Effective selection of the monoclonal antibodies inhibiting the enzyme activity of the bifunctional amylase-pullulanase produced by B. circulans: Possible presence of two enzymatic active sites on one polypeptide

Amylase-pullulanase enzyme (APE) hydrolyzes not only the α-1,6-glycosidic linkage but also the α-1,4-glycosidic linkage to the same extent. It is not clear whether a single site on the enzyme is responsible for the expression of both activities or not. Hybridomas that secrete monoclonal antibodies (MABs) specific to APE have been established. Amylase or pullulanase activity-inhibiting antibodies were specifically selected by incubating culture supernatant of hybridomas with the electrophoretically separated APE. Out of 54 MABs tested, MAP-12 specifically inhibited the amylase activity (about 94%) and MAP-17 showed 97% inhibition with 1 μg of each antibody, but inhibited the pullulanase activity slightly. On the other hand, MAP-5 inhibited mainly the pullulanase activity (35% inhibition with 1 μg and 86% with 2 μg of the antibody), and MAP-3 inhibited the pullulanase activity (about 88%) only with 1 μg of the antibody. These results clearly show the successful selection of the monoclonal antibodies which specifically inhibit each enzyme activity and suggest that APE may have two different active sites responsible for the expression of amylase and pullulanase activities.ope