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Journal of Bacteriology, August 2009, p. 4835-4844, Vol. 191, No. 15
0021-9193/09/$08.00+0     doi:10.1128/JB.00176-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Role of Maltogenic Amylase and Pullulanase in Maltodextrin and Glycogen Metabolism of Bacillus subtilis 168{triangledown} ,{dagger}

Jae-Hoon Shim,1,{ddagger} Jong-Tae Park,1,{ddagger} Jung-Sun Hong,1 Ki Woo Kim,2 Myo-Jeong Kim,3 Jung-Hyuk Auh,4 Young-Wan Kim,5 Cheon-Seok Park,6 Winfried Boos,7 Jung-Wan Kim,8* and Kwan-Hwa Park1*

Center for Agricultural Biomaterials and Department of Food Science and Biotechnology,1 National Instrumental Center for Environmental Management, Seoul National University, Seoul 151-921,2 Food Research Institute, School of Food and Life Science, and Biohealth Products Research Center, Inje University, Gimhae 621-749,3 Department of Food Science and Technology, Chungang University, Anseong 456-756,4 Department of Food and Biotechnology, Korea University, Jochiwon, Chungnam 339-700,5 Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Yongin 449-701, Republic of Korea,6 Department of Biology, University of Konstanz, 78457 Konstanz, Germany,7 Department of Biology, University of Incheon, Incheon 402-749, Republic of Korea8

Received 10 February 2009/ Accepted 13 May 2009

The physiological functions of two amylolytic enzymes, a maltogenic amylase (MAase) encoded by yvdF and a debranching enzyme (pullulanase) encoded by amyX, in the carbohydrate metabolism of Bacillus subtilis 168 were investigated using yvdF, amyX, and yvdF amyX mutant strains. An immunolocalization study revealed that YvdF was distributed on both sides of the cytoplasmic membrane and in the periplasm during vegetative growth but in the cytoplasm of prespores. Small carbohydrates such as maltoheptaose and β-cyclodextrin (β-CD) taken up by wild-type B. subtilis cells via two distinct transporters, the Mdx and Cyc ABC transporters, respectively, were hydrolyzed immediately to form smaller or linear maltodextrins. On the other hand, the yvdF mutant exhibited limited degradation of the substrates, indicating that, in the wild type, maltodextrins and β-CD were hydrolyzed by MAase while being taken up by the bacterium. With glycogen and branched β-CDs as substrates, pullulanase showed high-level specificity for the hydrolysis of the outer side chains of glycogen with three to five glucosyl residues. To investigate the roles of MAase and pullulanase in glycogen utilization, the following glycogen-overproducing strains were constructed: a glg mutant with a wild-type background, yvdF glg and amyX glg mutants, and a glg mutant with a double mutant (DM) background. The amyX glg and glg DM strains accumulated significantly larger amounts of glycogen than the glg mutant, while the yvdF glg strain accumulated an intermediate amount. Glycogen samples from the amyX glg and glg DM strains exhibited average molecular masses two and three times larger, respectively, than that of glycogen from the glg mutant. The results suggested that glycogen breakdown may be a sequential process that involves pullulanase and MAase, whereby pullulanase hydrolyzes the {alpha}-1,6-glycosidic linkage at the branch point to release a linear maltooligosaccharide that is then hydrolyzed into maltose and maltotriose by MAase.

* Corresponding author. Mailing address for K.-H. Park: Center for Agricultural Biomaterials and Department of Food Science and Biotechnology, Seoul National University, Seoul 151-921, Republic of Korea. Phone: 82-2-880-4852. Fax: 82-2-873-5095. E-mail: parkkh{at}snu.ac.kr. Mailing address for J.-W. Kim: Department of Biology, University of Incheon, Incheon 402-749, Republic of Korea. Phone: 82-32-770-8244. Fax: 82-32-770-4424. E-mail: kjw5864{at}incheon.ac.kr

{triangledown} Published ahead of print on 22 May 2009.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

{ddagger} These authors contributed equally.

Journal of Bacteriology, August 2009, p. 4835-4844, Vol. 191, No. 15
0021-9193/09/$08.00+0     doi:10.1128/JB.00176-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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