Skip to main content
SpringerLink
Log in

Importance of the residue Asp 290 on chain length selectivity and catalytic efficiency of recombinant Staphylococcus simulans lipase expressed in E. coli

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

Abstract

In addition to their physiological importance, microbial lipases, like staphylococcal ones, are of considerable commercial interest for biotechnological applications such as detergents, food production, and pharmaceuticals and industrial synthesis of fine chemicals. The gene encoding the extracellular lipase of Staphylococcus simulans (SSL) was subcloned in the pET-14b expression vector and expressed in Esherichia coli BL21 (DE3). The wild-type SSL was expressed as amino terminal His6-tagged recombinant protein. One-step purification of the recombinant lipase was achieved with nickel metal affinity column. The purified His-tagged SSL (His6-SSL) is able to hydrolyse triacylglycerols without chain length selectivity. The major differences among lipases are reflected in their chemical specificity in the hydrolysis of peculiar ester bonds, and their respective capacity to hydrolyse substrates having different physico-chemical properties. It has been proposed, using homology alignment, that the region around the residue 290 of Staphylococcus hyicus lipase could be involved in the selection of the substrate. To evaluate the importance of this environment, the residue Asp290 of Staphylococcus simulans lipase was mutated to Ala using site-directed mutagenesis. The mutant expression plasmid was also overexpressed in Esherichia coli and purified with a nickel metal affinity column. The substitution of Asp290 by Ala was accompanied by a significant shift of the acyl-chain length specificity of the mutant towards short chain fatty acid esters. Kinetic studies of wild-type SSL and its mutant D290A were carried out, and show essentially that the catalytic efficiency (k cat /K M ) of the mutant was affected. Our results confirmed that Asp290 is important for the chain length selectivity and catalytic efficiency of Staphylococcus simulans lipase.

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

Similar content being viewed by others

Abbreviations

SSL:

Staphylococcus simulans lipase

SAL NCTC 8530:

Staphylococcus aureus NCTC 8530 lipase

SAL PS54:

Staphylococcus aureus PS54 lipase

SEL:

Staphylococcus epidermis lipase

SHL:

Staphylococcus hyicus lipase

LB:

Luria–Bertani

IPTG:

Isopropyl-β-d-thiogalactopyranoside

NTA:

Nitrilotriacetate

BSA:

Bovine serum albumin

DTT:

Dithiothreitol

EDTA:

Ethylene Diamine Tetraacetic Acid

PC:

Phosphatidylcholine

EGTA:

Ethylene Glycol-bis (β-aminoethyl Ether) N,N,N′,N′-Tetraacetic Acid

NaDC:

Sodium deoxycholate

NaTDC:

Sodium taurodeoxycholate

DrPLA2 :

Dromedary pancreatic phospholipase A2

PCR:

Polymerase chain reaction

SDS/PAGE:

Sodium dodecyl sulfate/polyacrylamide gel electrophoresis

TC4 :

Tributyrin (tributyryl glycerol)

TC18 :

Triolein (trioleyl glycerol)

References

  1. Ben Bacha, A., Gargouri, Y., Bezzine, S., & Mejdoub, H. (2006). Purification and characterization of phospholipase A2 from dromedary pancreas. Biochimica et Biophysica Acta, 1760, 1202–1209.

    CAS  Google Scholar 

  2. Birnboim, H. C. (1983). A rapid alkaline extraction method for the isolation of plasmid DNA. Methods in Enzymology, 100, 243–255.

    Article  PubMed  CAS  Google Scholar 

  3. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of quantities of protein utilising the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.

    Article  PubMed  CAS  Google Scholar 

  4. Christensson, B., Fehrenbach, F. J., & Hedstrom, S. A. (1985). A new serological assay for Staphylococcus aureus infections: detection of IgG antibodies to S. aureus lipase with an enzyme-linked immunosorbent assay. Journal of Infectious Disease, 152, 286–292.

    CAS  Google Scholar 

  5. Davies, M. E. (1954). A study of the diffusible lipase produced by staphylococci and of its immunological activity. Journal of General Microbiology, 11, 37–44.

    PubMed  CAS  Google Scholar 

  6. Egloff, M.-P., Ransac, S., Marguet, F., Rogalska, E., van Tilbeurgh, H., Buono, G., Cambillau, C., & Verger, R. (1995). Enzymes lipolytiques et lipolyse. Les lipases: cinétiques, spécificités et aspect structuraux. OCL, 2, 52–67.

    CAS  Google Scholar 

  7. Gargouri, Y., Ben Salah, A., Douchet, I., & Verger, R. (1995). Kinetic behaviour of pancreatic lipase in five species using emulsion and monomolecular films of synthetic glycerides. Biochimica et Biophysica Acta, 1257, 223–229.

    PubMed  Google Scholar 

  8. Gargouri, Y., Moreau, H., & Verger, R. (1989). Gastric lipases: biochemical and physiological studies. Biochimica et Biophysica Acta, 1006, 255–271.

    PubMed  CAS  Google Scholar 

  9. Gargouri, Y., Julien, R., Sugihara, A., Verger, R., & Sarda, L. (1984). Inhibition of pancreatic and microbial lipases by proteins. Biochimica et Biophysica Acta, 795, 326–331.

    PubMed  CAS  Google Scholar 

  10. Gargouri, Y., Pieroni, G., Rivière, C., Sugihara, A., Sarda, L., Verger, R. (1985). Inhibition of lipases by proteins. A kinetic study with dicaprin monolayers. Journal of Biological Chemistry, 260, 2268–2273.

    PubMed  CAS  Google Scholar 

  11. Jaeger, K. E., Dijkstra, B. W., & Reetz, M. T. (1999). Bacterial biocatalysts: molecular biology, three-dimentional structures, and biotechnological applications of lipases. Annual Review of Microbiology, 53, 315–351.

    Article  PubMed  CAS  Google Scholar 

  12. Jaeger, K., & Reetz, M. (1998). Microbial lipases form versatile tools for biotechnology. Trends in Biotechnology, 16, 396–403.

    Article  PubMed  CAS  Google Scholar 

  13. Jürgens, D., & Huser, H. (1981). Large-scale purification of Staphylococcal lipase by interaction chromatography. Journal of Chromatography, 216, 295–301.

    Article  Google Scholar 

  14. Jürgens, D., Huser, H., & Fehrenbach, F. J. (1981). Purification and characterisation of Staphylococcus aureus lipase. FEMS Microbiology Letters, 12, 195–199.

    Google Scholar 

  15. Kloos, W. E., Schleifer, K., & Götz, F. (1991). The genus Staphylococcus. In A. Balows, H. G. Trüper, M. Dworkin, W. Harder, & K. H. Schleifer (Eds.), The prokaryotes (pp. 1369–1420). NY: Springer-Verlag.

  16. Kötting, J., Jürgens, D., & Huser, H. (1983). Separation and characterisation of two lipases from Staphylococcus aureus (ten 5). Journal of Chromatography, 281, 253–261.

    Article  PubMed  Google Scholar 

  17. Laemmli, U.K. (1970). Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.

    Article  PubMed  CAS  Google Scholar 

  18. Lee, C.Y., & Iandolo, J.J. (1986). Lysogenic conversion of Staphylococcal lipase is caused by insertion of the bacteriophage L54a genome into the lipase structural gene. Journal of Bacteriology, 166, 385–391.

    PubMed  CAS  Google Scholar 

  19. Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. Journal of Molecular Biology, 3, 208–218.

    Article  CAS  Google Scholar 

  20. Mosbah, H., Sayari, A., Bezzine, S., & Gargouri Y. (2006). Expression, purification, and characterization of His-tagged Staphylococcus xylosus lipase wild-type and its mutant Asp290Ala. Protein Express. Purification, 47, 516–523.

    CAS  Google Scholar 

  21. Oh, B., Kim, H., Lee, J., Kang, S., & Oh, T. (1999) Staphylococcus haemolyticus lipase: biochemical properties, substrate specificity and gene cloning. FEMS Microbiology Letter, 179, 385–392.

    Article  CAS  Google Scholar 

  22. Pablo, G., Hammons, A., Bradley, S., & Fulton, J. E. (1974). Characterisation of the extracellular lipases from Corynebacterium acnes and Staphylococcus epidermidis. The Journal of investigative Dermatology, 63, 231–238.

    Article  PubMed  CAS  Google Scholar 

  23. Pieroni, G., Gargouri, Y., Sarda, L., & Verger, R. (1990). Interactions of lipases with lipid monolayers. Facts and Questions. Advances in Colloid and Interface Science, 32, 341–378.

    Article  PubMed  CAS  Google Scholar 

  24. Rathelot, J., Julien, R., Canioni, P., Coeroli, C., & Sarda, L. (1975). Studies on the effect of bile salt and colipase on enzymatic lipolysis. Improvement method for the determination of pancreatic lipase and colipase. Biochimie, 57, 1117–1122.

    PubMed  CAS  Google Scholar 

  25. Rollof, J., Braconier, J. H., Soderstrom, C., & Nilsson-Ehle, P. (1988). Interference of Staphylococcus aureus lipase with human granulocyte function. European Journal of Clinical Microbiology & Infectious Diseases, 7, 505–510.

    Article  CAS  Google Scholar 

  26. Rosenstein, R., & Gotz, F. (2000). Staphylococcal lipases: biochemical and molecular characterisation. Biochimie, 82, 1005–10014.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  28. Sayari, A., Verger, R., & Gargouri, Y. (2002) Comparative kinetic studies of two staphylococcal lipases using the monomolecular film technique. Journal of Biochemistry and Molecular Biology, 34, 457–462.

    Google Scholar 

  29. Simons, J. W. F. A., Adams, H., Cox, R. C., Dekker, N., Gotz, F., Slotboom, A. J., & Verheij, H. M. (1996). The lipase from Staphylococcus aureus. Expression in Escherischia coli, large-scale purification and comparison of substrate specificity to Staphylococcus hyicus lipase. Eur. Journal of Biochemistry, 242, 760–769.

    CAS  Google Scholar 

  30. Simons, J. W. F. A., van Kampen, M. D., Ubarretxena-Belandia, I., Cox, R. C., Alves dos Santos, C. M., Egmond, M. R., & Verheij, H. M. (1999) Identification of a calcium binding site in Staphylococcus hyicus lipase: Generation of calcium-independent variants. Biochemistry, 38, 2–10.

    Article  PubMed  CAS  Google Scholar 

  31. Tysky, S., Hryniewicz, W., & Jeljaszewicz, J. (1983). Purification and properties of the staphylococcal extracellular lipase. Biochimica et Biophysica Acta, 749, 312–317.

    Google Scholar 

  32. Van Kampen, M. D., Dekker, N., Egmond, M. R., & Verheij, H. M. (1998). Substrate specificity of Staphylococcus hyicus lipase and Staphylococcus aureus lipase as studied by in vivo chimeragenesis. Biochemistry, 37, 3459–3466.

    Google Scholar 

  33. Weld, J. T., & O’Leray, W. M. (1963). Events associated with the development of lipid plaques on plasma agar. Nature, 199, 510–511.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported financially by « Ministère de la recherche scientifique, de la technologie et de développement des compétences—Tunisia » through a grant to « Laboratoire de Biochimie et de Génie Enzymatique des Lipases—ENIS ».

Author information

Authors and Affiliations

  1. Laboratoire de biochimie et de génie enzymatique des lipases, ENIS BPW, Sfax, 3038, Tunisia

    Adel Sayari, Habib Mosbah & Youssef Gargouri

Authors
  1. Adel Sayari
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Habib Mosbah
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Youssef Gargouri
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Youssef Gargouri.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sayari, A., Mosbah, H. & Gargouri, Y. Importance of the residue Asp 290 on chain length selectivity and catalytic efficiency of recombinant Staphylococcus simulans lipase expressed in E. coli . Mol Biotechnol 36, 14–22 (2007). https://doi.org/10.1007/s12033-007-0008-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-007-0008-2

Keywords

Search

Navigation