Skip to main content
SpringerLink
Log in

Heterologous Expression and Purification of a Heat-Tolerant Staphylococcus xylosus Lipase

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

Staphylococcus xylosus is a microorganism involved in fermentation of meat products and also a natural producer of extracellular lipases. The aim of the present work was to clone and express in E. coli a lipase from S. xylosus (AF208229). This lipase gene (1084 bp) was amplified from a S. xylosus strain isolated from naturally fermented salami and introduced in pET14b expression vector in order to express the recombinant fusion protein (histidine-tagged lipase) in E. coli. Recombinant histidine-tagged S. xylosus lipase was purified by affinity chromatography in an HPLC system. The histidine-tagged lipase is a monomer in solution, as determined by size-exclusion chromatography. It presents a high lipase activity at pH 9.0 and 42°C for p-nitrophenyl acetate and p-nitrophenyl butyrate, among seven different esters assayed (pNPC2, pNPC4, pNPC10, pNPC12, pNPC14, pNPC16, pNPC18). Moreover, the enzyme presented a quite interesting thermal stability, after an incubation period of 10 min at 95°C, 77% of the initial activity was retained.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Secundo, F., Carrea, G., Tarabiono, C., Gatti-Lafranconi, P., Brocca, S., Lotti, M., et al. (2006). The lid is a structural and functional determinant of lipase activity and selectivity. Journal of Molecular Catalysis B Enzymatic, 39, 166–170.

    Article  CAS  Google Scholar 

  2. Hasan, F., Shah, A. A., & Hameed, A. (2006). Industrial applications of microbial lipases. Enzyme and Microbial Technology, 39, 235–251.

    Article  CAS  Google Scholar 

  3. Roustan, J. L., Chu, A. R., Moulin, G., & Bigey, F. (2005). A novel lipase/acyltransferase from the yeast Candida albicans: Expression and characterization of the recombinant enzyme. Applied Microbiology and Biotechnology, 68, 203–212.

    Article  CAS  Google Scholar 

  4. Tyndall, J. D. A., Sinchaikul, S., Fothergill-Gilmore, L. A., Taylor, P., & Walkinshaw, M. D. (2002). Crystal structure of a thermostable lipase from Bacillus stearothermophilus P1. Journal of Molecular Biology, 323, 859–869.

    Article  CAS  Google Scholar 

  5. Tiesinga, J. J. W., van Pouderoyen, G., Nardini, M., Ransac, S., & Dijkstra, B. W. (2007). Structural basis of phospholipase activity of Staphylococcus hyicus lipase. Journal of Molecular Biology, 371, 447–456.

    Article  CAS  Google Scholar 

  6. Rosenstein, R., & Götz, F. (2000). Staphylococcal lipases: Biochemical and molecular characterization. Biochimie, 82, 1005–1014.

    Article  CAS  Google Scholar 

  7. Gupta, R., Gupta, N., & Rathi, P. (2004). Bacterial lipases: An overview of production, purification and biochemical properties. Applied Microbiology and Biotechnology, 64, 763–781.

    Article  CAS  Google Scholar 

  8. Lumor, S. E., & Akoh, C. C. (2008). Esterification and hydrolytic activities of Candida rugosa lipase isoform 1 (LIP1) immobilized on celite 545, duolite A7, and sephadex G-25. Journal of Agriculture and Food Chemistry, 56, 10396–10398.

    Article  Google Scholar 

  9. Chakraborty, K., & Raj, R. P. (2008). An extra-cellular alkaline metallolipase from Bacillus licheniformis MTCC 6824: Purification and biochemical characterization. Food Chemistry, 109, 727–736.

    Article  CAS  Google Scholar 

  10. Ha, S. H., Lan, M. N., Lee, S. H., Hwang, S. M., & Koo, Y. M. (2007). Lipase catalyzed biodiesel production from soybean oil in ionic liquids. Enyzme and Microbial Technology, 41, 480–483.

    Article  CAS  Google Scholar 

  11. Yao, H., Yu, S., Zhang, L., Zuo, K., Ling, H., Zhang, F., et al. (2008). Isolation of a novel lipase gene from Serratia liquefaciens S33 DB-1, functional expression in Pichia pastoris and its properties. Molecular Biotechnology, 38, 99–107.

    Article  CAS  Google Scholar 

  12. Quintana-Castro, R., Díaz, P., Gerardo Valerio-Alfaro, G., Hugo, S., García, H. S., & Rosamaría Oliart-Ros, R. (2009). Gene cloning, expression, and characterization of the Geobacillus thermoleovorans CCR11 thermoalkaliphilic lipase. Molecular Biotechnology, 42, 75–83.

    Article  CAS  Google Scholar 

  13. Jiang, Z., Zheng, Y., Luo, Y., Wang, G., Wang, W., Ma, Y., et al. (2005). Cloning and expression of a novel lipase gene from Pseudomonas fluorescens B52. Molecular Biotechnology, 31, 95–101.

    Article  CAS  Google Scholar 

  14. Lui, B., Qian, X., Wu, Q., & Lin, X. (2008). Two lipase-catalyzed sequential synthesis of drug derivatives in organic media. Enyzme and Microbial Technology, 43, 375–380.

    Article  Google Scholar 

  15. Morot-Bizot, S., Talon, R., & Leroy-Setrin, S. (2003). Development of specific PCR primers for a rapid and accurate identification of Staphylococcus xylosus, a species used in food fermentation. Journal of Microbiol Methods, 55, 279–286.

    Article  CAS  Google Scholar 

  16. Iacumin, L., Cocolin, L., Cantoni, C., & Comi, G. (2007). Preliminary analysis of the lipase gene (gehM) expression of Staphylococcus xylosus in vitro and during fermentation of naturally fermented sausages (in situ). Journal of Food Protection, 70, 2665–2669.

    CAS  Google Scholar 

  17. Rantsiou, K., Iacumin, L., Cantoni, C., Comi, G., & Cocolin, L. (2005). Ecology and characterization by molecular methods of Staphylococcus species isolated from fresh sausages. International Journal of Food Microbiology, 97, 277–284.

    Article  CAS  Google Scholar 

  18. Simonová, M., Strompfová, V., Marcináková, M., Lauková, A., Vesterlund, S., Latorre-Moratalla, M., et al. (2006). Characterization of Staphylococcus xylosus and Staphylococcus carnosus isolated from Slovak meat products. Meat Science, 73, 559–564.

    Article  Google Scholar 

  19. Kozacinski, L., Drosinos, E., Caklovica, F., Cocolin, F., Gasparik-Reichardt, J., & Veskovic, S. (2008). Investigation of microbial association of traditionally fermented sausages. Food Technology and Biotechnology, 46, 93–108.

    CAS  Google Scholar 

  20. Talon, R., Walter, D., Chariter, S., Barrière, C., & Montel, M. C. (1999). Effect of nitrate and incubation conditions on the production of catalase and nitrate reductase by staphylococci. International Journal of Food Microbiology, 52, 47–56.

    Article  CAS  Google Scholar 

  21. Iacumin, L., Comi, G., Cantoni, C., & Cocolin, L. (2006). Molecular and technological characterization of Staphylococcus xylosus isolated from naturally fermented Italian sausages by RAPD, Rep-PCR and Sau-PCR analysis. Meat Science, 74(2), 281–288.

    Article  CAS  Google Scholar 

  22. Mosbah, H., Sayari, A., Mejdoub, H., Dhouib, H., & Gargouri, Y. (2005). Biochemical and molecular characterization of Staphylococcus xylosus lipase. Biochimica et Biophysica Acta, 1723, 282–291.

    CAS  Google Scholar 

  23. Fiorentini, A. M., Sawitzki, M. C., Bertol, T. M., Brod, F. C. A., Pelisser, M. R., Arisi, A. C. M., et al. (2009). Phenotypic and molecular characterization of Staphylococcus xylosus: Technological potential for use in fermented sausage. Brazilian Archives of Biology and Technology, 52, 737–746.

    Article  CAS  Google Scholar 

  24. Maia, M. D. D., Heasley, A., Camargo de Morais, M. M., Melo, E. H. M., Morais, M. A., Jr, Ledingham, W. M., et al. (2001). Effect of culture conditions on lipase production by Fusarium solani in batch fermentation. Bioresource Technology, 76, 23–27.

    Article  CAS  Google Scholar 

  25. Woody, R. W. (1995). Circular dichroism. Methods in Enzymology, 246, 34–71.

    Article  CAS  Google Scholar 

  26. Kelly, S. M., & Price, N. C. (2000). The use of circular dichroism in the investigation of protein structure and function. Current Protein and Peptide Science, 1(4), 349–384.

    Article  CAS  Google Scholar 

  27. Ollis, D. L., Cheah, E., Cygler, M., Dijkstra, B., Frolow, F., Franken, S. M., et al. (1992). The alpha/beta hydrolase fold. Protein Engineering, 5, 197–211.

    Article  CAS  Google Scholar 

  28. Jaeger, K. E., Dijkstra, B. W., & Reetz, M. T. (1999). Bacterial biocatalysts: Molecular biology, three-dimensional structures and biotechnological applications of lipases. Annual Review of Microbiology, 53, 315–335.

    Article  CAS  Google Scholar 

  29. Svendsen, A. (2000). Lipase protein engineering. Biochimica et Biophysica Acta, 1543, 223–238.

    CAS  Google Scholar 

  30. Horchani, H., Mosbah, H., Salem, N. B., Gargouri, Y., & Sayari, A. (2009). Biochemical and molecular characterization of a thermoactive, alkaline and detergent-stable lipase from a newly isolated Staphylococcus aureus strain. Journal of Molecular Catalysis B Enzymatic, 56(4), 237–245.

    Article  CAS  Google Scholar 

  31. Sakinç, T., Kleine, B., & Gatermann, S. G. (2007). Biochemical characterization of the surface-associated lipase of Staphylococcus saprophyticus. FEMS Microbiology Letters, 274, 335–341.

    Article  Google Scholar 

  32. Sayari, A., Agrebi, N., Jaoua, S., & Gargouri, Y. (2001). Biochemical and molecular characterization of Staphylococcus simulans lipase. Biochimie, 83, 863–871.

    Article  CAS  Google Scholar 

  33. Demirijan, D., Moris-Varas, F., & Cassidy, C. (2001). Enzymes from extremophiles. Current Opinion in Chemical Biology, 5, 144–151.

    Article  Google Scholar 

  34. Haki, G. D., & Rakshit, S. K. (2003). Developments in industrially important thermostable enzymes: A review. Bioresource Technology, 89, 17–34.

    Article  CAS  Google Scholar 

  35. Palma-Fernandez, E. R., Gomes, E., & Silva, R. (2002). Purification and characterization of two b-glucosidases from the thermophilic fungus Thermoascus aurantiacus. Folia Microbiologica, 47, 685–690.

    Article  Google Scholar 

  36. Van Kampen, M. D., Rosenstein, R., Götz, F., & Egmond, M. R. (2001). Cloning, purification and characterization of Staphylococcus warneri lipase 2. Biochimica et Biophysica Acta, 1554, 229–241.

    Google Scholar 

  37. Chang, R. C., Chou, S. J., & Shaw, J. F. (2000). Site-directed mutagenesis of a highly active Staphylococcus epidermidis lipase fragment identifies residues essential for catalysis. Journal of the American Oil Chemists’ Society, 77, 1021–1025.

    Article  CAS  Google Scholar 

  38. Hotelier, T., Renault, L., Cousin, X., Negre, V., Marchot, P., & Chatonnet, A. (2004). ESTHER, the database of the α/β-hydrolase fold superfamily of proteins. Nucleic Acid Research, 32, 145–147.

    Article  Google Scholar 

  39. Macedo, G. A., Lozano, M. M. S., & Pastore, G. M. (2003). Enzymatic synthesis of short chain citronellyl esters by a new lipase from Rhizopus sp. Electronic Journal of Biotechnology, 6, 72–75.

    Google Scholar 

  40. Larios, A., García, H. S., Oliart, R. S., & Valerio-Alfaro, G. (2004). Synthesis of flavor and fragrance esters using Candida antartica lipase. Applied Microbiology and Biotechnology, 65, 373–376.

    Article  CAS  Google Scholar 

  41. Salah, R. B., Ghamghui, H., Miled, N., Mejdoub, H., & Gargouri, Y. (2007). Production of butyl acetate ester by lipase from novel strain of Rhizopus oryzae. Journal of Bioscience and Bioengineering, 103(4), 368–372.

    Article  Google Scholar 

  42. Chang, R. C., Chou, S. J., & Shaw, J. F. (2001). Synthesis of fatty esters by recombinant Staphylococcus epidermidis lipases in aqueous environment. Journal of Agriculture and Food Chemistry, 49, 2619–2622.

    Article  CAS  Google Scholar 

  43. Chen, J. P. (1996). Production of ethyl butyrate using gel-entrapped Candida cylindracea lipase. Journal of Fermentation and Bioengineering, 82(4), 404–407.

    Article  CAS  Google Scholar 

  44. Liu, S. Q., Holland, R., & Crow, V. (2003). Synthesis of ethyl butanoate by a commercial lipase in aqueous media under conditions relevant to cheese ripening. Journal of Dairy Research, 70, 359–363.

    Article  CAS  Google Scholar 

  45. Talon, R., Montel, M. C., & Berdague, J. L. (1996). Production of flavor esters by lipases of Staphylococcus warneri and Staphylococcus xylosus. Enyzme and Microbial Technology, 19, 620–622.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The present work was financially supported by CNPq processes 476285/2007-0 and 552508/2007-1, and Rede Proteoma de Santa Catarina (FAPESC/FINEP/MCT). MRP was supported by a fellowship from Universidade do Contestado (UnC) and FCAB was recipient of a fellowship from CAPES, Ministry of Education, Brazil. We also express our gratitude to Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul for sequence data.

Author information

Authors and Affiliations

  1. Depto de Ciência e Tecnologia de Alimentos, Universidade Federal de Santa Catarina, Rod. Admar Gonzaga, 1346, Florianopolis, SC, 88034-001, Brazil

    Fábio Cristiano Angonesi Brod, Márcia Regina Pelisser & Ana Carolina Maisonnave Arisi

  2. Centro de Biologia Molecular Estrutural, Depto de Bioquímica, Universidade Federal de Santa Catarina, Florianopolis, SC, 88040-900, Brazil

    Jean Borges Bertoldo, Javier Vernal & Hernán Terenzi

  3. Laboratório de Espectrometria de Massa, EMBRAPA, Recursos Genéticos e Biotecnologia, Brasilia, DF, Brazil

    Carlos Bloch Jr

Authors
  1. Fábio Cristiano Angonesi Brod
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Márcia Regina Pelisser
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jean Borges Bertoldo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Javier Vernal
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Carlos Bloch Jr
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hernán Terenzi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ana Carolina Maisonnave Arisi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ana Carolina Maisonnave Arisi.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 998 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Brod, F.C.A., Pelisser, M.R., Bertoldo, J.B. et al. Heterologous Expression and Purification of a Heat-Tolerant Staphylococcus xylosus Lipase. Mol Biotechnol 44, 110–119 (2010). https://doi.org/10.1007/s12033-009-9218-0

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-009-9218-0

Keywords

Search

Navigation