Annals of Microbiology

, 57:623 | Cite as

Cloning and expression of a mannanase gene fromErwinia carotovora CXJZ95-198

  • Yunxiong Zhang
  • Zhengchu Liu
  • Xinbo Chen
Molecular Biology Original Article


A gene encoding a mannanase (ManA) was cloned from the genomic library ofErwinia carotovora CXJZ95-198 and expressed inEscherichia coli cells. A 1783 bp DNA fragment containing amanA gene was sequenced. An open reading frame (ORF) of 1137 bp encoded a protein of 378 amino acids. The expressed enzyme had a molecular mass of approximately 42 KD determined by SDS-PAGE. The optimal pH and temperature for the expressed enzyme was 7.5 and 55 °C, respectively. The nucleotide sequence ofmanA had remarkably low homology with other sequences reported. No typical promoter was found but a palindrome sequence existed downstream of the stop codon. The deduced amino acid sequence from mature ManA showed homology of about 53% with those fromBacillus sp., but much lower homology with those from other strains. The ManA was presumably classified as family 26 of glycosidases. It was also clarified that the 1.3 kb fragment up the locus nt 4449729 ofErwinia carotovora genomic DNA was a mannanase gene.

Key words

cloning Erwinia carotovora expression gene location mannanase gene 


  1. Aspinall G.O., Hirst E.L., Percival E.G.V., Willianson I.R. (1953). The mannans of ivory nut, part I. The methylation of mannan A and mannan B. J. Chem. Soc., 3184–3188.Google Scholar
  2. Cann I.K.O., Kocherginskaya S., King M.R., White B.A., Mackie R.I. (1999). Molecular cloning, sequencing, and expression of a novel multidomain mannanase gene fromThermoanaerobacterium polysaccharolyticum. J. Bacteriol., 181: 1643–1651.PubMedGoogle Scholar
  3. Gibbs M.D., Reeves R.A., Sunna A., Bergquist P.L. (1999). Sequencing and expression of a mannanase gene from the extreme thermophileDictyoglomus thermophilum Rt46B.1, and characteristics of the recombinant enzyme. Curr. Microbiol., 39: 351–357.CrossRefPubMedGoogle Scholar
  4. Khanongnuch C., Ooi T., Klinoshita S. (1999). Cloning and nucleotice sequence of β-mannanase and cellulase genes fromBacillus sp. 5H. World J. Microbiol. Biotechnol., 15: 249–258.CrossRefGoogle Scholar
  5. Laemmli U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680–685.CrossRefPubMedGoogle Scholar
  6. Li B.K. (2005). Purification and Characterization of Mannanase fromErwinia carotovora CXJZ95-198. Master Degree Dissertation. Xiangjiang Agricultural University, Urumq, Xinjiang, China.Google Scholar
  7. Liu Z., Peng Y.D. (2004). Study on Techniques of Bio-pulping Based on Kenaf Bark,Eulaliopsis binata, In: Liu A., Ed., International Development of Kenaf and Allied Fibers: Proceedings of the International Kenaf Symposium held at Beijing, China, Minneapolis, MN, CCG International, pp. 311–317.Google Scholar
  8. Mendoza N.S., Arai M., Sugimoto K., Ueda M., Kawaguchi T., Joson L.M. (1995). Cloning and sequencing of β-mannanase gene fromBacillus subtilis NM-39. Biochim. Bioph Acta, 1243: 552–554.Google Scholar
  9. Miller G.L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem., 31: 426–428.CrossRefGoogle Scholar
  10. Nagarajan N., Yona G. (2004). Automatic prediction of protein domains from sequence information using a hybrid learning system. Bioinformatics, 20: 1335–1360.CrossRefPubMedGoogle Scholar
  11. Nomura N., Yamashita M., Murooka Y. (1996). Genetic organization of a DNA-processing region required for mobilization of a non-self-transmissible plasmid, pEC3, isolated fromErwinia carotovora subsp.carotovora. Gene, 170: 57–62.CrossRefPubMedGoogle Scholar
  12. Ohnishi H., Nishida T., Yoshida A., Kamio Y., Izaki K. (1991). Nucleotide sequence ofpnl gene fromErwinia carotovora. Biochem. Bioph. Res. Co., 176: 321–327.CrossRefGoogle Scholar
  13. Ovos D.R., George H., Cairns J.J. (1990). Genetically engineeredErwinia carotovora: survival, intraspecific competition, and effects upon selected bacterial genera. Appl. Environ. Microbiol., 56: 1689–1694.Google Scholar
  14. Paul J. (1997). Chemistry and biochemistry of hemicellulose-relationship between hemicellulose structure and enzymes required for hydrolysis. Macromol. Symp., 120: 183–196.Google Scholar
  15. Politz O., Krah M., Thomsen K.K., Borris R. (2000). A highly thermostable endo-(β)-mannanase from the marine bacteriumRhodothermus marinus. Appl. Microbiol. Biotechnol., 53: 715–72.CrossRefPubMedGoogle Scholar
  16. Stalbrand H., Saloheimo A., Vehmaanperä J., Henrissat H., Penttilä M. (1995). Cloning and expression inSaccharomyces cerevisiae of aTrichoderma reesei β-mannanase gene containing a cellulose binding domain. Appl. Microbiol. Biotechnol., 61 (3): 1090–1097.Google Scholar
  17. Tamaru Y., Araki T., Morishita T., Kimura T., Sakka K., Ohmiya K. (1997). Cloning, DNA sequencing, and expression of the β-mannanase gene from a marine bacterium.Vibrio sp. strain MA-138. J. Ferment. Bioeng., 83: 201–205.CrossRefGoogle Scholar
  18. Zhang Y.X., Liu Z.C. (2006). The genomic DNA library construction ofErwinia carotovora CXJZ95-198. Plant Fibers Products, 28 (4): 176–181.Google Scholar
  19. Wolfrom M.L., Laver M.L., Patin D.L. (1961). Carbohydrate of the coffee beans II. Isolation and characterization of a mannan. J. Org. Chem., 26: 4533–4535.CrossRefGoogle Scholar

Copyright information

© University of Milan and Springer 2007

Authors and Affiliations

  1. 1.College of Chemistry and Environmental EngineeringChangsha University of Science and TechnologyChangsha, HunanChina
  2. 2.Institute of Bast Fibre CropsCAASChangsha, HunanChina

Personalised recommendations