM ESCHERICHIA-COLI - CHARACTERIZATION AND IMMOBILIZATION ON SPHERISORB

1. Trehalase was partially purified from Escherichia coli and characte rized. The Km for trehalose was 0.78 mM, the pH optimum 5.5 and the te mperature optimum 30 degrees C. 2. Trehalase represented approximately 50% of the total protein released by osmotic shock. The preparation w as free of nonspecific carbohydrate hydrolases, which act on sucrose, galactose and maltose, permitting trehalose determination in biologica l samples, such as insect hemolymph and free cell extracts among other s. 3. The enzyme was stable in 50 mM malcate buffer, pH 16.2, at -8 de grees 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 ach ieved by covalent linkage to spherisorb-5NH(2) (spherical silica gel). Retention of total catalytic activity averaged 32%. 5. The reactor, s tored for one month at -5 degrees C, retained 98% of its initial immob ilized activity. 6. This immobilized form of the enzyme could be used routinely for specific determinations of trehalose.