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

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• 作者：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)
  
• 关键词：lipAMS8 ; Cold ; adapted ; Signal peptide ; RTX repeats
• 刊名：The Protein Journal
• 出版年：2013
• 出版时间：April 2013
• 年：2013
• 卷：32
• 期：4
• 页码：317-325
• 全文大小：348KB
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• 作者单位：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. Enzyme and Microbial Technology Research Center, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Selangor, 43400, Malaysia
  2. Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Selangor, 43400, Malaysia
  3. Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Selangor, 43400, Malaysia
  
• ISSN：1573-4943

文摘

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.