Skip to main content
SpringerLink
Log in

Cloning and expression of a β-glycosidase gene from Thermus thermophilus. Sequence and biochemical characterization of the encoded enzyme

  • Published:
Glycoconjugate Journal Aims and scope Submit manuscript

Abstract

A 3.2 kilobase pair DNA fragment from Thermus thermophilus HB27 coding for a β-galactosidase activity was cloned and sequenced. A gene and a truncated open reading frame orf1 encoding respectively a β-glycosidase (ttβ-gly) and probably a sugar permease were located directly adjacent to each other. The deduced aminoacid sequence of the enzyme Ttβ-gly showed strong identity with those of β-glycosidases belonging to the glycosyl hydrolase family 1. The enzyme was overexpressed in Escherichia coli and was purified by a two-step purification procedure. The recombinant enzyme is monomeric with a molecular mass of 49-kDa. It catalyzes the hydrolysis of β-D-galactoside, β-D-glucoside and β-D-fucoside derivatives. However, the kcat/Km ratio is much higher for p-nitrophenyl-β-D-glucoside and p-nitrophenyl-β-D-fucoside than for p-nitrophenyl-β-D-galactoside. The specificity towards linkage positions of the disaccharides tested decreased in the following order: β1-3 (100%) < β1-2 (71%) < β1-4 (40%) < β1-6 (10%). Ttβ-gly is a thermostable enzyme displaying an optimum temperature of 88°C and a half life of 10 min at 90°C. It performs transglycosylation reactions at high temperature with a yield exceeding 63% for transfucosylation reactions. On the basis of this work, the enzyme appears to be an attractive tool in the synthesis of fucosyl adducts and fucosyl sugars.

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

Similar content being viewed by others

References

  1. Davies G, Henrissat B (1995) Structure 3: 853-59.

    Google Scholar 

  2. Cote G L, Tao BY (1990) Glycoconjugate J 7: 145-62.

    Google Scholar 

  3. Ichikawa Y, Look GC, Wong C-H (1992) Anal Biochem 202: 215-38.

    Google Scholar 

  4. Yoon J-H, Ajisaka K (1996) Carbohydr Res 292: 153-63.

    Google Scholar 

  5. Nilsson, KGI (1991) In Enzymes in Carbohydrate Synthesis (Bednarski MD, Simon E S, eds) pp 51-62, ACS Symposium Series 466, Washington, D.C.

  6. Attal S, Bay S, Cantacuzene D (1992) Tetrahedron 48: 9251-60.

    Google Scholar 

  7. Leparoux S, Padrines M, Fortun Y, Colas B (1996) Biotechnol Lett 18: 135-38.

    Google Scholar 

  8. Leparoux S, Padrines M, Placier G, Colas B (1997) Biochim Biophys Acta 1336: 522-32.

    Google Scholar 

  9. Adams M W W, Perler F B, Kelly RM (1995) Bio/technology 13: 662-68.

    Google Scholar 

  10. Bauer MW, Halio, SB, Kelly RM (1996) Adv Protein Chem 48: 271-310.

    Google Scholar 

  11. Moracci M, Nucci R, Febbraio F, Vaccaro C, Vespa N, La Cara F, Rossi M (1995) Enzyme Microb Technol 17: 992-97.

    Google Scholar 

  12. Aguilar CF, Sanderson I, Moracci M, Ciaramella M, Nucci R, Rossi M, Pearl LH (1997) J Mol Biol 271: 789-802.

    Google Scholar 

  13. Trincone A, Improta R, Nucci R, Rossi M, Gambacorta A (1994) Biocatalysis 10: 195-210.

    Google Scholar 

  14. Dion M, Loussouarn F, Batisse N, Rabiller C, Sakanyan V (1995) Biotechnol Lett 17: 905-10.

    Google Scholar 

  15. Lee SG, Lee DC, Kim HS (1997) Appl Biochem Biotechnol 62: 251-66.

    Google Scholar 

  16. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Science 239: 487-91.

    Google Scholar 

  17. Breves R, Bronnenmeier K, Wild N, Lottspeich F, Staudenbauer WL, Hofemeister J (1997) Appl Environ Microbiol 63: 3902-10.

    Google Scholar 

  18. Berger J-L, Lee BH, Lacroix C (1995) Appl Microbiol Biotechnol 44: 81-87.

    Google Scholar 

  19. Nakao M, Nakayama T, Harada M, Kakudo A, Ikemoto H, Kobayashi S, Shibano Y (1994) Appl Microbiol Biotechnol 41: 337-43.

    Google Scholar 

  20. Henrissat B, (1991) Biochem J 280: 309-16.

    Google Scholar 

  21. Cowan DA, Daniel RM, Martin AM, Morgan HW (1984) Biotechnol Bioeng 26: 1141-45.

    Google Scholar 

  22. Koyama Y, Okamoto S, Furukawa K (1990) Appl Environ Microbiol 56: 2251-54.

    Google Scholar 

  23. Tabata K, Hoshino T (1996) Microbiology 142: 401-10.

    Google Scholar 

  24. Sakaki Y, Oshima T (1975) J Virol 15: 1449-53.

    Google Scholar 

  25. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1993) Current Protocols in Molecular Biology. New York: John Wiley.

    Google Scholar 

  26. Miller JH (1992) A Short Course in Bacterial Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

    Google Scholar 

  27. Laemmli UK (1970) Nature 227: 680-85.

    Google Scholar 

  28. Kunst A, Draeger B, Ziegenhorn J (1984) In Methods of Enzymatic Analysis (Bergmeyer H U, ed) vol 6, pp 178-85. New York and London: Verlag Chemie, Weinheim and Academic Press.

    Google Scholar 

  29. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Anal Biochem 150: 76-85.

    Google Scholar 

  30. Leparoux S, Fortun Y, Colas B (1994) Biotechnol Lett 16: 677-82.

    Google Scholar 

  31. Oshima T, Imahori K (1974) Int J Syst Bacteriol 24: 102-12.

    Google Scholar 

  32. Maseda H, Hoshino T (1995) FEMS Microbiol Lett 128: 127-34.

    Google Scholar 

  33. Lieb W, Gabelsberger J, Schleifer K-H (1994) Mol Gen Genet 242: 111-15.

    Google Scholar 

  34. Love DR, Fisher R, Bergquist PL (1988) Mol Gen Genet 213: 84-92.

    Google Scholar 

  35. Wright RM, Yablonsky MD, Shalita ZP, Goyal AK, Eveleigh DE (1992) Appl Environ Microbiol 58: 3455-65.

    Google Scholar 

  36. Perez-Pons JA, Cayetano A, Rebordosa X, Lloberas J, Guasch A, Querol E (1994) Eur J Biochem 223: 557-65.

    Google Scholar 

  37. Wakarchuk WW, Greenberg NM, Kilburn DG, Miller RC Jr, Warren RAJ (1988) J Bacteriol 170: 301-07.

    Google Scholar 

  38. Cubellis MV, Rozzo C, Montecucchi P, Rossi M (1990) Gene 94: 89-94.

    Google Scholar 

  39. Voorhorst WG, Eggen RI, Luesink EJ, de Vos WM (1995) J Bacteriol 177: 7105-11.

    Google Scholar 

  40. Oxtoby E, Dunn MA, Pancoro A, Hughes MA (1991) Plant Mol Biol 17: 209-19.

    Google Scholar 

  41. Moracci M, Cabaldo L, Ciaramella M, Rossi M (1996) Protein Eng 9: 1191-95.

    Google Scholar 

  42. Withers SG, Warren RAJ, Street IP, Rupitz K, Kempton JB, Aebersold R (1990) J Am Chem Soc 112: 5887-89.

    Google Scholar 

  43. Wang Q, Trimbur D, Graham R, Warren RAJ, Withers SG (1995) Biochemistry 34: 14554-62.

    Google Scholar 

  44. Barret T, Suresh CG, Tolley SP, Dodson EJ, Hughes MA (1995) Structure 3: 853-59.

    Google Scholar 

  45. Wiesmann C, Beste G, Hengstenberg W, Schulz GE (1995) Structure 3: 961-68.

    Google Scholar 

  46. Studier FW, Moffat BA (1986) J Mol Biol 189: 113-30.

    Google Scholar 

  47. Hatch FT, Bruce AL (1968) Nature 218: 1166-68.

    Google Scholar 

  48. Colas B (1980) Biochim Biophys Acta 613: 448-58.

    Google Scholar 

  49. Mahler HR (1961) Ann NY Acad Sci 92: 426-39.

    Google Scholar 

  50. Dixon M, Webb EC (1979) Enzymes, 3rd ed., pp 72-75. London: Longman Group Limited.

    Google Scholar 

  51. Fortun Y, Colas B (1991) Biotechnol Lett 13: 863-66.

    Google Scholar 

  52. Leparoux S, Colas B (1994) Int J Biochem 26: 247-54.

    Google Scholar 

  53. Prisco A, Moracci M, Rossi M, Ciaramella M (1995) J Bacteriol 177: 1614-19.

    Google Scholar 

  54. Gräbnitz F, Seiss M, Rücknagel KP, Standenbauer WL (1991) Eur J Biochem 200: 301-09.

    Google Scholar 

  55. Gabelsberger J, Liebl W, Schleifer K-H (1993) FEMS Microbiol Lett 109: 131-38.

    Google Scholar 

  56. Pisani FM, Rella R, Raia C, Rozzo C, Nucci R, Gambacorta A, De Rosa M, Rossi M (1990) Eur J Biochem 187: 321-28.

    Google Scholar 

  57. Kengen SWM, Luesink EJ, Stams AJM, Zehnder AJB (1993) Eur J Biochem 213: 305-12.

    Google Scholar 

  58. Nucci R, Moracci M, Vaccaro C, Vespa N, Rossi M (1993) Biotechnol Appl Biochem 17: 239-50.

    Google Scholar 

  59. Leah R, Kigel J, Svendsen I, Mundy J (1995) J Biol Chem 270: 15789-97.

    Google Scholar 

  60. Sinnott ML (1990) Chem Rev 90: 1171-202.

    Google Scholar 

  61. Trincone A, Pagnotta E (1995) Biotechnol Lett 17: 45-48.

    Google Scholar 

  62. Onishi N, Tanaka T (1995) Appl Environ Microbiol 61: 4026-30.

    Google Scholar 

  63. Fischer L, Bromann R, Kengen SWM, De Vos WM, Wagner F (1996) Bio/technology 14: 88-91.

    Google Scholar 

  64. Nunoura N, Ohdan K, Yano T, Yamamoto K, Kumagai H (1996) Biosci Biotechnol Biochem 60: 188-93.

    Google Scholar 

  65. Vieille C, Zeikus JG (1996) Trends Biotechnol 14: 183-90.

    Google Scholar 

  66. Volkin DB, Middaugh CR (1992) In Stability of Protein Pharmaceuticals, Part A: Chemical and Physical Pathways of Protein Degradation (Ahern TJ, Manning MC, eds) pp 215-47, New York: Plenum Press.

    Google Scholar 

  67. Ishikawa K, Kimura S, Kanaya S, Morikawa K, Nakamura H (1993) Protein Eng 6: 85-91.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Unité de Recherche sur la Biocatalyse, Faculté des Sciences et Techniques, Université de Nantes, 2, rue de la Houssinière, B.P. 92208, F-44322, Nantes, Cedex 3, France

    Michel Dion, Laurent Fourage, Jean-Noël Hallet & Bernard Colas

Authors
  1. Michel Dion
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Laurent Fourage
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jean-Noël Hallet
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bernard Colas
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bernard Colas.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Dion, M., Fourage, L., Hallet, JN. et al. Cloning and expression of a β-glycosidase gene from Thermus thermophilus. Sequence and biochemical characterization of the encoded enzyme. Glycoconj J 16, 27–37 (1999). https://doi.org/10.1023/A:1006997602727

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1006997602727

Search

Navigation