Efficient production of Thermus protease aqualysin I in Escherichia coli: effects of cloned gene structure and two-stage culture

  • S. Sakamoto
  • I. Terada
  • Y. -C. Lee
  • K. Uehara
  • H. Matsuzawa
  • M. Iijima
Original Paper Applied Genetics and Regulations


The DNA sequence encoding Thermus protease aqualysin I was inserted downstream from a bacteriophage T7 promoter in an expression vector. In the T7 expression system, using a strain lacking an F′ episome, aqualysin I was produced in soluble form without chemical induction. The deletions of part (30 amino acid residues) or all (105 residues) of the C-terminal pro-sequence from the C terminus significantly affected both cellular growth and the production of the enzyme. Complete deletion adversely affected both. In contrast, the 30-residue deletion markedly improved productivity by approximately four times compared to non-deletion, and shortened the time needed for the activation of a precursor to active enzyme. The concentration of inducer isopropyl β-D-thiogalactopyrano-side (IPTG) was varied to examine its effects, and it was found that a low concentration of IPTG improved aqualysin I production. To avoid the inhibitory effects of acetic acid accumulation in the culture medium, the use of other carbon sources besides glucose was examined. When cells were cultivated with glycerol, the acetic acid level remained relatively low, and both good cellular growth and a high level of production were attained. The aqualysin I


Specific Growth Rate IPTG Induction Acetic Acid Concentration Final Cell Concentration Acetic Acid Formation 
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Copyright information

© Springer-Verlag 1996

Authors and Affiliations

  • S. Sakamoto
    • 1
  • I. Terada
    • 2
  • Y. -C. Lee
    • 3
  • K. Uehara
    • 1
  • H. Matsuzawa
    • 3
  • M. Iijima
    • 1
  1. 1.Life Science Research LaboratoryJapan Tobacco Inc.Aoba-kuJapan
  2. 2.Sea Water Science Research LaboratoryJapan Tobacco Inc.OdawaraJapan
  3. 3.Department of Agricultural ChemistryThe University of TokyoBunkyo-kuJapan

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