Hydrolysis of the inulin from jerusalem artichoke by immobilized inulase

Abstract

$\beta$-Fructosidase (inulase) from $\mbox{\underline{Kluyveromyces}}$ fragilis was purified 106 folds using Sepharose 6B column (5.0 X 95 cm). The purified enzyme was immobilized on Tygon tube and aminoethyl cellulose for the purpose of studies on production of fructose from Jerusalem artichoke tuber extract and mass transfer limitation. Silanization in chloroform at $65\,^\circ\!C$ and 10\% glutaraldehyde treatment were found to be critical points for the immobilization on Tygon tube, while 2\% glutaradehyde was the most effective for the immobilization of the enzyme on aminoethyl cellulose. The characteristics of the immobilized enzymes were examined. Optimal pH``s of invertase and inulase activity were 4.0 and 5.5, respectively, for the enzyme immobilized on aminoethyl cellulose, while 3.5 and 4.5, respectively, for the immobilized enzyme on Tygon tube. Optimal temperatures of invertase and inulase activities were shown to be $60\,^\circ\!C$ and $45\,^\circ\!C$, respectively for the enzyme immobilized on amonoethyl cellulose. Compared with soluble enzyme, both activities were unstable in this temperature. The activation energies were 5.9 kcal/mole for the enzyme immobilized on Tygon tube and 7.0 kcal/mole for the immobilized enzyme on aminoethyl cellulose with sucrose as substrate. With inulin as substrate these were 3.4 kcal/mole for the former and 5.6 kcal/mole for the latter. Km for sucrose hydrolysis was 10 mM for the immobilized enzyme on Tygon tube, and 7.7 mM for the enzyme immobilized on aminoethyl cellulose. When 2\% inulin was used instead of 5\% inulin, the activity ratios (invertase/inulase) were 6.8 in soluble enzyme, 7.5 in the immobilized enzyme on aminoethyl cellulose, and 10 in the case of the enzyme immobilized on Tygon tube. Considering the higher $K_m$ value, decrease in activation energy and the activity ratio, there existed a significant limitation of internal diffusion in the case of the enzyme immobilized on Tygon tube using the inul...