Food Research Institute, NARO

Applied Bacteriology Laboratory


There are a lot of useful bacteria which are utilized for the production of fermented food or for other food industries. We focus on two bacterial products: poly-γ-DL-glutamate, an amino acid homopolymer produced by Bacillus subtilis natto; and cyclic isomaltooligosaccharides (cyclodextrans or CIs) produced by Bacillus circulans. We aim to elucidate the regulatory and metabolic systems that control the synthesis of these bacterial products and to improve their productivity.

Primary research themes

  • Biosynthesis of bacterial oligosaccharides
     Cyclodextrans (CIs) are cyclic oligosaccharides consisted of 7 to 17 molecules of α-1,6 linked glucose residues. CIs are extremely water-soluble, they inhibit the activity of glucansucrases strongly and this characteristic can be useful for anti-plaque substances. CIs with ten or more glucose molecules show strong inclusion ability. We are trying to clarify the structures and mechanisms of those enzymes that are involved in producing CIs in order to increase the productivity of CIs and to develop the product specificity.
  • Mechanism by which Bacillus subtilis natto produces a sticky substance
     We are examining the gene function related to the production of poly-γ-DL-glutamate, the main ingredient of the sticky substance produced by natto bacteria. If we can clarify how this polymer is produced, it will be possible to vary the degree of polymerization and thus the stickiness of the substance. The findings will have important implications in the food industry.

Introduction of members

   Kazumi Funane
  Specialization - applied biochemistry

Senior Researcher
  Keitarou Kimura
  Specialization - applied microbiology

Contract members: 4 (as of February 2013)

Main publications

  • M. Hachinohe, K. Kimura, Y. Kubo, K. Tanji, S. Hamamatsu, S. Hagiwara, D. Nei, H. Kameya, R. Nakagawa, U. Matsukura, S. Todoriki, S. Kawamoto, Distribution of Radioactive Cesium (134Cs plus 137Cs) of the Contaminated Japanese Soybean cultivar during the preparation of Tofu, Natto, and Nimame (boiled soybean). J. Food Prot., (2013) in press
  • Y. Kubo, T. Inaoka, T. Hachiya, M. Miyake, S. Hase, R. Nakagawa, H. Hasegawa, K. Funane, Y. Sakakibara, K. Kimura, Development of a rifampicin-resistant Bacillus subtilis strain for natto-fermentation showing enhanced exoenzyme production. J. Biosci. Bioeng., doi 10.1016/j.jbiosc.2012.12.012 (2013)
  • K. Kimura, H. Kameya, D. Nei, Y. Kakihara, S. Hagiwara, H. Okadome, K. Tanji, S. Todoriki, U. Matsukura, S. Kawamoto, Dynamics of radioactive cesium (134Cs and 137Cs) during the milling of contaminated Japanese wheat cultivars and during the cooking of udon noodle made from wheat flour. J. Food Prot., 75 (10), 1823-1828 (2012)
  • R. Suzuki, K. Terasawa, K. Kimura, Z. Fujimoto, M. Momma, M. Kobayashi, A. Kimura, K. Funane, Biochemical characterization of a novel cycloisomaltooligosaccharide glucanotransferase from Paenibacillus sp. 598K. Biochim. Biophys. Acta, 1824 (7), 919-924 (2012)
  • Z. Fujimoto, K. Kimura, Crystal structure of bacteriophage ΦNIT1 zinc peptidase PghP that hydrolyzes γ-glutamyl linkage of bacterial poly-γ-glutamate. Proteins, 80 (3), 722-732 (2012)
  • Y.-M. Kim, E. Yamamoto, M.-S. Kang, H. Nakai, W. Saburi, M. Okuyama, H. Mori, K. Funane, M. Momma, Z. Fujimoto, M. Kobayashi, D. Kim, A. Kimura, Bacteroides thetaiotaomicron VPI-5482 glycoside hydrolase family 66 homolog catalyzes dextranolytic and cyclization reactions. FEBS J., 279 (17), 3185-3191 (2012)
  • Y.-M. Kim, Y. Kiso, T. Muraki, M.-S. Kang, H. Nakai, W. Saburi, W. Lang, H.-K. Kang, N. Okuyama, H. Mori, R. Suzuki, K. Funane, N. Suzuki, M. Momma, A. Fujimoto, T. Oguma, M. Kobayashi, D. Kim, A. Kimura, Novel dextranase catalyzing cycloisomaltooligosaccharide-formation and identification of catalytic amino acids and their functions using chemical rescue approach. J. Biol. Chem., 287 (24), 19927-19935 (2012)
  • N. Suzuki, Y.-M. Kim, Z. Fujimoto, M. Momma, K. Funane, A. Kimura, Structural elucidation of dextran degradation mechanism by Streptococcus mutans dextranase belonging to glycoside hydrolase family 66. J. Biol. Chem., 287 (24), 19916-19926 (2012)
  • T.-H. Do, Y. Suzuki, N. Abe, J. Kaneko, Y. Itoh, K. Kimura, Suppressor mutants of loss of DegQ function in Bacillus subtilis (natto) poly-gamma-glutamate synthesis. Appl. Enviorn. Microbiol. 77 (23), 8249-8258 (2011)
  • Y. Kawabata, K. Kimura, K. Funane, Extracellular production of cycloisomaltooligosaccharide glucanotransferase and cyclodextran by a protease-deficient Bacillus subtilis host-vector system. Appl. Microbiol. Biotech. 93 (5), 1877-1884 (2011)
  • N. Suzuki, Y.-M. Kim, Z. Fujimoto, M. Momma, K. Funane, A. Kimura, Crystallization and preliminary crystallographic analysis of dextranase from Streptococcus mutans. Acta Crystallographica Section F, F67, 1542-1544, (2011)
  • Y. Kubo, A. P. Rooney, Y. Tsukakoshi, R. Nakagawa, H. Hasegawa, K. Kimura. Phylogenetic analysis of Bacillus subtilis strains applicable to natto (fermented soybean) production. Appl. Environ. Microbiol. 77 (18), 6463-6469 (2011)
  • Y.-M. Kim, R. Shimizu, H. Nakai, H. Mori, M. Okuyama, M.-S. Kang, Z. Fujimoto, K. Funane, D. Kim, A. Kimura, Truncation of N- and C-terminal regions of Streptococcus mutans dextranase enhances catalytic activity. Appl. Microbiol. Biotechnol. 91 (2), 329-339 (2011)
  • K. Kimura, L.-S. P. Tran, K. Funane. Loss of poly-γ-glutamic acid synthesis of Bacillus subtilis (natto) due to IS4Bsu1 translocation to swrA gene. Food Sci. Technol. Res. 17 (5), 447-451 (2011)
  • K. Funane, Y. Kawabata, R. Suzuki, Y.-M. Kim, H.-K. Kang, N. Suzuki, Z. Fujimoto, A. Kimura, M. Kobayashi, Deletion analysis of regions at the C-terminal part of cycloisomaltooligosaccharide glucanotransferase from Bacillus circulans. Biochim. Biophys. Acta, 1814 (3), 428-434 (2011)
  • T. Konishi, M. Ohnishi-K., K. Funane, Y. Miyazaki, T. Konishi, T. Ishii, An arginyl residue in rice UDP-arabinopyranose mutase is required for catalytic activity and autoglycosylation. Carbohydr. Res., 345 (6), 787-791 (2010)
  • K. Kimura, L.-S. P. Tran, T.-H. Do, Y. Itoh, Expression of the pgsB Encoding the Poly-gamma-DL-glutamate Synthetase of Bacillus subtilis (natto). Biosci. Biotechnol. Biochem., 73 (5), 1149-1155 (2009)
  • K. Funane, K. Terasawa, Y. Mizumo, H. Ono, S. Gubu, T. Tokashiki, Y. Kawabata, Y.-M. Kim, A. Kimura, M. Kobayashi, Isolation of Bacillus and Paenibacillus Bacterial strains that produce large molecules of cyclic isomaltooligosaccharides. Biosci. Biotechnol. Biochem., 72 (12), 3277- 3280 (2008)
  • K. Funane, K. Terasawa, Y. Mizuno, H. Ono, T. Miyagi, S. Gibu, T. Tokashiki, Y. Kawabata, Y.-M. Kim, A. Kimura, M. Kobayashi, A novel cyclic isomaltooligosaccharide (cycloisomaltodecaose, CI-10) produced by Bacillus circulans T-3040 displays remarkable inclusion ability compared with cyclodextrins. J. Biotechnol., 130 (2), 188-192 (2007)
  • K. Kimura and Y. Itoh, Determination and characterization of IS4Bsu1-insertion loci and identification of a new insertion sequence element of the IS256 family in Natto starter. Biosci. Biotechnol. Biochem., 71 (10), 2458-2464 (2007)
  • T. Yamamoto, K. Terasawa, Y.-M. Kim, A. Kimura, Y. Kitamura, M. Kobayashi, K. Funane, Identification of catalytic amino acids of cyclodextran glucanotransferase from Bacillus circulans T-3040. Biosci. Biotechnol. Biochem., 70 (8), 1947-1953 (2006)
  • K. Funane, T. Ishii, H. Ono, M. Kobayashi, Changes in linkage pattern of glucan products induced by substitution of Lys residues in the dextransucrase. FEBS Lett., 579 (21), 4739-4745 (2005)
  • K. Kimura, Tran. P. L.-S., I. Uchida, Y. Itoh. Characterization of Bacillus subtilis gamma-glutamyl transferase and its involvement in capsule poly-gamma-glutamate degradation. Microbiology, 150 (Pt 12), 4115-4123 (2004)
  • K. Funane, T. Ishii, K. Terasawa, T. Yamamoto, M. Kobayashi, Construction of chimeric glucansucrase for analyzing substrate-binding regions that affect the structure of glucan products. Biosci. Biotechnol. Biochem., 68 (9), 1912-1920 (2004)
  • K. Kimura, Tran. P. L.-S., Y. Itoh. Roles and regulation of the glutamate racemase isogenes, racE and yrpC, in Bacillus subtilis. Microbiology, 150 (Pt 9), 2911-2920 (2004)
  • K. Kimura, Y. Itoh. Characterization of poly-gamma-glutamate hydrolase encoded by a bacteriophage genome: Possible role in phage infection of Bacillus subtilis encapsulated with poly-gamma-glutamate. Appl. Environ. Microbiol., 69 (5), 2491-2497 (2003)
  • Y. Nakada, T. Nishijyo, and Y. Itoh. Divergent Structure and Regulatory Mechanism of Proline Catabolic Systems: Characterization of the putAP Proline Catabolic Operon of Pseudomonas aeruginosa PAO1 and Its Regulation by PruR, an AraC/XylS Family Protein. J. Bacteriol., 184 (20), 5633-5640 (2002)
  • K. Kimura, Y. Inatsu, and Y. Itoh. Frequency of the Insertion Sequence IS4Bsu1 among Bacillus subtilis strains isolated from Fermented Soybean Foods in Southeast Asia. Biosci. Biotechnol. Biochem., 66 (9), 1994-1996 (2002)
  • K. Kimura, A. Nagasawa, M. Fujii, and Y. Itoh. Cloning of the pepX Gene of Lactobacillus helveticus IFO3809 Encoding Salt-tolerant X-Prolyl Dipepetidyl Aminopeptidase and Characterization of the Enzyme. J. Biosci. Biotechnol., 93 (6), 587-594 (2002)