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Applied Microbiology and Biotechnology

, Volume 49, Issue 3, pp 277–284 | Cite as

Cloning, sequencing and overexpression of a Rhodothermus marinus gene encoding a thermostable cellulase of glycosyl hydrolase family 12

  • S. Halldórsdóttir
  • E. T. Thórólfsdóttir
  • R. Spilliaert
  • M. Johansson
  • S. H. Thorbjarnardóttir
  • A. Palsdottir
  • G. Ó. Hreggvidsson
  • J. K. Kristjánsson
  • O. Holst
  • G. Eggertsson
ORIGINAL PAPER

Abstract

A gene library from the thermophilic eubacterium Rhodothermus marinus, strain ITI 378, was constructed in pUC18 and transformed into Escherichia coli. Of 5400 transformants, 3 were active on carboxymethylcellulose. Three plasmids conferring cellulase activity were purified and were all found to contain the same cellulase gene, celA. The open reading frame for the celA gene is 780 base pairs and encodes a protein of 260 amino acids with a calculated molecular mass of 28.8 kDa. The amino acid sequence shows homology with cellulases in glycosyl hydrolase family 12. The celA gene was overexpressed in E. coli when the pET23, T7 phage RNA polymerase system was used. The enzyme showed activity on carboxymethylcellulose and lichenan, but not on birch xylan or laminarin. The expressed enzyme had six terminal histidine residues and was purified by using a nickel nitrilotriacetate column. The enzyme had a pH optimum of 6–7 and its highest measured initial activity at 100 °C. The heat stability of the enzyme was increased by removal of the histidine residues. It then retained 75% of its activity after 8 h at 90 °C.

Keywords

Cellulase Carboxymethylcellulose Histidine Residue celA Calculated Molecular Mass 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer-Verlag Berlin Heidelberg 1998

Authors and Affiliations

  • S. Halldórsdóttir
    • 1
  • E. T. Thórólfsdóttir
    • 1
  • R. Spilliaert
    • 1
  • M. Johansson
    • 3
  • S. H. Thorbjarnardóttir
    • 1
  • A. Palsdottir
    • 1
  • G. Ó. Hreggvidsson
    • 2
  • J. K. Kristjánsson
    • 2
  • O. Holst
    • 3
  • G. Eggertsson
    • 1
  1. 1.Laboratory of Molecular Genetics, Institute of Biology, University of Iceland, IS-108 Reykjavik, Iceland Tel.: +354 525 4603 Fax: +354 525 4069 e-mail: gudmegg@rhi.hi.isIS
  2. 2.Department of Biotechnology, Technological Institute of Iceland, Keldnaholt, IS-112 Reykjavik, IcelandIS
  3. 3.Center for Chemistry and Chemical Engineering, Lund University, S-22100 Lund, SwedenSE

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