Advertisement

Extremophiles

, 11:115 | Cite as

A highly thermostable trehalase from the thermophilic bacterium Rhodothermus marinus

  • Carla D. Jorge
  • Maria Manuel Sampaio
  • Gudmundur Ó. Hreggvidsson
  • Jakob K. Kristjánson
  • Helena SantosEmail author
Original Paper

Abstract

Trehalases play a central role in the metabolism of trehalose and can be found in a wide variety of organisms. A periplasmic trehalase (α,α-trehalose glucohydrolase, EC 3.2.1.28) from the thermophilic bacterium Rhodothermus marinus was purified and the respective encoding gene was identified, cloned and overexpressed in Escherichia coli. The recombinant trehalase is a monomeric protein with a molecular mass of 59 kDa. Maximum activity was observed at 88°C and pH 6.5. The recombinant trehalase exhibited a K m of 0.16 mM and a V max of 81 μmol of trehalose (min)−1 (mg of protein)−1 at the optimal temperature for growth of R. marinus (65°C) and pH 6.5. The enzyme was highly specific for trehalose and was inhibited by glucose with a K i of 7 mM. This is the most thermostable trehalase ever characterized. Moreover, this is the first report on the identification and characterization of a trehalase from a thermophilic bacterium.

Keywords

Rhodothermus marinus Periplasmic trehalase Glycosidase Thermostability 

Notes

Acknowledgments

This work was funded by the European Commission Contracts QLK3-CT-2000-00640 and COOP-CT-2003-508644 and Fundação para a Ciência e a Tecnologia and FEDER, Portugal, POCI/59310/2004. We thank Winfried Boos, Konstanz, for a fruitful collaboration that led to the discovery of trehalase activity in R. marinus. M. Regalla from Analytical Services performed the N-terminal sequencing at the Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal. C. Jorge acknowledges a PhD grant from PRAXIS XXI (SFRH/BD/10572/2002).

References

  1. de Aquino AC, Peixoto-Nogueira SC, Jorge JA, Terenzi HF, Polizeli ML (2005) Characterization of an acid trehalase produced by the thermotolerant fungus Rhizopus microsporus var. rhizopodiformis: Biochemical properties and immunochemical localisation. FEMS Microbiol Lett 251:169–175PubMedCrossRefGoogle Scholar
  2. Berthelot K, Delmotte FM (1999) Purification and characterization of an α-glucosidase from Rhizobium sp. (Robinia pseudoacacia L.) strain USDA 4280. Appl Environ Microbiol 65:2907–2911PubMedGoogle Scholar
  3. Bjornsdottir SH, Blondal T, Hreggvidsson GO, Eggertsson G, Petursdottir S, Hjorleifsdottir S, Thorbjarnardottir SH, Kristjansson JK (2006) Rhodothermus marinus: physiology and molecular biology. Extremophiles 10:1–16PubMedCrossRefGoogle Scholar
  4. Blücher A, Karlsson EN, Holst O (2000) Substrate-dependent production and some properties of a thermostable α-galactosidase from Rhodothermus marinus. Biotechnol Lett 22:663–669CrossRefGoogle Scholar
  5. Boos W, Ehmann U, Bremer E, Middendorf A, Postma P (1987) Trehalase of Escherichia coli. Mapping and cloning of its structural gene and identification of the enzyme as a periplasmic protein induced under high osmolarity growth conditions. J Biol Chem 262:13212–13218PubMedGoogle Scholar
  6. Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  7. Dahlberg L, Holst O, Kristjánsson JK (1993) Thermostable xylanolytic enzymes from Rhodothermus marinus grown on xylan. Appl Microbiol Biotechnol 40:63–68CrossRefGoogle Scholar
  8. Edman P, Begg G (1967) A protein sequenator. Eur J Biochem 1:80–91PubMedCrossRefGoogle Scholar
  9. Elbein AD (1974) The metabolism of α,α-trehalose. Adv Carbohydr Chem Biochem 30:227–256PubMedCrossRefGoogle Scholar
  10. Elbein AD, Pan YT, Pastuszak I, Carroll D (2003) New insights on trehalose: a multifunctional molecule. Glycobiology 13:17–27CrossRefGoogle Scholar
  11. Gomes J, Steiner W (1998) Production of a high activity of an extremely thermostable β-mannanase by the thermophilic eubacterium Rhodothermus marinus, grown on locust beam gum. Biotechnol Lett 20:729–733CrossRefGoogle Scholar
  12. Gomes J, Gomes I, Terler K, Gubala N, Ditzelmüller G, Steiner W (2000) Optimization of culture medium and conditions for α-L-arabinofuranosidase production by the extreme thermophilic eubacterium Rhodothermus marinus. Enzyme Microb Technol 27:414–422PubMedCrossRefGoogle Scholar
  13. Gomes I, Gomes J, Steiner W (2003) Highly thermostable amylase and pullulanase of the extreme thermophilic eubacterium Rhodothermus marinus: production and partial characterization. Bioresource Technol 90:207–214CrossRefGoogle Scholar
  14. Henrissat B, Bairoch A (1993) New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 293:781–788PubMedGoogle Scholar
  15. Herzog RM, Galinski EA, Trüber HG (1990) Degradation of the compatible solute trehalose in Ectothiorhodospira halochloris: isolation and characterization of trehalase. Arch Microbiol 153:600–606CrossRefGoogle Scholar
  16. Hiller K, Grote A, Scheer M, Münch R, Jahn D (2004) PrediSi: prediction of signal peptides and their cleavage positions. Nucleic Acids Res 32:375–379CrossRefGoogle Scholar
  17. Horlacher R, Uhland K, Klein W, Ehrmann M, Boos W (1996) Characterization of a cytoplasmic trehalase of Escherichia coli. J Bacteriol 178:6250–6257PubMedGoogle Scholar
  18. Hreggvidsson GO, Kaiste E, Holst O, Eggertsson G, Palsdottir A, Kristjánsson JK (1996) An extremely thermostable cellulase from the thermophilic eubacterium Rhodothermus marinus. Appl Environ Microbiol 62:3047–3049Google Scholar
  19. Im H, Henson CA (1995) Characterization of a high pI α-glucosidase from germinated barley seeds: substrate specificity subsite affinities and active-site residues. Carbohydr Res 277:145–159CrossRefGoogle Scholar
  20. Inagaki K, Ueno N, Tamura T, Tanaka H (2001) Purification and characterization of an acid trehalase from Acidobacterium capsulatum. J Biosci Bioeng 91:141–146PubMedCrossRefGoogle Scholar
  21. Jahagirdar AP, Seligy VL (1992) A transfer membrane method for in situ detection and quantification of trehalase. Anal Biochem 202:96–99PubMedCrossRefGoogle Scholar
  22. Jorge JA, Polizeli ML, Thevelein JM, Terenzi HF (1997) Trehalases and trehalose hydrolysis in fungi. FEMS Microbiol Lett 154:165–171PubMedCrossRefGoogle Scholar
  23. Kadowaki MK, Polizeli ML, Terenzi HF, Jorge JA (1996) Characterization of trehalase activities from the thermophilic fungus Scytalidium thermophilum. Biochim Biophys Acta 1291:199–205PubMedGoogle Scholar
  24. Kalf GF, Rieder SV (1958) The purification and properties of trehalase. J Biol Chem 230:691–698PubMedGoogle Scholar
  25. Lopez MF, Torrey JG (1985) Purification and properties of trehalase in Frankia ArI3. Arch Microbiol 143:209–215CrossRefGoogle Scholar
  26. Manelius A, Dahlberg L, Holst O (1994) Some properties of a thermostable β-xylosidase from Rhodothermus marinus. Appl Biochem Biotechnol 44:39–48CrossRefGoogle Scholar
  27. Manjunath P, Shenoy BC, Raghavendra Roa MR (1983) Fungal glucoamylases. J Appl Biochem 5:235–260PubMedGoogle Scholar
  28. Mansure JJ, Silva JT, Panek AD (1992) Characterization of trehalase in Rhodotorula rubra. Biochem Int 28:693–700PubMedGoogle Scholar
  29. Marmur LJ (1961) A procedure for the isolation of deoxy-ribonucleic acid from microorganisms. J Mol Biol 3:208–218CrossRefGoogle Scholar
  30. Murakami S, Yagami M, Suzuki Y (1998) Purification and some properties of an extremely thermostable trehalose-hydrolyzing α-glucosidase from Bacillus flavocaldarius KP1228. Starch 50:100–103CrossRefGoogle Scholar
  31. Nakao M, Nakayama T, Harada M, Kakudo A, Ikemoto H, Kobayashi S, Shibano Y (1994) Purification and characterization of a Bacillus sp. SAM1606 thermostable α-glucosidase with transglucosylation activity. Appl Microbiol Biotechnol 41:337–343PubMedCrossRefGoogle Scholar
  32. Nunes OC, Donato MM, da Costa MS (1992) Isolation and characterization of Rhodothermus strains from S. Miguel, Azores. System Appl Microbiol 15:92–97Google Scholar
  33. Parrou JL, Jules M, Beltran G, François J (2005) Acid trehalase in yeast and filamentous fungi: localization, regulation and physiological function. FEMS Yeast Res 5:503–511PubMedCrossRefGoogle Scholar
  34. Prasad ARS, Maheshwari R (1978) Purification and properties of trehalase from the thermophilic fungus Humicola lanuginosa. Biochim Biophys Acta 525:162–170PubMedGoogle Scholar
  35. Robinson-Rechavi M, Alibés A, Godzik A (2006) Contribution of electrostatic interactions, compactness and quaternary structure to protein thermostability: lessons from structural genomics of Thermotoga maritima. J Mol Biol 356:547–557PubMedCrossRefGoogle Scholar
  36. Saha BC, Bothast RJ (1993) Production and characteristics of an intracellular α-glucosidase from a color variant strain of Aureobasidium pullulans. Curr Microbiol 27:73–77CrossRefGoogle Scholar
  37. Silva Z, Borges N, Martins LO, Wait R, da Costa MS, Santos H (1999) Combined effect of the growth temperature and salinity of the medium on the accumulation of compatible solutes by Rhodothermus marinus and Rhodothermus obamensis. Extremophiles 3:163–172PubMedCrossRefGoogle Scholar
  38. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24:4876–4882CrossRefGoogle Scholar
  39. Van Assche JA, Carlier AR (1975) Some properties of trehalase from Phycomyces blakesleeanus. Biochim Biophys Acta 391:154–161PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Carla D. Jorge
    • 1
  • Maria Manuel Sampaio
    • 1
  • Gudmundur Ó. Hreggvidsson
    • 2
    • 3
  • Jakob K. Kristjánson
    • 2
  • Helena Santos
    • 1
    Email author
  1. 1.Instituto de Tecnologia Química e BiológicaUniversidade Nova de LisboaOeirasPortugal
  2. 2.Prokaria Ltd112 ReykjavikIceland
  3. 3.University of Iceland101 ReykjavikIceland

Personalised recommendations