Springer Nature is making Coronavirus research free. View research | View latest news | Sign up for updates

Characterization of the third chitinase Chi18C of Clostridium paraputrificum M-21

  • 134 Accesses

  • 9 Citations


A novel chitinase gene chiC of Clostridium paraputrificum M-21, a chitinolytic and hydrogen-gas-producing bacterium, was characterized along with its translated product. The chi18C gene encodes 683 amino acids (signal peptide included) with a deduced molecular weight of 74,651. Chi18C is a modular enzyme composed of a family-18 catalytic module of glycoside hydrolases, two reiterated modules of unknown function, and a family-12 carbohydrate-binding module. Recombinant Chi18C was active toward soluble and insoluble chitin preparations, and synthetic substrates such as 4-methylumbelliferyl-β-d-N-N-N″-triacetylchitotriose, but not active toward 4-MU-N-acetylglucosamine or 4-MU-β-d-N-N′-diacetylchitobioside. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunological analyses suggested that the expression of chi18C was inducible with chitinous substrates and that Chi18C was secreted into the culture medium. A possible role of Chi18C in the chitinolytic system of C. paraputrificum M-21 is discussed.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5


  1. Alam MM, Nikaido N, Tanaka H, Watanabe T (1995) Cloning and sequencing of the chiC gene of Bacillus circulans WL-12 and relationship of its product to some other chitinases and chitinase-like proteins. J Ferment Bioeng 80:454–461

  2. Bradford MM (1976) A rapid and sensitive method for the quantitation of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

  3. Brun E, Moriaud F, Gans PM, Blackledge J, Barras F, Marion D (1997) Solution structure of the cellulose-binding domain of the endoglucanase Z secreted by Erwinia chrysanthemi. Biochemistry 36:16074–16086

  4. Evvyernie D, Yamazaki S, Morimoto K, Karita S, Kimura T, Sakka K, Ohmiya K (2000) Identification and characterization of Clostridium paraputrificum M-21, a chitinolytic, mesophilic and hydrogen-producing bacterium. J Biosci Bioeng 89:596–601

  5. Evvyernie D, Morimoto K, Karita S, Kimura T, Sakka K, Ohmiya K (2001) Conversion of chitinous wastes to hydrogen gas by Clostridium paraputrificum M-21. J Biosci Bioeng 91:339–343

  6. Hashimoto M, Ikegami T, Seino S, Ohuchi N, Fukada H, Sugiyama J, Shirakawa M, Watanabe T (2000) Expression and characterization of the chitin-binding domain of Chitinase A1 from Bacillus circulans WL-12. J Bacteriol 182:3045–3054

  7. Henrissat B, Bairoch A (1996) Updating the sequence-based classification of glycosyl hydrolases. Biochem J 316:695–696

  8. Imoto T, Yagishita K (1971) A simple activity measurement of lysozyme. Agric Biol Chem 35:1154–1156

  9. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

  10. Li H, Morimoto K, Katagiri N, Kimura T, Sakka K, Lun S, Ohmiya K (2002) A novel β-N-acetylglucosaminidase of Clostridium paraputrificum M-21 with high activity on chitobiose. Appl Microbiol Biotechnol 60:420–427

  11. Li H, Morimoto K, Kimura T, Sakka K, Ohmiya K (2003) A new type of β-N-acetylglucosaminidase from hydrogen-producing Clostridium paraputrificum M-21. J Biosci Bioeng 95:268–274

  12. Morimoto K, Karita S, Kimura T, Sakka K, Ohmiya K (1997) Cloning, sequencing, and expression of the gene encoding Clostridium paraputrificum chitinase ChiB and analysis of the functions of novel cadherin-like domains and a chitin-binding domain. J Bacteriol 179:7306–7314

  13. Morimoto K, Karita S, Kimura T, Sakka K, Ohmiya K (1999) Sequencing, expression, and transcription analysis of the Clostridium paraputrificum chiA gene encoding chitinase ChiA. Appl Microbiol Biotechnol 51:340–347

  14. Morimoto K, Karita S, Kimura T, Sakka K, Ohmiya K (2001) Characterization of Clostridium paraputrificum chitinase A from a recombinant Escherichia coli. J Biosci Bioeng 92:466–468

  15. Neu HC, Heppel LA (1965) The release of enzyme from Escherichia coli by osmotic shock and during the formation of spheroplast. J Biol Chem 240:3685–3692

  16. O’brien M, Colwell RR (1987) A rapid test for chitinase activity that uses 4-methylumbelliferyl-N-acetyl-β-d-glucosaminide. Appl Environ Microbiol 53:1718–1720

  17. Rosenberg M, Court D (1979) Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet 13:319–353

  18. Sasaki C, Yokoyama A, Itoh Y, Hashimoto M, Watanabe T, Fukamizo T (2002) Comparative study of the reaction mechanism of family 18 chitinases from plants and microbes. J Biochem 131:557–564

  19. Shine J, Dalgarno L (1975) Determinant of cistron specificity in bacterial ribosomes. Nature 254:34–38

  20. Shiro M, Ueda M, Kawaguchi T, Arai M (1994) Cloning of a cluster of chitinase genes from Aeromonas sp. No. 10S-24. Biochim Biophys Acta 1305:44–48

  21. Sidhu SS, Kalmar GB, Willis LG, Borgford TJ (1994) Streptomyces griseus protease C. A novel enzyme of the chymotrypsin superfamily. J Biol Chem 269:20167–20171

  22. Tsujibo H, Orikoshi H, Shiotani K, Hayashi M, Umeda J, Miyamoto K, Imada C, Okami Y, Inamori Y (1988) Characterization of chitinase C from a marine bacterium, Alteromonas sp. strain O-7, and its corresponding gene and domain structure. Appl Environ Microbiol 64:472–478

  23. Tsujibo H, Orikoshi H, Tanno H, Fujimoto K, Miyamoto K, Imada C, Okami Y, Inamori Y (1993) Cloning, sequence, and expression of a chitinase gene from a marine bacterium, Alteromonas sp. strain O-7. J Bacteriol 175:176–181

  24. Ueda M, Shiro M, Kawaguchi T, Arai M (1994) Molecular cloning and nucleotide sequence of the gene encoding chitinase II from Aeromonas sp. No.10S-24. J Ferment Bioeng 78:205–211

  25. Ueda M, Shiro M, Kawaguchi T, Arai M (1996) Expression of the chitinase III gene of Aeromonas sp. No. 10S-24 in Escherichia coli. Biosci Biotechnol Biochem 60:1195–1197

  26. von Heijne G (1986) A new method for predicting signal sequence cleavage sites. Nucleic Acids Res 14:4683–4690

  27. Watanabe T, Suzuki K, Oyanagi W, Ohnishi K, Tanaka H (1990) Gene cloning of chitinase A1 from Bacillus circulans WL-12 revealed its evolutionary relationship to Serratia chitinase and to the type III homology units of fibronectin. J Biol Chem 265:15659–15665

  28. Watanabe T, Oyanagi W, Suzuki W, Ohnishi K, Tanaka H (1992) Structure of the gene encoding chitinase D of Bacillus circulans WL-12 and possible homology of the enzyme to other prokaryotic chitinases and class III plant chitinase. J Bacteriol 174:408–414

Download references


This study was in part carried out as part of “High Efficiency Bioenergy Conversion Project/Development of High Efficiency Hydrogen–Methane Processing,” which was entrusted by the New Energy and Industrial Technology Development Organization (NEDO). We thank Mr. M. Benson Wamalwa for critical reading of the manuscript.

Author information

Correspondence to Kazuo Sakka.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Morimoto, K., Yoshimoto, M., Karita, S. et al. Characterization of the third chitinase Chi18C of Clostridium paraputrificum M-21. Appl Microbiol Biotechnol 73, 1106–1113 (2007).

Download citation


  • Chitinase
  • Clostridium
  • Paraputrificum