Abstract
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 via an institution to check access.
Similar content being viewed by others
References
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
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
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
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
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
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
Henrissat B, Bairoch A (1996) Updating the sequence-based classification of glycosyl hydrolases. Biochem J 316:695–696
Imoto T, Yagishita K (1971) A simple activity measurement of lysozyme. Agric Biol Chem 35:1154–1156
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
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
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
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
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
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
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
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
Rosenberg M, Court D (1979) Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet 13:319–353
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
Shine J, Dalgarno L (1975) Determinant of cistron specificity in bacterial ribosomes. Nature 254:34–38
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
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
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
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
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
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
von Heijne G (1986) A new method for predicting signal sequence cleavage sites. Nucleic Acids Res 14:4683–4690
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
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
Acknowledgments
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
Authors and Affiliations
Corresponding author
Rights 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). https://doi.org/10.1007/s00253-006-0582-4
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-006-0582-4