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World Journal of Microbiology and Biotechnology

, Volume 27, Issue 2, pp 431–441 | Cite as

Gene cloning, expression and characterization of a cold-adapted lipase from a psychrophilic deep-sea bacterium Psychrobacter sp. C18

  • Ruipeng Chen
  • Lizhong Guo
  • Hongyue Dang
Original Paper

Abstract

A psychrophilic bacterium Psychrobacter sp. C18 previously isolated from the Southern Okinawa Trough deep-sea sediments showed extracellular lipolytic activity towards tributyrin. A genomic DNA library was constructed and screened to obtain the corresponding lipase gene. The sequenced DNA fragment contains an open reading frame of 945 bp, which was denoted as the lipX gene, from which a protein sequence LipX was deduced of 315 amino acid residues with a molecular mass of 35,028 Da. This protein contained the bacterial lipase GNSMG (GxSxG, x represents any amino acid residue) and HG consensus motifs. The recombinant pET28a(+)/lipX gene was overexpressed in heterologous host Escherichia coli BL21 (DE3) cells to overproduce the lipase protein LipXHis with a 6× histidine tag at its C-terminus. Nickel affinity chromatography was used for purification of the expressed recombinant lipase. The maximum lipolytic activity of the purified recombinant lipase was obtained at temperature of 30°C and pH 8.0 with p-nitrophenyl myristate (C14) as a substrate. Thermostability assay indicated that the recombinant LipXHis is a cold-adapted lipase, which was active in 10% methanol, ethanol, acetone and 30% glycol, and inhibited partially by Zn2+, Co2+, Mn2+, Fe3+ and EDTA. Most non-ionic detergents, such as DMSO, Triton X-100, Tween 60 and Tween 80 enhanced the lipase activity but 1% SDS completely inhibited the enzyme activity. Additionally, the highest lipolytic rate of the recombinant LipXHis lipase was achieved when p-nitrophenyl myristate was used as a substrate, among all the p-nitrophenyl esters tested.

Keywords

Cold-adapted lipase Genomic DNA library Recombinant lipase Psychrobacter Deep-sea sediment 

Notes

Acknowledgments

The authors thank Hu Zhu for his assistance in the project. This work was supported by China Ocean Mineral Resources R&D Association grants DYXM-115-02-2-20 and DYXM-115-02-2-6, Hi-Tech Research and Development Program of China grant 2007AA091903, China National Natural Science Foundation grant 40576069, Fundamental Research Funds for the Central Universities of China grant 09CX05005A, and Foundation of the State Key Laboratory of Heavy Oil Processing, China University of Petroleum grant SKL2010-02.

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Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  1. 1.College of Life SciencesQingdao Agricultural UniversityQingdaoChina
  2. 2.State Key Laboratory of Heavy Oil Processing and Centre for Bioengineering and BiotechnologyChina University of Petroleum (East China)QingdaoChina

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