K-069. Role of Maltogenic Amylase and Pullulanase in the Metabolism of Maltodextrin and Glycogen in Bacillus subtilis 168

J-W. Kim1, C-H. Cha1, J-T. Park2, J-H. Shim2, W. Boos3, K-H. Park2;
1Univ. of Incheon, Incheon, REPUBLIC OF KOREA, 2Seoul Natl. Univ., Seoul, REPUBLIC OF KOREA, 3Univ. of Konstanz, Konstanz, GERMANY.

Two amylolytic enzymes, a maltogenic amylase (MAase) encoded by yvdF and a pullulanase (PNase) encoded by amyX, were investigated for their functions in carbohydrate metabolism in B. subtilis by constructing three mutants, yvdF-, amyX-, and yvdF-/amyX-. Transmission electron microscopy using immunochemical labeling and fluorescent microscopy using green fluorescence protein revealed that YvdF was localized close to the cell envelope area during vegetative growth but redistributed to the cytoplasm of forespore during sporulation. Inactivation of yvdF promoted sporulation by more than two folds than wild type, indicating that MAase was involved in the process determining life cycle of the bacteria. Maltoheptaose and β-cyclodextrin (β-CD) were taken up via two distinct transporters, by the Mdx and Cyc ABC transporter, respectively. They were hydrolyzed immediately to linear maltodextrin in wild type B. subtilis. On the other hand, the yvdF mutant (yvdF-) could not degrade those substrates well. This indicated that maltodextrins and β-CD were hydrolyzed by MAase after being taken up by the bacteria. The role of MAase and PNase in glycogen utilization also was monitored in vitro and in vivo. When the cell extract of wild type, yvdF, amyX- or amyX/yvdF- double mutant was incubated with glycogen individually, the overall numbers of the side chains decreased greatly by the extract of wild type whereas slowly decreased by those of the mutants. The breakdown rate of glycogen was in the order of wild type > yvdF- > amyX- > amyX-/yvdF-, indicating that PNase and MAase might play an important role in glycogen degradation. Furthermore, PNase showed high specificity for the hydrolysis of the outer side chain of glycogen with 3-5 glucosyl residues. The results suggested that glycogen breakdown was a sequencial process of PNase and MAase in which PNase hydrolysed α-1,6-glycosidic linkage at the branch point to release the linear maltooligosaccharides that are further hydrolysed into maltose and maltotriose by MAase.