Characterization of a monoclonal antibody that specifically inhibits pullulanase activity ofbacillus circulans amylase-pullulanase enzyme |
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Authors: | Cheorl-Ho Kim Tae-Kyun Lee |
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Institution: | (1) Department of Biochemistry and Molecular Biology, Dong Guk University, 707 Sukjang Dong, Kyung Ju City, 780-714 Kyungpook, Korea;(2) Department of College of Oriental Medicine and Gynecology, Dong Guk University, 707 Sukjang Dong, Kyung Ju City, 780-714 Kyungpook, Korea |
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Abstract: | A monoclonal antibody (MAb) against amylase-pullulanase enzyme fromBacillus circulons, which hydrolyzes not only theα-1,6-glycosidic linkage but also theα-1,4-glycosidic linkage to the same extent, has been produced by the fusion of BALB/c mouse spleen cells immunized with the
native enzyme and P3x63Ag8U1 myeloma cells, and examined for inhibition of pullulanase activity in order to characterize the
catalytic site of the pullulanase. The MAb recognizes active enzyme, but not the SDS-denatured or heat-inactivated protein,
indicating that the antibody is highly conformational-dependent, specific for active enzyme. The antibody inhibited the pullulanase
activity, but not amylase activity. The monoclonal antibody immunoblotted the enzyme and immunoprecipitated the enzyme. The
immunoprecipitation was inhibited in the presence of substrate, pullulan, and the MAb competitively inhibited the binding
of pullulan to the enzyme. The MAb, therefore, recognizes the pullulanbinding site of the enzyme. Kinetic analysis showed
that the MAb inhibited pullulanase activity with inhibition constant (K
i
,) of 0.77Μg/mL, providing evidence that the antibody decreases the catalytic rate of enzyme activity and has an effect on
substrate binding. These results strongly confirm the previous observations that APE may have two different active sites responsible
for the expression of amylase and pullulanase activities (Kim, C. H. and Kim, Y. S.Eur. J. Biochem. 1995,227, 687–693). |
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Keywords: | Monoclonal antibody bifunctional enzyme amylasepullulanase enzyme selective inhibition active conformation recognition Bacillus circulans |
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