, Volume 9, Issue 1, pp 53–64 | Cite as

Cloning, expression, and characterization of a highly thermostable family 18 chitinase from Rhodothermus marinus

  • Cédric F. V. Hobel
  • Gudmundur Ó. Hreggvidsson
  • Viggó T. Marteinsson
  • Farah Bahrani-Mougeot
  • Jón M. Einarsson
  • Jakob K. Kristjánsson
Original Paper


A family 18 chitinase gene chiA from the thermophile Rhodothermus marinus was cloned and expressed in Escherichia coli. The gene consisted of an open reading frame of 1,131 nucleotides encoding a protein of 377 amino acids with a calculated molecular weight of 42,341 Da. The deduced ChiA was a non-modular enzyme with one unique glycoside hydrolase family 18 catalytic domain. The catalytic domain exhibited 43% amino acid identity with Bacillus circulans chitinase C. Due to poor expression of ChiA, a signal peptide-lacking mutant, chiAΔsp, was designed and used subsequently. The optimal temperature and pH for chitinase activity of both ChiA and ChiAΔsp were 70°C and 4.5–5, respectively. The enzyme maintained 100% activity after 16 h incubation at 70°C, with half-lives of 3 h at 90°C and 45 min at 95°C. Results of activity measurements with chromogenic substrates, thin-layer chromatography, and viscosity measurements demonstrated that the chitinase is an endoacting enzyme releasing chitobiose as a major end product, although it acted as an exochitobiohydrolase with chitin oligomers shorter than five residues. The enzyme was fully inhibited by 5 mM HgCl2, but excess ethylenediamine tetraacetic acid relieved completely the inhibition. The enzyme hydrolyzed 73% deacetylated chitosan, offering an attractive alternative for enzymatic production of chitooligosaccharides at high temperature and low pH. Our results show that the R. marinus chitinase is the most thermostable family 18 chitinase isolated from Bacteria so far.


Cloning Expression Family 18 chitinase Highly thermostable Rhodothermus marinus Thin layer chromatography Viscosity measurements 



We would like to thank Anna Gudny Hermansdottir and Jon Oskar Jonsson for their help designing and optimizing the protein purification steps. We gratefully acknowledge Bernard Henrissat, Daan Van Aalten, Wolfgang Schwarz, and particularly Hakon Birgisson for very valuable discussions. This work was supported by a grant from the Icelandic Center for Research, contract number 030840003.


  1. van Aalten DMF, Komander D, Synstad B, Gaseidnes S, Peter MG, Eijsink VGH (2001) Structural insights into the catalytic mechanism of a family 18 exo-chitinase. Proc Natl Acad Sci USA 98:8979–8984CrossRefPubMedGoogle Scholar
  2. Alfredsson GA, Kristjansson JK, Hjorleifsdottir S, Stetter KO (1988) Rhodothermus marinus, gen. nov., sp. nov., a thermophilic, halophilic bacterium from submarine hot springs in Iceland. J Gen Microbiol 134:299–306Google Scholar
  3. Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedGoogle Scholar
  4. Andronopoulou E, Vorgias CE (2003) Purification and characterization of a new hyperthermostable, allosamidin-insensitive and denaturation-resistant chitinase from the hyperthermophilic archaeon Thermococcus chitonophagus. Extremophiles 7:43–53PubMedGoogle Scholar
  5. Beynon RJ, Easterby JS (1996) Buffer solutions: the basics. BIOS Scientific, OxfordGoogle Scholar
  6. Bhushan B (2000) Production and characterization of a thermostable chitinase from a new alkalophilic Bacillus sp. BG-11. J Appl Microbiol 88:800–808CrossRefPubMedGoogle Scholar
  7. Bhushan B, Hoondal GS (1998) Isolation, purification and properties of a thermostable chitinase from an alkalophilic Bacillus sp BG-11. Biotechnol Lett 20:157–159Google Scholar
  8. Boer W de, Gunnewiek P, Kowalchuk GA, Van Veen JA (2001) Growth of chitinolytic dune soil beta-subclass Proteobacteria in response to invading fungal hyphae. Appl Environ Microbiol 67:3358–3362Google Scholar
  9. Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  10. Christodoulou E, Duffner F, Vorgias CE (2001) Overexpression, purification, and characterization of a thermostable chitinase (Chi40) from Streptomyces thermoviolaceus OPC-520. Protein Expr Purif 23:97–105CrossRefPubMedGoogle Scholar
  11. Cohen-Kupiec R, Chet I (1998) The molecular biology of chitin digestion. Curr Opin Biotechnol 9:270–277CrossRefPubMedGoogle Scholar
  12. Cottrell MT, Moore JA, Kirchman DL (1999) Chitinases from uncultured marine microorganisms. Appl Environ Microbiol 65:2553–2557Google Scholar
  13. Ewing B, Green P (1998) Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res 8:186–194PubMedGoogle Scholar
  14. Felse PA, Panda T (1999) Regulation and cloning of microbial chitinase genes. Appl Microbiol Biotechnol 51:141–151CrossRefPubMedGoogle Scholar
  15. Gao J, Bauer MW, Shockley KR, Pysz MA, Kelly RM (2003) Growth of hyperthermophilic archaeon Pyrococcus furiosus on chitin involves two family 18 chitinases. Appl Environ Microbiol 69:3119–3128Google Scholar
  16. Gomes J, Gomes I, Terler K, Gubala N, Ditzelmuller G, Steiner W (2000) Optimisation of culture medium and conditions for alpha-L-arabinofuranosidase production by the extreme thermophilic eubacterium Rhodothermus marinus. Enzyme Microb Technol 27:414–422Google Scholar
  17. Haki GD, Rakshit SK (2003) Developments in industrially important thermostable enzymes: a review. Bioresour Technol 89:17–34CrossRefPubMedGoogle Scholar
  18. Halldorsdottir S, Thorolfsdottir ET, Spilliaert R, Johansson M, Thorbjarnardottir SH, Palsdottir A, Hreggvidsson GO, Kristjansson JK, Holst O, Eggertsson G (1998) Cloning, sequencing and overexpression of a Rhodothermus marinus gene encoding a thermostable cellulase of glycosyl hydrolase family 12. Appl Microbiol Biotechnol 49:277–284CrossRefPubMedGoogle Scholar
  19. Henrissat B (1999) Classification of chitinase modules. In: Muzzarelli RAA (ed) Chitin and chitinases. Birkhäuser, Basel, pp 137–159Google Scholar
  20. Howard MB, Ekborg NA, Taylor LE, Weiner RM, Hutcheson SW (2003) Genomic analysis and initial characterization of the chitinolytic system of Microbulbifer degradans strain 2–40. J Bacteriol 185:3352–3360CrossRefPubMedGoogle Scholar
  21. Hsu SC, Lockwood JL (1975) Powdered chitin agar as a selective medium for enumeration of Actinomycetes in water and soil. Appl Microbiol 29:422–426PubMedGoogle Scholar
  22. Huber R, Stohr J, Hohenhaus S, Rachel R, Burggraf S, Jannasch HW, Stetter KO (1995) Thermococcus chitonophagus sp. nov., a novel, chitin-degrading, hyperthermophilic archaeum from deep-sea hydrothermal vent environment. Arch Microbiol 164:255–264CrossRefGoogle Scholar
  23. Krah M, Misselwitz R, Politz O, Thomsen KK, Welfe H, Borriss R (1998) The laminarinase from thermophilic eubacterium Rhodothermus marinus—conformation, stability, and identification of active site carboxylic residues by site-directed mutagenesis. Eur J Biochem 257:101–111CrossRefPubMedGoogle Scholar
  24. Laemmli UK (1970) Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature 227:680PubMedGoogle Scholar
  25. Mabuchi N, Araki Y (2001) Cloning and sequencing of two genes encoding chitinases A and B from Bacillus cereus CH. Can J Microbiol 47:895–902CrossRefPubMedGoogle Scholar
  26. Nawani NN, Kapadnis BP, Das AD, Rao AS, Mahajan SK (2002) Purification and characterization of a thermophilic and acidophilic chitinase from Microbispora sp V2. J Appl Microbiol 93:965–975CrossRefPubMedGoogle Scholar
  27. Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–6CrossRefGoogle Scholar
  28. Nordberg-Karlsson E, Bartonek-Roxa E, Holst O (1997) Cloning and sequence of a thermostable multidomain xylanase from the bacterium Rhodothermus marinus. BBA Gene Structure Expr 1353:118–124Google Scholar
  29. Patil RS, Ghormade V, Deshpande MV (2000) Chitinolytic enzymes: an exploration. Enzyme Microb Technol 26:473–483CrossRefPubMedGoogle Scholar
  30. Politz O, Krah M, Thomsen KK, Borriss R (2000) A highly thermostable endo-(1,4)-beta-mannanase from the marine bacterium Rhodothermus marinus. Appl Microbiol Biotechnol 53:715–721CrossRefPubMedGoogle Scholar
  31. Price NC, Stevens L (1989) Fundamentals of enzymology, 2nd edn. Oxford University Press, OxfordGoogle Scholar
  32. Ramaiah N, Hill RT, Chun J, Ravel J, Matte MH, Straube WL, Colwell RR (2000) Use of a chiA probe for detection of chitinase genes in bacteria from the Chesapeake Bay. FEMS Microbiol Ecol 34:63–71CrossRefPubMedGoogle Scholar
  33. Ramchuran SO, Nordberg Karlsson E, Velut S, De Mare L, Hagander P, Holst O (2002) Production of heterologous thermostable glycoside hydrolases and the presence of host-cell proteases in substrate limited fed-batch cultures of Escherichia coli BL21(DE3). Appl Microbiol Biotechnol 60:408–416CrossRefPubMedGoogle Scholar
  34. Rice P, Longden I, Bleasby A (2000) EMBOSS: the European molecular biology open software suite. Trends Genet 16:276–277CrossRefPubMedGoogle Scholar
  35. Sakai K, Yokota A, Kurokawa H, Wakayama M, Moriguchi M (1998) Purification and characterization of three thermostable endochitinases of a noble Bacillus strain, MH-1, isolated from chitin-containing compost. Appl Environ Microbiol 64:3397–3402Google Scholar
  36. Sambrook K, Fritsch EF, Maniatis T (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  37. Souza RF, Gomes RC, Coelho RRR, Alviano CS, Soares RMA (2003) Purification and characterization of an endochitinase produced by Colletotrichum gloeosporioides. FEMS Microbiol Lett 222:45–50CrossRefPubMedGoogle Scholar
  38. Spilliaert R, Hreggvidsson GO, Kristjansson JK, Eggertsson G, Palsdottir A (1994) Cloning and sequencing of a Rhodothermus marinus gene, bglA, coding for a thermostable beta-glucanase and its expression in Escherichia coli. Eur J Biochem 224:923–930PubMedGoogle Scholar
  39. Sutrisno A, Ueda M, Abe Y, Nakazawa M, Miyatake K (2004) A chitinase with high activity toward partially N-acetylated chitosan from a new, moderately thermophilic, chitin-degrading bacterium Ralstonia sp. A-471. Appl Microbiol Biotechnol 63:398–406CrossRefPubMedGoogle Scholar
  40. Takayanagi T, Ajisaka K, Takiguchi Y, Shimahara K (1991) Isolation and characterization of thermostable chitinases from Bacillus licheniformis X-7u. Biochim Biophys Acta 1078:404–410CrossRefPubMedGoogle Scholar
  41. Tanaka T, Fujiwara S, Nishikori S, Fukui T, Takagi M, Imanaka T (1999) A unique chitinase with dual active sites and triple substrate binding sites from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1. Appl Environ Microbiol 65:5338–5344PubMedGoogle Scholar
  42. Tanaka T, Fukui T, Imanaka T (2001) Different cleavage specificities of the dual catalytic domains in chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. J Biol Chem 276:35629–35635CrossRefPubMedGoogle Scholar
  43. Tanaka T, Fukui T, Atomi H, Imanaka T (2003) Characterization of an exo-beta-D-glucosaminidase involved in a novel chitinolytic pathway from the hyperthermophilic archaeon Thermococcus kodakamensis KOD1. J Bacteriol 185:5175–5181CrossRefPubMedGoogle Scholar
  44. Thorbjarnardottir SH, Jonsson ZO, Andresson OS, Kristjansson JK, Eggertsson G, Palsdottir A (1995) Cloning and sequence analysis of the DNA ligase-encoding gene of Rhodothermus marinus, and overproduction, purification and characterization of 2 thermophilic DNA ligases. Gene 161:1–6CrossRefPubMedGoogle Scholar
  45. Tsujibo H, Minoura K, Miyamoto K, Endo H, Moriwaki M, Inamori Y (1993) Purification and properties of a thermostable chitinase from Streptomyces thermoviolaceus OPC-520. Appl Environ Microbiol 59:620–622PubMedGoogle Scholar
  46. Wang SL, Chang WT (1997) Purification and characterization of two bifunctional chitinases/lysozymes extracellularly produced by Pseudomonas aeruginosa K-187 in a shrimp and crab shell powder medium. Appl Environ Microbiol 63:380–386PubMedGoogle Scholar
  47. Wang SL, Hwang JR (2001) Microbial reclamation of shellfish wastes for the production of chitinases. Enzyme Microb Technol 28:376–382CrossRefPubMedGoogle Scholar
  48. Watanabe T, Kobori K, Miyashita K, Fujii T, Sakai H, Uchida M, Tanaka H (1993) Identification of glutamic acid-204 and aspartic acid-200 in chitinase A1 of Bacillus circulans Wl-12 as essential residues for chitinase Activity. J Biol Chem 268:18567–18572PubMedGoogle Scholar
  49. Williamson N, Brian P, Wellington EMH (2000) Molecular detection of bacterial and streptomycete chitinases in the environment. Antonie Leeuwenhoek 78:315–321CrossRefPubMedGoogle Scholar
  50. Wilms B, Hauck A, Reuss M, Syldatk C, Mattes R, Siemann M, Altenbuchner J (2001) High-cell-density fermentation for production of L-N-carbamoylase using an expression system based on the Escherichia coli rhaBAD promoter. Biotechnol Bioeng 73:95–103CrossRefPubMedGoogle Scholar
  51. Wong C, Sridhara S, Bardwell JCA, Jakob U (2000) Heating greatly speeds coomassie blue staining and destaining. Biotechniques 28:426PubMedGoogle Scholar
  52. Xia GQ, Jin CS, Zhou J, Yang SJ, Zhang SZ, Jin C (2001) A novel chitinase having a unique mode of action from Aspergillus fumigatus YJ-407. Eur J Biochem 268:4079–4085CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Cédric F. V. Hobel
    • 1
  • Gudmundur Ó. Hreggvidsson
    • 1
    • 2
  • Viggó T. Marteinsson
    • 1
  • Farah Bahrani-Mougeot
    • 1
    • 3
  • Jón M. Einarsson
    • 4
  • Jakob K. Kristjánsson
    • 1
  1. 1.Prokaria Ltd.ReykjavikIceland
  2. 2.Institute of BiologyUniversity of IcelandReykjavikIceland
  3. 3.Department of Oral MedicineCarolinas Medical CenterCharlotteUSA
  4. 4.Primex R and D DivisionReykjavikIceland

Personalised recommendations