Molecular Biotechnology

, Volume 26, Issue 2, pp 101–109 | Cite as

Cloning of the thermostable α-amylase gene from Pyrococcus woesei in Escherichia coli

Isolation and some properties of the enzyme
  • Beata Grzybowska
  • Piotr Szweda
  • Józef Synowiecki
Research

Abstract

Pyrococcus woesei (DSM 3773) α-amylase gene was cloned into pET21d(+) and pYTB2 plasmids, and the pET21d(+)α-amyl and pYTB2α-amyl vectors obtained were used for expression of thermostable α-amylase or fusion of α-amylase and intein in Escherichia coli BL21(DE3) or BL21(DE3)pLysS cells, respectively. As compared with other expression systems, the synthesis of α-amylase in fusion with intein in E. coli BL21(DE3)pLysS strain led to a lower level of inclusion bodies formation—they exhibit only 35% of total cell activity—and high productivity of the soluble enzyme form (195,000 U/L of the growth medium). The thermostable α-amylase can be purified free of most of the bacterial protein and released from fusion with intein by heat treatment at about 75°C in the presence of thiol compounds. The recombinant enzyme has maximal activity at pH 5.6 and 95°C. The half-life of this preparation in 0.05 M acetate buffer (pH 5.6) at 90°C and 110°C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following incubation for 2 h at 120°C. Maltose was the main end product of starch hydrolysis catalyzed by this α-amylase. However, small amounts of glucose and some residual unconverted oligosaccharides were also detected. Furthermore, this enzyme shows remarkable activity toward glycogen (49.9% of the value determined for starch hydrolysis) but not toward pullulan.

Index entries

α-amylase overexpression starch liquefaction thermostable enzymes Pyrococcus woesei Escherichia coli 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Koch, R., Spreinat, A., Lemke, K., and Antranikian, G. (1991) Purification and properties of a hyperthermoactive α-amylase from the archaebacterium P. woesei. Arch. Microbiol. 155, 572–578.CrossRefGoogle Scholar
  2. 2.
    Linden, A., Niehaus F., and Antranikian, G. (2000) Single-step purification of a recombinant thermostable α-amylase after solubilization of the enzyme from insoluble aggregates. J. Chromatogr. 737, 253–259.CrossRefGoogle Scholar
  3. 3.
    Fischer, B., Sumner, I., and Goodenough, P. (1993) Isolation, renaturation, and formation of disulfide bonds of eukaryotic proteins expressed in Escherichia coli as inclusion bodies. Biotechnol. Bioeng. 41, 3–13.CrossRefGoogle Scholar
  4. 4.
    Uemori, T., Ishino, Y., Toh, H., Asada, K., and Kato, I. (1993) Organization and nucleotide sequence of the DNA polymerase from the archaeon Pyrococcus furiosus. Nucleic Acids Res. 21, 259–265.PubMedCrossRefGoogle Scholar
  5. 5.
    Bernfeld, P. (1955) Amylases α and β. Methods Enzymol. 1, 149–158.CrossRefGoogle Scholar
  6. 6.
    Bradford, M. M. (1976) A rapid and sensitive method for quantication of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.PubMedCrossRefGoogle Scholar
  7. 7.
    Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.PubMedCrossRefGoogle Scholar
  8. 8.
    Hansen, S. A. (1975) Thin-layer chromatography method for identification of oligosaccharides in starch hydrolysates. J. Chromatogr. 105, 388–390.CrossRefGoogle Scholar
  9. 9.
    Laderman, K. A., Asada, K., Uemori, T., et al. (1993) α-Amylase from the hyperthermophilic archaebacterium Pyrococcus furiosus. J. Biol. Chem. 268, 24,402–24,407.Google Scholar
  10. 10.
    Dong, G., Vieille, C., Savchenko, A., and Zeikus, J. G. (1997) Cloning, sequencing, and expression of the gene encoding extracellular α-amylase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme. Appl. Environ. Microbiol. 63, 3569–3576.PubMedGoogle Scholar
  11. 11.
    Pisani, F. M., Rella, R., Raja, C. A., et al. (1990) Thermostable β-galactosidase from the archaebacterium sulfolobus solfatoricus: purification and porperties. Eur. J. Blochem. 187, 321–328.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc 2004

Authors and Affiliations

  • Beata Grzybowska
  • Piotr Szweda
  • Józef Synowiecki
    • 1
  1. 1.Department of Food Chemistry and TechnologyGdansk University of TechnologyGdanskPoland

Personalised recommendations