Skip to main content
SpringerLink
Log in

Functional expression of an alkaline lipase inEscherichia coli

  • Industrial Microbiology
  • Original Articles
  • Published:
Annals of Microbiology Aims and scope Submit manuscript

Abstract

The aim of the present study was to express and evaluate a previously cloned lipase gene. In this study, the cloned gene was subcloned in the pET15bEscherichia coli BL21(DE3) expression system. The expression of the recombinant lipase was induced using 1 mM IPTG for 3 hours. The enzyme activity was measured using p-nitrophenyl-decanoate as substrate. The recombinant lipase showed a molecular weight of 26 kDa by SDS-PAGE. Maximum activity was found at pH 9-10 and 40-50 °C. ZnCl2 at 1, 0.3, 0.1, 0.03, and 0.01 mM concentrations were found to be inhibitory to the enzyme activity and did not improve enzyme thermostability. The recombinant lipase showed an optimum temperature higher than lipase ofBacillus subtilis (its closest relative in primary structure) while similar activity in the alkaline pH.

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

Similar content being viewed by others

References

  • Abdou A.M. (2003). Purification and partial characterization of psychrotrophicSerratia marcescens lipase. J. Dairy Sci., 86: 127–132.

    Article  CAS  PubMed  Google Scholar 

  • Alvarez-Macarie E., Augier-Magro V., Baratti J. (1999). Characterization of a thermostable esterase activity from the moderate thermophilebacillus licheniformis. Biosci. Biotechnol. Biochem., 63: 1865–1870.

    Article  CAS  PubMed  Google Scholar 

  • Alvarez-Macarie E., Baratti J. (2000). Short chain flavour ester synthesis by a new esterase fromBacillus licheniformis. Journal of Molecular Catalysis — B Enzymatic, 10: 377–383.

    Article  CAS  Google Scholar 

  • Arpigny J.L., Jaeger K.E. (1999). Bacterial lipolytic enzymes: Classification and properties. Biochem. J., 343: 177–183.

    Article  CAS  PubMed  Google Scholar 

  • Brune K.A., Goetz F. (1992). Degradation of lipids by bacterial lipases. Microbial Degradation of Natural Products, 243–266.

  • Cho A.R., Yoo S.K., Kim E.J. (2000). Cloning, sequencing and expression inEscherichia coli of a thermophilic lipase fromBacillus thermoleovorans ID-1. FEMS Microbiol. Lett., 186: 235–238.

    Article  CAS  PubMed  Google Scholar 

  • Choi W.C., Kim M.H., Ro H.S., Ryu S.R., Oh T.K., Lee J.K. (2005). Zinc in lipase L1 fromGeobacillus stearothermophilus L1 and structural implications on thermal stability. FEBS Lett., 579: 3461–3466.

    Article  CAS  PubMed  Google Scholar 

  • Dartois V., Baulard A., Schanck K., Colson C. (1992). Cloning, nucleotide sequence and expression inEscherichia coli of a lipase gene fromBacillus subtilis 168. Biochimica et Biophysica Acta — Gene Structure and Expression, 1131: 253–260.

    Article  CAS  Google Scholar 

  • Dharmsthiti S., Pratuangdejkul J., Theeragool G.T., Luchai S. (1998). Lipase activity and gene cloning ofAcinetobacter calcoaceticus LP009. J. Gen. Appl. Microbiol., 44: 139–145.

    Article  CAS  PubMed  Google Scholar 

  • Dharmsthiti S., Luchai S. (1999). Production, purification and characterization of thermophilic lipase fromBacillus sp. THL027. FEMS Microbiol. Lett., 179: 241–246.

    Article  CAS  PubMed  Google Scholar 

  • Eggert T., Pencreac’h G., Douchet I., Verger R., Jaeger K.E. (2000). A novel extracellular esterase fromBacillus subtilis and its conversion to a monoacylglycerol hydrolase. Eur. J. Biochem., 267: 6459–6469.

    Article  CAS  PubMed  Google Scholar 

  • Ghanem E.H., Al-Sayed H.A., Saleh K.M. (2000). An alkalophilic thermostable lipase produced by a new isolate ofBacillus alcalophilus. World Journal of Microbiology and Biotechnology, 16: 459–464.

    Article  CAS  Google Scholar 

  • Gilbert E.J., Cornish A., Jones C.W. (1991). Purification and properties of extracellular lipase fromPseudomonas aeruginosa EF2. J. Gen. Microbiol., 137: 2223–2229.

    CAS  PubMed  Google Scholar 

  • Gupta R., Gupta N., Rathi P. (2004). Bacterial lipases: an overview of production, purification and biochemical properties. Appl Microbiol Biotechnol, 64: 763–781.

    Article  CAS  PubMed  Google Scholar 

  • Iizumi T., Nakamura K., Fukase T. (1990). Purification and characterization of a thermostable lipase from newly isolatedPseudomonas sp. KWI-56. Agric. Biol. Chem., 54: 1253–1258.

    CAS  Google Scholar 

  • Jaeger K.E., Ransac S., Dijkstra B.W., Colson C., Van Heuvel M., Misset O. (1994). Bacterial lipases. FEMS Microbiology Reviews, 15: 29–63.

    Article  CAS  PubMed  Google Scholar 

  • Jaeger K.E., Reetz M.T. (1998). Microbial lipases form versatile tools for biotechnology. Trends in Biotechnology, 16: 396–403.

    Article  CAS  PubMed  Google Scholar 

  • Jaeger K.E., Dijkstra B.W., Reetz M.T. (1999). Bacterial biocatalysts: Molecular biology, three-dimensional structures, and biotechnological applications of lipases. Annual Reviews of Microbiology, 53: 315–351.

    Article  CAS  Google Scholar 

  • Jaeger K.E., Eggert T. (2002). Lipases for biotechnology. Curr. Opin. Biotechnol., 13: 390–397.

    Article  CAS  PubMed  Google Scholar 

  • Jeong S.T., Kim H.K., Kim S.J., Chi S.W., Pan J.G., Oh T.K., Ryu S.E. (2002). Novel zinc-binding center and a temperature switch in theBacillus stearothermophilus L1 lipase. Journal of Biological Chemistry, 277: 17041–17047.

    Article  CAS  PubMed  Google Scholar 

  • Jorgensen S., Skov K.W., Diderichsen B. (1991). Cloning, sequence, and expression of a lipase gene fromPseudomonas cepacia: Lipase production in heterologous hosts requires twoPseudomonas genes. J. Bacteriol., 173: 559–567.

    CAS  PubMed  Google Scholar 

  • Kademi A., Ait-Abdelkader N., Fakhreddine L., Baratti J.C. (2000a). Characterization of a new thermostable esterase from the moderate thermophilic bacteriumBacillus circulans. Journal of Molecular Catalysis — B Enzymatic, 10: 395–401.

    Article  CAS  Google Scholar 

  • Kademi A., Ait-Abdelkader N., Fakhreddine L., Baratti J. (2000b). Purification and characterization of a thermostable esterase from the moderate thermophileBacillus circulans. Appl. Microbiol. Biotechnol., 54: 173–179.

    Article  CAS  PubMed  Google Scholar 

  • Kazlauskas R.J., Bornscheuer U.T. (1998). Biotransformations with lipases. Biotechnology, 8 A: 37–191.

    Google Scholar 

  • Kim H.K., Park S.Y., Lee J.K., Oh T.K. (1998). Gene cloning and characterization of thermostable lipase fromBacillus stearothermophilus L1. Biosci. Biotechnol. Biochem., 62: 66–71.

    Article  CAS  PubMed  Google Scholar 

  • Kim H.K., Sung M.H., Kim H.M., Oh T.K. (1994). Occurrence of thermostable lipase in thermophilicBacillus sp. Strain 398. Biosci. Biotech. BioChem., 58: 961–962.

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Lee D.W., Koh Y.S., Kim K.J., Kim B.C., Choi H.J., Kim D.S.et al. (1999). Isolation and characterization of a thermophilic lipase fromBacillus thermoleovorans ID-1. FEMS Microbiol. Lett., 179: 393–400.

    Article  CAS  PubMed  Google Scholar 

  • Lee S.W., Won K., Lim H.K., Kim J.C., Choi G.J., Cho K.Y. (2004). Screening for novel lipolytic enzymes from uncultured soil microorganisms. Appl. Microbiol. Biotechnol., 65: 720–726.

    Article  CAS  PubMed  Google Scholar 

  • Lesuisse E., Schanck K., Colson C. (1993). Purification and preliminary characterization of the extracellular lipase ofBacillus subtilis 168, an extremely basic pH-tolerant enzyme. Eur. J. Biochem., 216: 155–160.

    Article  CAS  PubMed  Google Scholar 

  • Liu W.H., Beppu T., Arima K. (1973). Purification and general properties of the lipase of thermophilic fungusHumicola lanuginosa. Agric. Biol. Chem., 37: 157–163.

    CAS  Google Scholar 

  • Moller B., Vetter R., Wilke D., Foullois B. (1992). Patent application number WO, 91/16422.

  • Mosbah H., Sayari A., Bezzine S., Gargouri Y. (2006). Expression, purification, and characterization of HistaggedStaphylococcus xylosus lipase wild-type and its mutant Asp 290 Ala. Protein Expr. Purif., 47: 516–523.

    Article  CAS  Google Scholar 

  • Nardini M., Dijkstra B.W. (1999). a/b hydrolase fold enzymes: The family keeps growing. Curr Opin Struct Biol, 9: 732–737.

    Article  CAS  PubMed  Google Scholar 

  • Nishio T., Chikano T., Kamimura M. (1987). Studies onPseudomonas fragi 22.39B lipase. Part II purification and some properties of lipase produced byPseudomonas tragi. Agric. Biol. Chem., 51: 181–186.

    CAS  Google Scholar 

  • Nthangeni M.B., Patterton H.G., Van Tonder A., Vergeer W.P., Litthauer D. (2001). Over-expression and properties of a purified recombinantBacillus licheniformis lipase: A comparative report onBacillus lipases. Enzyme Microb. Technol., 28: 705–712.

    Article  CAS  PubMed  Google Scholar 

  • Oh B.C., Kim H.K., Lee J.K., Kang S.C., Oh T.K. (1999).Staphylococcus haemolyticus lipase: Biochemical properties, substrate specificity and gene cloning. FEMS Microbiol. Lett., 179: 385–392.

    Article  CAS  PubMed  Google Scholar 

  • Pandey A., Benjamin S., Soccol C.R., Nigam P., Krieger N., Soccol V.T. (1999). The realm of microbial lipases in biotechnology. Biotechnology and Applied Biochemistry, 29: 119–131.

    CAS  PubMed  Google Scholar 

  • Pratuangdejkul J., Dharmsthiti S. (2000). Purification and characterization of lipase from psychrophilicAcinetobacter calcoaceticus LP009. Microbiological Research, 155: 95–100.

    CAS  PubMed  Google Scholar 

  • Rabbani M., Mirmohammadsadeghi H., Ani M., goodarzvand chegini K., Etemadifar Z., Moazen F. (2009). Cloning and nucleotide sequence of a lipase gene from a soil isolate. (Submitted for publication).

  • Rua M.L., Atomi H., Schmidt-Dannert C., Schmid R.D. (1998). High-level expression of the thermoalkalophilic lipase fromBacillus thermocatenulatus inEscherichia coli. Appl. Microbiol. Biotechnol., 49: 405–410.

    Article  CAS  PubMed  Google Scholar 

  • Schlieben N.H., Niefind K., Schomburg D. (2004). Expression, purification, and aggregation studies of His-tagged thermoalkalophilic lipase fromBacillus thermocatenulatus. Protein Expr. Purif., 34: 103–110.

    Article  CAS  PubMed  Google Scholar 

  • Schmidt-Dannert C., Sztajer H., Stocklein W., Menge U., Schmid R.D. (1994). Screening, purification and properties of a thermophilic lipase fromBacillus thermocatenulatus. Biochim. Biophys. Acta — Lipids and Lipid Metabolism, 1214: 43–53.

    Article  CAS  Google Scholar 

  • Schmidt-Dannert C, Rua M.L., Atomi H., Schmid R.D. (1996). Thermoalkalophilic lipase ofBacillus thermocatenulatus. I. Molecular cloning, nucleotide sequence, purification and some properties. Biochim. Biophys. Acta., 1301: 105–114.

    PubMed  Google Scholar 

  • Schmidt-Dannert C., Rua M.L., Schmid R.D. (1997). Two novel lipases from thermophilebacillus thermocatenulatus: Screening, purification, cloning, overexpression, and properties. Methods in Enzymology, 284: 194–220.

    Article  CAS  PubMed  Google Scholar 

  • Simons J.W.F.A., Van Kampen M.D., Riel S., Goêtz F., Egmond M.R., Verheij H.M. (1998). Cloning, purification and characterisation of the lipase fromStaphylococcus epidermidis: Comparison of the substrate selectivity with those of other microbial lipases. Eur. J. Biochem., 253: 675–683.

    Article  CAS  PubMed  Google Scholar 

  • Stocklein W., Sztajer H., Menge U., Schmid R.D. (1993). Purification and properties of a lipase fromPenicillium expansum. Biochim. Biophys. Acta — Lipids and Lipid Metabolism, 1168: 181–189.

    Article  CAS  Google Scholar 

  • Sugihara A., Tani T., Tominaga Y (1991). Purification and characterization of a novel thermostable lipase fromBacillus sp. J. Biochem. (Tokyo), 109: 211–216.

    CAS  Google Scholar 

  • Sugihara A., Ueshima M., Shimada Y, Tsunasawa S., Tominaga Y. (1992). Purification and characterization of a novel thermostable lipase fromPseudomonas cepacia. J. Biochem. (Tokyo), 112: 598–603.

    CAS  Google Scholar 

  • Sztajer H., Lunsdorf H., Erdmann H., Menge U., Schmid R. (1992). Purification and properties of lipase fromPenicillium simplicissimum. Biochim. Biophys. Acta — Lipids and Lipid Metabolism, 1124: 253–261.

    Article  CAS  Google Scholar 

  • Wattiau P., Renard M.E., Ledent P., Debois V, Blackman G., Agathos S.N. (2001). A PCR test to identifyBacillus subtilis and closely related species and its application to the monitoring of wastewater biotreatment. Appl. Microbiol. Biotechnol., 56: 816–819.

    Article  CAS  PubMed  Google Scholar 

  • Wei P., Bai L, Song W., Hao G. (2009). Characterization of two soil metagenome-derived lipases with high specificity for p-nitrophenyl palmitate. Archives of Microbiology, 191: 233–240.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran

    Mohammed Rabbani, Hamid Mirmohammadsadeghi & Fatemeh Moazen

  2. Department of Clinical Biochemistry and Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran

    Mohsen Ani & Koorosh Goodarzvand Chegini

  3. Department of Biology, School of Basic Sciences, University of Isfahan, Isfahan, Iran

    Zahra Etemadifar

Authors
  1. Mohammed Rabbani
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hamid Mirmohammadsadeghi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mohsen Ani
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Koorosh Goodarzvand Chegini
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zahra Etemadifar
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Fatemeh Moazen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mohammed Rabbani.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rabbani, M., Mirmohammadsadeghi, H., Ani, M. et al. Functional expression of an alkaline lipase inEscherichia coli . Ann. Microbiol. 59, 763–769 (2009). https://doi.org/10.1007/BF03179221

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF03179221

Keywords

Search

Navigation