Abstract
For the cross-linking of Taka-amylase A (TAA) to carboxymethyl dextran (CM-Dex), the use of water-soluble carbodiimide (EDC, l-ethyl-3-(3-dimethylaminopropyl)carbodiimide) was examined. CM-Dex was treated with EDC and the carboxyl groups were converted into the activated form, and then, the activated CM-Dex was attached to TAA. Purification of the modified TAA was done by a column of DEAE-Sephacel, where 80% of the enzyme activity was eluted at the breakthrough fraction (M-TAA I). About 14% of the activity was eluted with 0.25 MNaCl (M-TAA II). Polyacrylamide gel electrophoresis showed an increase in the carbohydrate at the position of native TAA. The Km values of M-TAA I and II for the soluble starch were 2 - 6-fold higher than that of native TAA, while Km values for γ-cyclodextrin were at the same level as the native TAA. Although the pH stability of M-TAA I at the acidic limb of pH 3.0 - 4.5 decreased slightly, the stability at the alkaline limb of pH 10.0 - 11.0 was improved. There were some increase in the stability against the denaturation by methanol.
