Current Genetics

, Volume 59, Issue 3, pp 129–137 | Cite as

A gene encoding a new cold-active lipase from an Antarctic isolate of Penicillium expansum

  • Suja MohammedEmail author
  • Junior Te’o
  • Helena Nevalainen
Research Article


Cold-active lipases are of significant interest as biocatalysts in industrial processes. We have identified a lipase that displayed activity towards long carbon-chain-p-nitrophenyl substrates (C12–C18) at 25 °C from the culture supernatant of an Antarctic Penicillium expansum strain assigned P. expansum SM3. Zymography revealed a protein band of around 30 kDa with activity towards olive oil. DNA fragments of a lipase gene designated as lipPE were isolated from the genomic DNA of P. expansum SM3 by genomic walking PCR. Subsequently, the complete genomic lipPE gene was amplified using gene-specific primers designed from the 5′- and 3′-regions. Reverse transcription PCR was used to amplify the lipPE cDNA. The deduced amino acid sequence consisted of 285 residues that included a predicted signal peptide. Three peptides identified by LC/MS/MS analysis of the proteins in the culture supernatant of P. expansum were also present in the deduced amino acid sequence of the lipPE gene suggesting that this gene encoded the lipase identified by initial zymogram activity analysis. Full analysis of the nucleotide and the deduced amino acid sequences indicated that the lipPE gene encodes a novel P. expansum lipase. The lipPE gene was expressed in E. coli for further characterization of the enzyme with a view of assessing its suitability for industrial applications.


Cold-active lipase Antarctic Penicillium expansum Genomic walking PCR 



We thank Paul Worden from the Macquarie University Sequencing Facility for DNA sequencing. We also thank Dr. Ron Bradner for sharing information about the Antarctic fungal strains and identification of strains. S. Mohammed would like to express her sincere gratitude to Prof. Paul Pilowsky for his enormous support and valuable advice on manuscript preparation. This research has been facilitated by access to the Australian Proteome Analysis Facility established under the Australian Government’s Major National Research Facilities Program. S. Mohammed is the recipient of a RAACE (Research Award in Areas and Centres of Excellence) Scholarship awarded by Macquarie University.

Supplementary material

294_2013_394_MOESM1_ESM.doc (841 kb)
Supplementary material 1 (DOC 841 kb)


  1. Alford JA, Pierce DA (1961) Lipolytic activity of microorganisms at low and intermediate temperatures. III. Activity of microbial lipases at temperatures below 0 °C. J Food Sci 26:518–524CrossRefGoogle Scholar
  2. Andualema B, Gessesse A (2012) Microbial lipases and their industrial applications: review. Biotechnology 11:100–118CrossRefGoogle Scholar
  3. Bell PJL, Sunna A, Gibbs MD, Curach NC, Nevalainen H, Bergquist PL (2002) Prospecting for novel lipase genes using PCR. Microbiology 148:2283–2291PubMedGoogle Scholar
  4. Bian C, Yuan C, Chen L, Meehan EJ, Jiang L, Huang Z, Lin L, Huang M (2010) Crystal structure of a triacylglycerol lipase from Penicillium expansum at 1.3 Å determined by sulfur SAD. Proteins 78:1601–1605PubMedGoogle Scholar
  5. Bradner JR, Bell PL, Te′o VSJ, Nevalainen KMH (2003) The application of PCR for the isolation of a lipase gene from the genomic DNA of an Antarctic microfungus. Curr Genet 44:224–230PubMedCrossRefGoogle Scholar
  6. Castro-Ochoa LD, Rodríguez-Gómez C, Valerio-Alfaro G, Oliart Ros R (2005) Screening, purification and characterization of the thermoalkalophilic lipase produced by Bacillus thermoleovorans CCR11. Enzyme Microb Technol 37:648–654CrossRefGoogle Scholar
  7. Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387–395PubMedCrossRefGoogle Scholar
  8. Feller G, Gerday C (2003) Psychrophilic enzymes: hot topics in cold adaptation. Nat Rev Microbiol 1:200–208PubMedCrossRefGoogle Scholar
  9. Feller G, Narinx E, Arpigny JL, Aittaleb M, Baise E, Genicot S, Gerday C (1996) Enzymes from psychrophilic organisms. FEMS Microbiol Rev 18:189–202CrossRefGoogle Scholar
  10. Florczak T, Makowski K, Turkiewicz M (2007) The cold-adapted lipase of an Antarctic fungus Beauveria sp. P7 as an effective catalyst of enantioselective 1-phenylethanol trasesterification. J Biotech 131:S89–S90CrossRefGoogle Scholar
  11. Gerday C, Aittaleb M, Bentahir M, Chessa J, Claverie P, Collins T, D’Amico S, Dumont J, Garsoux G, Georlette D, Hoyoux A, Lonhienne T, Meuwis M, Feller G (2000) Cold-adapted enzymes: from fundamentals to biotechnology. Trends Biotechnol 18:103–107PubMedCrossRefGoogle Scholar
  12. Jaegar KE, Eggert T (2002) Lipases for biotechnology. Curr Opin Biotech 13:390–397CrossRefGoogle Scholar
  13. Joseph B, Ramteke PW, Thomas G, Shrivastava N (2007) Standard review cold-active microbial lipases: a versatile tool for industrial applications. Biotechnol Mol Biol Rev 2:039–048Google Scholar
  14. Joseph B, Ramteke PW, Thomas G (2008) Cold active microbial lipases: some hot issues and recent developments. Biotechnol Adv 26:457–470PubMedCrossRefGoogle Scholar
  15. Kim H-R, Kim I-H, Hou CT, Kwon K-I, Shin B-S (2010) Production of a novel cold-active lipase from Pichia lynferdii Y-7723. J Agric Food Chem 58:1322–1326PubMedCrossRefGoogle Scholar
  16. Lebendiker M (2002) Small and large scale MBP-fusion protein purification. The Hebrew University of Jerusalem. Accessed 10 June 2010
  17. Li N, Zong MH (2010) Lipases from the genus Penicillium: production, purification, characterization and applications. J Mol Catal B Enzym 66:43–54CrossRefGoogle Scholar
  18. Lin L, Xie BF, Yang GZ, Shi QQ, Lin QY, Xie LH, Wu X, Wu SG (2002) Cloning and sequence analysis of cDNA encoding alkaline lipase from Penicillium expansum PF898. Chin J Biochem Mol Biol 18:32–37Google Scholar
  19. Liu R, Jiang X, Mou H, Guan H, Hwang H, Li X (2009) A novel low-temperature resistant alkaline lipase from a soda lake fungus strain Fusarium solani N4-2 for detergent formulation. Biochem Eng J 46:265–270CrossRefGoogle Scholar
  20. Mala JG, Takeuchi S (2008) Understanding structural features of microbial lipases—an overview. Anal Chem Insights 3:9–19PubMedGoogle Scholar
  21. Morris DD, Reeves RA, Gibbs MD, Saul DJ, Bergquist PL (1995) Correction of the beta-mannanase domain of the celC pseudogene from Caldocellulosiruptor saccharolyticus and activity of the gene product on kraft pulp. Appl Environ Microbiol 61:2262–2269PubMedGoogle Scholar
  22. Nevalainen H, Bradner R, Wadud S, Mohammed S, McRae C, Te’o J (2012) Enzyme activities and biotechnological applications of cold-active microfungi. In: Anitori RP (ed) Extremophiles: microbiology and biotechnology. Caister Academic Press, PortlandGoogle Scholar
  23. Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–6PubMedCrossRefGoogle Scholar
  24. Nthangeni MB, Ramagoma F, Tlou MG, Litthauer D (2005) Development of a versatile cassette for directional genome walking using cassette ligation-mediated PCR and its application in the cloning of complete lipolytic genes from Bacillus species. J Microbiol Methods 61:225–234PubMedCrossRefGoogle Scholar
  25. Patkar SA, Björkling F, Zundel M, Schülein M, Svendsen A, Heldt-Hansen HP, Gormsen E (1993) Purification of two lipases from Candida antarctica and their inhibition by various inhibitors. Indian J Chem 32B:76–80Google Scholar
  26. Penttilä M, Nevalainen H, Rättö M, Salminen E, Knowles J (1987) A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. Gene 61:155–164PubMedCrossRefGoogle Scholar
  27. Peterson R, Grinyer J, Nevalainen H (2011) Secretome of the coprophilous fungus Doratomyces stemonitis C8, isolated from koala feces. Appl Environ Microbiol 77:3793–3801PubMedCrossRefGoogle Scholar
  28. Pleiss J, Fischer M, Peiker M, Thiele C, Schmid RD (2000) Lipase engineering database: understanding and exploiting sequence–structure–function relationships. J Mol Catal B Enzym 10:491–508CrossRefGoogle Scholar
  29. Reis P, Holmberg K, Watzke H, Leser ME, Miller R (2009) Lipases at interfaces: a review. Adv Coll Interface Sci 147–148:237–250CrossRefGoogle Scholar
  30. Ruiz C, Falcocchio S, Xoxi E, Javier Pastor FI, Diaz P, Saso L (2004) Activation and inhibition of Candida rugosa and Bacillus-related lipases by saturated fatty acids evaluated by a new colorimetric microassay. Biochim Biophys Acta (BBA)—Gen Subj 1672:184–191CrossRefGoogle Scholar
  31. Sharma R, Chisti Y, Banerjee UC (2001) Production, purification, characterization, and applications of lipases. Biotechnol Adv 19:627–662PubMedCrossRefGoogle Scholar
  32. Wu M, Qian Z, Jiang P, Min T, Sun C, Huang W (2003) Cloning of an alkaline lipase gene from Penicillium cyclopium and its expression in Escherichia coli. Lipids 38:191–199PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Suja Mohammed
    • 1
    Email author
  • Junior Te’o
    • 2
  • Helena Nevalainen
    • 2
  1. 1.Australian School of Advanced Medicine, Faculty of Human SciencesMacquarie UniversitySydneyAustralia
  2. 2.Department of Chemistry and Biomolecular Sciences, Biomolecular Frontiers Research CentreMacquarie UniversitySydneyAustralia

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