The Protein Journal

, Volume 32, Issue 4, pp 317–325 | Cite as

Cold-Adapted RTX Lipase from Antarctic Pseudomonas sp. Strain AMS8: Isolation, Molecular Modeling and Heterologous Expression

  • Mohd Shukuri Mohamad Ali
  • Menega Ganasen
  • Raja Noor Zaliha Raja Abd Rahman
  • Adam Leow Thean Chor
  • Abu Bakar Salleh
  • Mahiran Basri


A new strain of psychrophilic bacteria (designated strain AMS8) from Antarctic soil was screened for extracellular lipolytic activity and further analyzed using molecular approach. Analysis of 16S rDNA showed that strain AMS8 was similar to Pseudomonas sp. A lipase gene named lipAMS8 was successfully isolated from strain AMS8, cloned, sequenced and overexpressed in Escherichia coli. Sequence analysis revealed that lipAMS8 consist of 1,431 bp nucleotides that encoded a polypeptide consisting of 476 amino acids. It lacked an N-terminal signal peptide and contained a glycine- and aspartate-rich nonapeptide sequence at the C-terminus, which are known to be the characteristics of repeats-in-toxin bacterial lipases. Furthermore, the substrate binding site of lipAMS8 was identified as S207, D255 and H313, based on homology modeling and multiple sequence alignment. Crude lipase exhibited maximum activity at 20 °C and retained almost 50 % of its activity at 10 °C. The molecular weight of lipAMS8 was estimated to be 50 kDa via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal expression level was attained using the recombinant plasmid pET32b/BL21(DE3) expressed at 15 °C for 8 h, induced by 0.1 mM isopropyl β-D thiogalactoside (IPTG) at E. coli growth optimal density of 0.5.


lipAMS8 Cold-adapted Signal peptide RTX repeats 



Isopropyl β-D thiogalactosidase


Free fatty acid


Polymerase chain reaction




Sodium dodecyl sulfate-polyacrylamide gel electrophoresis





This research was funded by Ministry of Higher Education (Fundamental Research Grant Scheme 01-04-10-766FR).


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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Mohd Shukuri Mohamad Ali
    • 1
    • 2
  • Menega Ganasen
    • 1
    • 2
  • Raja Noor Zaliha Raja Abd Rahman
    • 1
  • Adam Leow Thean Chor
    • 1
    • 3
  • Abu Bakar Salleh
    • 1
    • 2
  • Mahiran Basri
    • 1
  1. 1.Enzyme and Microbial Technology Research Center, Faculty of Biotechnology and Biomolecular SciencesUniversiti Putra MalaysiaSerdangMalaysia
  2. 2.Department of Biochemistry, Faculty of Biotechnology and Biomolecular SciencesUniversiti Putra MalaysiaSerdangMalaysia
  3. 3.Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular SciencesUniversiti Putra MalaysiaSerdangMalaysia

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