Molecular Biotechnology

, Volume 26, Issue 2, pp 101–109

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

DOI: 10.1385/MB:26:2:101

Cite this article as:
Grzybowska, B., Szweda, P. & Synowiecki, J. Mol Biotechnol (2004) 26: 101. doi:10.1385/MB:26:2:101


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 

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

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