Advertisement

Biotechnology Letters

, Volume 34, Issue 2, pp 281–286 | Cite as

Cloning of an endoglycanase gene from Paenibacillus cookii and characterization of the recombinant enzyme

  • Suguru Shinoda
  • Shin Kanamasa
  • Motoo Arai
Original Research Paper

Abstract

An endoglycanase gene of Paenibacillus cookii SS-24 was cloned and sequenced. This Pgl8A gene had an open reading frame of 1,230 bp that encoded a putative signal sequence (31 amino acids) and mature enzyme (378 amino acids: 41,835 Da). The enzyme was most homologous to a β-1,3-1,4-glucanase of Bacillus circulans WL-12 with 84% identity. The recombinant enzyme hydrolyzed carboxymethyl cellulose, swollen celluloses, chitosan and lichenan but not Avicel, chitin powder or xylan. With chitosan as the substrate, the optimum temperature and hydrolysis products of the recombinant enzyme varied at pH 4.0 and 8.0. This is the first report that characterizes chitosanase activity under different pH conditions.

Keywords

Cellulase Chitosanase Gene cloning Paenibacillus cookii 

Supplementary material

10529_2011_759_MOESM1_ESM.docx (14 kb)
Supplementary Table 1. (DOCX 15 kb)
10529_2011_759_MOESM2_ESM.docx (93 kb)
Supplementary Fig. 1. SDS–PAGE analysis of the purified enzyme from P. cookii SS-24 and recombinant enzyme. Lane M, protein standard marker (Protein Ladder 10-250 kDa, New England Biolabs); Lane 1, purified enzyme from P. cookii; Lane 2, purified recombinant enzyme. The purified enzymes were applied to 12% SDS–PAGE. (DOCX 94 kb)
10529_2011_759_MOESM3_ESM.docx (1.3 mb)
Supplementary Fig. 2. Comparison of the amino acid sequence of Pgl8A and other glycosyl hydrolases. The amino acid sequences of GHF-8 enzymes from P. cookii SS-24 (Pgl8A), B. circulans WL-12 (B.WL-12), B. circulans KSM-N257 (B.N257), Lysobacter sp. IB-9374 (L.IB9374), Bacillus sp. KCTC0377BP (B. KCTC) and Bacillus sp. K17 (B.K17) are shown. The conserved amino acid sequence of GHF-8 catalytic module is underlined. Asterisks denote potential catalytic residues. (DOCX 1,305 kb)

References

  1. Adachi W, Sakihama Y, Shimizu S, Sunami T, Fukazawa T, Suzuki M, Yatsunami R, Nakamura S, Takenaka A (2004) Crystal structure of family GH-8 chitosanase with subclass II specificity from Bacillus sp. K17. J Mol Biol 343:785–795PubMedCrossRefGoogle Scholar
  2. Choi YJ, Kim EJ, Piao Z, Yun YC, Shin YC (2004) Purification and characterization of chitosanase from Bacillus sp. strain KCTC 0377BP and its application for the production of chitosan oligosaccharides. Appl Environ Microbiol 70:4522–4531PubMedCrossRefGoogle Scholar
  3. Hash JH, King KW (1958) On the nature of the β-glucosidases of Myrothecium verrucaria. J Biol Chem 232:381–393PubMedGoogle Scholar
  4. Hedges A, Wolfe RS (1974) Extracellular enzyme from Myxobacter AL-1 that exhibits both β-1, 4-glucanase and chitosanase activities. J Bacteriol 120:844–853PubMedGoogle Scholar
  5. Imoto T, Yagishita K (1971) Simple activity measurement of lysozyme. Agri Biol Chem 35:1154–1156CrossRefGoogle Scholar
  6. Kimoto H, Kusaoke H, Yamamoto I, Fujii Y, Onodera T, Taketo A (2002) Biochemical and genetic properties of Paenibacillus glycosyl hydrolase having chitosanase activity and discoidin domain. J Biol Chem 277:14695–14702PubMedCrossRefGoogle Scholar
  7. Mitsutomi M, Isono M, Uchiyama A, Nikaidou N, Ikegami T, Watanabe T (1998) Chitosanase activity of the enzyme previously reported as β-1, 3-1, 4-glucanase from Bacillus circulans WL-12. Biosci Biotechnol Biochem 62:2107–2114PubMedCrossRefGoogle Scholar
  8. Muzzarelli RAA (1977) Chitin. Pergamon Press, London, p 105Google Scholar
  9. Ogura J, Toyoda A, Kurosawa T, Chong AL, Chohnan S, Masaki T (2006) Purification, characterization, and gene analysis of cellulase (Cel8A) from Lysobacter sp. IB-9374. Biosci Biotechnol Biochem 70:2420–2428PubMedCrossRefGoogle Scholar
  10. Sakamoto R, Arai M, Murao S (1984) Enzymatic properties of hydrocellulase from Aspergillus aculeatus. J Ferment Technol 62:561–567Google Scholar
  11. Shahidi F, Arachchi JKV, Jeon YJ (1999) Food applications of chitin and chitosans. Trends Food Sci Technol 10:37–51CrossRefGoogle Scholar
  12. Shimosaka M, Nogawa M, Ohno Y, Okazaki M (1993) Chitosanase from the plant pathogenic fungus, Fusarium solani f. sp. phaseoli—purification and some properties. Biosci Biotechnol Biochem 57:231–235CrossRefGoogle Scholar
  13. Yabuki M, Uchiyama A, Suzuki K, Ando A, Fujii T (1988) Purification and properties of chitosanase from Bacillus circulans MH-K1. J Gen Appl Microbiol 34:255–270CrossRefGoogle Scholar
  14. Yatsunami R, Sakihama Y, Suzuki M, Fukazawa T, Shimizu S, Sunami T, Endo K, Takenaka A, Nakamura S (2002) A novel chitosanase from Bacillus sp. strain K17: gene cloning and expression in Escherichia coli. Nucl Acids Res Suppl 2:227–228CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  1. 1.Department of Environmental Biology, College of Bioscience and BiotechnologyChubu UniversityAichiJapan

Personalised recommendations