Skip to main content
SpringerLink
Log in

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

  • Original Paper
  • Published:
Extremophiles Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • 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 98:8979–8984

    Article  PubMed  Google Scholar 

  • 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 134:299–306

    Google Scholar 

  • 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 25:3389–3402

    CAS  PubMed  Google Scholar 

  • 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 7:43–53

    CAS  PubMed  Google Scholar 

  • Beynon RJ, Easterby JS (1996) Buffer solutions: the basics. BIOS Scientific, Oxford

    Google Scholar 

  • Bhushan B (2000) Production and characterization of a thermostable chitinase from a new alkalophilic Bacillus sp. BG-11. J Appl Microbiol 88:800–808

    Article  CAS  PubMed  Google Scholar 

  • Bhushan B, Hoondal GS (1998) Isolation, purification and properties of a thermostable chitinase from an alkalophilic Bacillus sp BG-11. Biotechnol Lett 20:157–159

    Google Scholar 

  • 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 67:3358–3362

    Google Scholar 

  • 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 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Christodoulou E, Duffner F, Vorgias CE (2001) Overexpression, purification, and characterization of a thermostable chitinase (Chi40) from Streptomyces thermoviolaceus OPC-520. Protein Expr Purif 23:97–105

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Cottrell MT, Moore JA, Kirchman DL (1999) Chitinases from uncultured marine microorganisms. Appl Environ Microbiol 65:2553–2557

    Google Scholar 

  • Ewing B, Green P (1998) Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res 8:186–194

    CAS  PubMed  Google Scholar 

  • Felse PA, Panda T (1999) Regulation and cloning of microbial chitinase genes. Appl Microbiol Biotechnol 51:141–151

    Article  CAS  PubMed  Google Scholar 

  • 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 69:3119–3128

    Google Scholar 

  • 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 27:414–422

    Google Scholar 

  • Haki GD, Rakshit SK (2003) Developments in industrially important thermostable enzymes: a review. Bioresour Technol 89:17–34

    Article  CAS  PubMed  Google Scholar 

  • 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 49:277–284

    Article  PubMed  Google Scholar 

  • Henrissat B (1999) Classification of chitinase modules. In: Muzzarelli RAA (ed) Chitin and chitinases. Birkhäuser, Basel, pp 137–159

    Google Scholar 

  • 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 185:3352–3360

    Article  CAS  PubMed  Google Scholar 

  • Hsu SC, Lockwood JL (1975) Powdered chitin agar as a selective medium for enumeration of Actinomycetes in water and soil. Appl Microbiol 29:422–426

    CAS  PubMed  Google Scholar 

  • 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 164:255–264

    Article  CAS  Google Scholar 

  • 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 257:101–111

    Article  CAS  PubMed  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature 227:680

    PubMed  Google Scholar 

  • Mabuchi N, Araki Y (2001) Cloning and sequencing of two genes encoding chitinases A and B from Bacillus cereus CH. Can J Microbiol 47:895–902

    Article  CAS  PubMed  Google Scholar 

  • 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 93:965–975

    Article  CAS  PubMed  Google Scholar 

  • 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 10:1–6

    Article  CAS  Google Scholar 

  • 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 1353:118–124

    CAS  Google Scholar 

  • Patil RS, Ghormade V, Deshpande MV (2000) Chitinolytic enzymes: an exploration. Enzyme Microb Technol 26:473–483

    Article  CAS  PubMed  Google Scholar 

  • 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 53:715–721

    Article  CAS  PubMed  Google Scholar 

  • Price NC, Stevens L (1989) Fundamentals of enzymology, 2nd edn. Oxford University Press, Oxford

    Google Scholar 

  • 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 34:63–71

    Article  CAS  PubMed  Google Scholar 

  • 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 60:408–416

    Article  CAS  PubMed  Google Scholar 

  • Rice P, Longden I, Bleasby A (2000) EMBOSS: the European molecular biology open software suite. Trends Genet 16:276–277

    Article  CAS  PubMed  Google Scholar 

  • 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 64:3397–3402

    Google Scholar 

  • Sambrook K, Fritsch EF, Maniatis T (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • Souza RF, Gomes RC, Coelho RRR, Alviano CS, Soares RMA (2003) Purification and characterization of an endochitinase produced by Colletotrichum gloeosporioides. FEMS Microbiol Lett 222:45–50

    Article  CAS  PubMed  Google Scholar 

  • 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 224:923–930

    CAS  PubMed  Google Scholar 

  • 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 63:398–406

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • 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 65:5338–5344

    CAS  PubMed  Google Scholar 

  • 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 276:35629–35635

    Article  CAS  PubMed  Google Scholar 

  • 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 185:5175–5181

    Article  CAS  PubMed  Google Scholar 

  • 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 161:1–6

    Article  PubMed  Google Scholar 

  • 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 59:620–622

    CAS  PubMed  Google Scholar 

  • 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 63:380–386

    CAS  PubMed  Google Scholar 

  • Wang SL, Hwang JR (2001) Microbial reclamation of shellfish wastes for the production of chitinases. Enzyme Microb Technol 28:376–382

    Article  CAS  PubMed  Google Scholar 

  • 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 268:18567–18572

    CAS  PubMed  Google Scholar 

  • Williamson N, Brian P, Wellington EMH (2000) Molecular detection of bacterial and streptomycete chitinases in the environment. Antonie Leeuwenhoek 78:315–321

    Article  CAS  PubMed  Google Scholar 

  • 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 73:95–103

    Article  CAS  PubMed  Google Scholar 

  • Wong C, Sridhara S, Bardwell JCA, Jakob U (2000) Heating greatly speeds coomassie blue staining and destaining. Biotechniques 28:426

    CAS  PubMed  Google Scholar 

  • 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 268:4079–4085

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

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.

Author information

Author notes

  1. Farah Bahrani-Mougeot

    Present address: Department of Oral Medicine, Carolinas Medical Center, P.O Box 32861, Charlotte, NC, 28232, USA

Authors and Affiliations

  1. Prokaria Ltd., Gylfaflöt 5, 112, Reykjavik, Iceland

    Cédric F. V. Hobel, Gudmundur Ó. Hreggvidsson, Viggó T. Marteinsson, Farah Bahrani-Mougeot & Jakob K. Kristjánsson

  2. Institute of Biology, University of Iceland, Sturlugata 7, 101, Reykjavik, Iceland

    Gudmundur Ó. Hreggvidsson

  3. Primex R and D Division, Myrargata 2, 101, Reykjavik, Iceland

    Jón M. Einarsson

Authors
  1. Cédric F. V. Hobel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gudmundur Ó. Hreggvidsson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Viggó T. Marteinsson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Farah Bahrani-Mougeot
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jón M. Einarsson
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jakob K. Kristjánsson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Viggó T. Marteinsson.

Additional information

Communicated by K. Horikoshi

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hobel, C.F.V., Hreggvidsson, G.Ó., Marteinsson, V.T. et al. Cloning, expression, and characterization of a highly thermostable family 18 chitinase from Rhodothermus marinus. Extremophiles 9, 53–64 (2005). https://doi.org/10.1007/s00792-004-0422-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00792-004-0422-3

Keywords

Search

Navigation