Skip to main content
Download PDF
  • Industrial Microbiology
  • Original Articles
  • Published:

Biochemical characterisation of two forms of halo- and thermo-tolerant chitinase C ofSalinivibrio costicola expressed inEscherichia coli

Annals of Microbiology volume 57, pages 249–257 (2007)Cite this article

Abstract

Two forms of chitinase C (Chi-I and Chi-II) were purified until homogeneity from the culture supernatant of a transformantEscherichia coli harbouringchitinase C gene from the halophilic bacteriumSalinivibrio costicola strain 5SM-1. Chi-II was derived from Chi-I by C-terminal processing. Chi-I and Chi-II showed similar salinity optimum at 1–2% NaCl and retained more than 80% of their activity at 3–5% NaCl and more than 50% residual activity at 14% NaCl. The two enzymes could also well function (activity > 95%) in the absence of NaCl. Both had highest activity at pH 7.0 and 50 °C and both were stable over a wide range of pH (3.0–10.0). More than 50% activity remained at 80 °C after 60 min treatment. Among 4 major cations contained in sea water, only Mg2+ at 10 mM increased activity about 10%. Usingp-nitrophenyl-N,N′-diacetylchitobiose as substrate, Chi-I and Chi-II hadK m of 30 and 31.8 μM andV max of 10 and 9.2 μmol/h/mg protein, respectively. Chi-I and Chi-II were classified as exochitinases by product analysis of theE. coli culture supernatant with high performance liquid chromatography (HPLC) and thin-layer chromatography (TLC).

References

  • Aunpad R., Panbangred W. (2003). Cloning and characterization of constitutively expressed chitinase C gene from marine bacteriumSalinivibrio costicola strain 5SM-1. J. Biosci. Bioeng., 96: 529–536.

    Article  CAS  PubMed  Google Scholar 

  • Barata R.A., Andrade M.H.G., Rodrigues R.D., Castro I.M. (2002). Purification and characterization of an extracellular trypsin-like protease ofFusarium oxysporum var.lini. J. Biosci. Bioeng., 94: 304–308.

    Article  CAS  Google Scholar 

  • Bendt A., Huller H., Kammel U., Helmke E., Schweder T. (2001). Cloning, expression and characterization of a chitinase gene from the Antarctic psychrotolerant bacteriumVibrio sp. strain Fi: 7. Extremophiles, 5: 119–126.

    Article  CAS  PubMed  Google Scholar 

  • Boggon T.J., Murray J., Chappuis-Flament S., Wong E., Gumbiner B.M., Shapiro L. (2002). C-cadherin ectodomain structure and implications for cell adhesion mechanisms. Science, 296: 1308–1313.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Brurber M.B., Nes I.F., Eijsink V.G.H. (1996). Comparative studies of chitinases A and B fromSerratia marcescens. Microbiology, 142: 1581–1589.

    Article  Google Scholar 

  • Collinge D.B., Kragh K.M., Mikkelsen J.D., Nielsen K.K., Rasmussen U., Vad K. (1993). Plant chitinase. Plant J., 3: 31–40.

    Article  CAS  PubMed  Google Scholar 

  • Gupta R, Saxena R.K., Chaturvedi P., Virdi J.S. (1995). Chitinase production byStreptomyces viridificans. J. Appl. Bacteriol., 78: 378–383.

    CAS  PubMed  Google Scholar 

  • Kim S.B., Park T.K. (1994). Isolation and characterization of chitin from crab shell. Korean J. Biotech. Bioeng., 9: 174–179.

    Google Scholar 

  • Konagay, Y., Tsuchiy, C., Sugit, H. (2006). Purification and characterization of chitinases fromClostridium sp. E-16 isolated from the intestinal tract of the South American sea lion (Otaria flavescens). Lett. Appl. Microbiol., 43: 187–193.

    Article  CAS  Google Scholar 

  • Kupiec R.C., Chet I. (1998). The molecular biology of chitin digestion. Curr. Opin. Biotech., 9: 270–277.

    Article  Google Scholar 

  • Laemmli U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680–685.

    Article  CAS  PubMed  Google Scholar 

  • Lee H.S., Han D.S., Choi S.J., Choi S.W., Kim D.S., Bai D.H., Yu J.H. (2000). Purification, characterization and primary structure of a chitinase fromPseudomonas sp. YHS-A2. Appl. Microbiol. Biotech., 54: 397–405.

    Article  CAS  Google Scholar 

  • Lin C., Chen H., Lin F. (1997). Expression and characterization of the recombinant gene encoding chitinase fromAeromonas caviae. Enzyme Microbiol. Technol., 21: 472–478.

    Article  CAS  Google Scholar 

  • Mellado E., Moore E.R., Nieto J.J., Ventosa A. (1996). Analysis of 16S rRNA gene sequences ofVibrio costicola strains: description ofSalinivibrio costicola gen. nov., comb. nov. Int. J. Syst. Bacteriol., 46: 817–821.

    CAS  PubMed  Google Scholar 

  • Monreal J., Reese E.T. (1969). The chitinase ofSerratia marcescens. Can. J. Microbiol., 15: 689–696.

    Article  CAS  PubMed  Google Scholar 

  • Morimoto K., Kanta S., Kimura T., Sakku K., Ohbiya K. (1997). Cloning, sequencing and expression of the gene encodingClostridium paraputrificum chitinase B and analysis of the functions of novel cadherin-like domains and a chitin binding domain. J. Bacteriol., 179: 7306–7314.

    CAS  PubMed  Google Scholar 

  • Ohishi K., Yamagishi M., Ohta T., Suzuki M., Izumida H., Sano H., Nishijima M., Miwa T. (1996). Purification and properties of two chitinases fromVibrio alginolyticus H-8. J. Ferment. Bioeng., 82: 598–600.

    Article  CAS  Google Scholar 

  • Ohtakara A., Mitsutomi M., Uchida Y. (1979). Purification and some properties of chitinase fromVibrio sp. J. Ferment. Technol., 57: 169–173.

    CAS  Google Scholar 

  • Okazaki K., Tagawa K. (1991). Purification and characterization of a chitinase fromStreptomyces cinereoruber. J. Ferment. Bioeng., 71: 237–238.

    Article  CAS  Google Scholar 

  • Okazaki K., Kato F., Watanabe N., Yasuda S., Masui Y., Hayakawa S. (1995). Purification and properties of two chitinases fromStreptomyces sp. J-13-3. Biosci. Biotechnol. Biochem., 59: 1586–1597.

    Article  CAS  PubMed  Google Scholar 

  • Patil R.S., Chormade V., Desphande M.V. (2000). Chitinolytic enzymes: an exploration. Enzyme Microbiol. Technol., 26: 473–483.

    Article  CAS  Google Scholar 

  • Reissig J.L., Strominer J.L., Leloi L.F. (1955). A modified colorimetric method for the estimation of N-acetylamino sugars. J. Biol. Chem., 27: 959–966.

    Google Scholar 

  • Suginta W., Robertson P.A.W., Austin B., Fry S.C., Fothergill-Gilmore L.A. (2000). Chitinases fromVibrio: activity screening and purification of ChiA fromVibrio carchariae. J. Appl. Microbiol., 89: 76–84.

    Article  CAS  PubMed  Google Scholar 

  • Takayanagi T., Ajisaka K., Takiguchi Y., Shimahara K. (1991). Isolation and characterization of thermostable chitinases fromBacillus licheniformis X-7u. Biochem. Biophys. Acta, 1078: 404–410.

    CAS  PubMed  Google Scholar 

  • Thamthiankul S., Moar W.J., Miller M.E., Panbangred W. (2004). Improving the insecticidal activity ofBacillus thuringiensis subsp.aizawai againstSpodopter exigua by chromosomal expression of a chitinase gene. Appl. Microbiol. Biotechnol., 65: 183–192.

    Article  CAS  PubMed  Google Scholar 

  • Tsujibo H., Yoshida Y., Miyamoto K. (1992). Purification, properties and partial amino acid sequence of chitinase from a marineAlteromonas sp. strain O-7. Can. J. Microbiol., 38: 891–897.

    Article  CAS  PubMed  Google Scholar 

  • Tsujibo H., Orikoshi H., Baba N., Miyahara M., Miyamoto K., Yasuda M., Inamori Y. (2002). Identification and characterization of the gene cluster involved in chitin degradation in a marine bacterium,Alteromonas sp. strain O-7. Appl. Environ. Microbiol., 68: 263–270.

    Article  CAS  PubMed  Google Scholar 

  • Ueda M., Fujiwara A., Kawaguchi T., Arai M. (1995). Purification and some properties of six chitinases fromAeromonas sp. No. 10S-24. Biosci. Biotech. Biochem., 59: 2162–2164.

    Article  CAS  Google Scholar 

  • Wang S., Chang W. (1997). Purification and characterization of two bifunctional chitinases/lysozymes extracellularly produced byPseudomonas aeruginosa K-187 in a shrimp and crab shell powder medium. Appl. Environ. Microbiol., 63: 380–386.

    CAS  PubMed  Google Scholar 

  • Watanabe T., Ito Y., Yamada T., Hashimoto M., Sekine S., Tanaka H. (1994). The role of the C-terminal domain and type III domains of chitinase A1 fromBacillus circulans WL-12 in chitin degradation. J. Bacteriol., 176: 4465–4472.

    CAS  PubMed  Google Scholar 

  • Yu C., Lee A.M., Bassler B.L., Roseman S. (1991). Chitin utilization by marine bacteria. A physiological function for bacterial adhesion to immobilized carbohydrate. J. Biol. Chem., 266: 24260–24267.

    CAS  PubMed  Google Scholar 

  • Zhu B.C.R., Lo J., Li Y., Li S., Jaynes J.M., Gildemeister O.S., Laine R.A., Ou C. (1992). Thermostable, salt tolerant, wide pH range novel chitobiase fromVibrio parahemolyticus: Isolation, characterization, molecular cloning and expression. J. Biochem., 112: 163–167.

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Graduate Program in Biomedical Sciences, Faculty of Allied Health Sciences, Thammasat University, Rangsit Campus, 12121, Pathumthani, Thailand

    Ratchaneewan Aunpad

  2. Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, The University of Sheffield, S10 2TN, Sheffield, England

    David W. Rice & Svetalana Sedelnikova

  3. Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Road, 10400, Bangkok, Thailand

    Watanalai Panbangred

Authors
  1. Ratchaneewan Aunpad
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David W. Rice
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Svetalana Sedelnikova
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Watanalai Panbangred
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Ratchaneewan Aunpad or Watanalai Panbangred.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Aunpad, R., Rice, D.W., Sedelnikova, S. et al. Biochemical characterisation of two forms of halo- and thermo-tolerant chitinase C ofSalinivibrio costicola expressed inEscherichia coli . Ann. Microbiol. 57, 249–257 (2007). https://doi.org/10.1007/BF03175215

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF03175215

Key words

Annals of Microbiology

ISSN: 1869-2044

Contact us