Applied Microbiology and Biotechnology

, Volume 68, Issue 2, pp 203–212 | Cite as

A novel lipase/acyltransferase from the yeast Candida albicans: expression and characterisation of the recombinant enzyme

  • Jean Louis Roustan
  • Agustin Rascon Chu
  • Guy Moulin
  • Frédéric BigeyEmail author
Biotechnologically Relevant Enzymes and Proteins


A gene encoding an extracellular lipase (CaLIP4) from Candida albicans was successfully expressed in Saccharomyces cerevisiae after mutagenesis of its unusual CUG serine codon into a universal one. The ability of this lipase, which shares 60% sequence homology with the lipase/acyltransferase from Candida parapsilosis, to synthesise esters was investigated. CaLIP4 behaved as a true lipase, displaying activity towards insoluble triglycerides and having no activity in the presence of short-chain fatty acid (FA) esters and phosphatidylcholine. Methyl, ethyl and propyl esters were efficiently used. The lipase exhibited highest selectivity for unsaturated FA. With saturated FAs, C14–C16 acyl chains were preferred. In a biphasic aqueous/lipid system, CaLIP4 displayed a high alcoholysis activity with a range of alcohols (e.g. methanol, ethanol, propanol and isopropanol) as acyl acceptor. During the course of the alcoholysis reaction, new esters are produced at concentrations above the thermodynamic equilibrium of the esterification reaction, indicating that ester synthesis does not proceed by esterification but mainly by direct acyltransfer. Ester synthesis is under kinetic control due to the high rate of alcoholysis. Unwanted hydrolysis is limited by competition between the acyl acceptor (alcohol) and water for the acyltransfer reaction, favouring the alcohol.


Lipase Lipase Activity Fatty Acid Ethyl Ester Ester Synthesis Acyl Acceptor 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This work was supported in part by Cognis Deutschland GmbH & Co. KG. We thank Johannes Crotogino and Ramses Obenga for their assistance in the cloning and mutagenesis experiments. We thank Professor Eric Dubreucq (UMR IR2B, ENSA.M.-INRA, Montpellier, France) for fruitful discussions throughout the research work and Guilhem Janbon (Institut Pasteur, Paris, France) for providing C. albicans DNA.


  1. Autryve P van, Ratomahenina R, Riaublanc A, Mitrani C, Pina M, Graille J, Galzy P (1991) Spectrometry assay of lipase activity using rhodamine 6G. Oléagineux 46:29–31Google Scholar
  2. Briand D (1995) Production d’esters méthyliques et éthyliques d’acides gras par bioconversion en milieu aqueux. PhD thesis, ENSAM, Montpellier, FranceGoogle Scholar
  3. Briand D, Dubreucq E, Galzy P (1994) Enzymatic fatty esters synthesis in aqueous medium with lipase from Candida parapsilosis (Ashford) Langeron and Talice. Biotechnol Lett 16:813–818CrossRefGoogle Scholar
  4. Briand D, Dubreucq E, Galzy P (1995a) Factors affecting the acyltransfer activity of the lipase from Candida parapsilosis in aqueous media. J Am Oil Chem Soc 72:1367–1373Google Scholar
  5. Briand D, Dubreucq E, Galzy P (1995b) Functioning and regioselectivity of the lipase of Candida parapsilosis (Ashford) Langeron and Talice in aqueous medium—new interpretation of regioselectivity taking acyl migration into account. Eur J Biochem 228:169–175Google Scholar
  6. Briand D, Dubreucq E, Grimaud J, Galzy P (1995c) Substrate specificity of the lipase from Candida parapsilosis. Lipids 30:747–754Google Scholar
  7. Brunel L, Neugnot V, Landucci L, Boze H, Moulin G, Bigey F, Dubreucq E (2004) High-level expression of Candida parapsilosis lipase/acyltransferase in Pichia pastoris. J Biotechnol 111:41–50CrossRefGoogle Scholar
  8. Fernandez-Lorente G, Palomo JM, Fuentes M, Mateo C, Guisan JM, Fernadez-Lafuente R (2003) Self-assembly of Pseudomonas fluorescens lipase into biomolecular aggregates dramatically affects functional properties. Biotechnol Bioeng 82:232–237CrossRefGoogle Scholar
  9. Fu Y, Ibrahim AS, Fonzi W, Zhou X, Ramos CF, Ghannoum MA (1997) Cloning and characterization of a gene (LIP1) which encodes a lipase from the pathogenic yeast Candida albicans. Microbiology 143:331–340Google Scholar
  10. Giacometti J, Milosevic A, Milin C (2002) Gas chromatographic determination of fatty acids contained in different lipid classes after their separation by solid-phase extraction. J Chromatogr A 976:47–54CrossRefGoogle Scholar
  11. Gietz DR, Schiestl RTH, Willems AR, Woods RA (1995) Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11:355–360Google Scholar
  12. Graupner M, Haalck L, Spener F, Lindner H, Glatter O, Paltauf F, Hermetter A (1999) Molecular dynamics of microbial lipases as determined from their intrinsic tryptophan fluorescence. Biophys J 77:493–504Google Scholar
  13. Hube B, Stehr F, Bossenz M, Mazur A, Kretschmar M, Schäfer W (2000) Secreted lipases of Candida albicans: cloning, characterisation and expression analysis of a new gene family with at least ten members. Arch Microbiol 174:362–374CrossRefGoogle Scholar
  14. Kaieda M, Samukawa T, Kondo A, Fukuda H (2001) Effect of methanol and water contents on production of biodiesel fuel from plant oil catalyzed by various lipases in a solvent-free system. J Biosci Bioeng 91:12–15CrossRefGoogle Scholar
  15. Kouker G, Jaeger K-E (1987) Specific and sensitive plate assay for bacterial lipases. Appl Environ Microbiol 59:211–213Google Scholar
  16. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedGoogle Scholar
  17. Lecointe C, Dubreucq E, Galzy P (1996) Ester synthesis in aqueous media in the presence of various lipases. Biotechnol Lett 18:869–874CrossRefGoogle Scholar
  18. Miled N, Beisson F, de Caro J, de Caro A, Arondel V, Verger R (2001) Interfacial catalysis by lipases. J Mol Catal B 11:165–171CrossRefGoogle Scholar
  19. Neugnot V, Moulin G, Dubreucq E, Bigey F (2002) The lipase/acyltransferase from Candida parapsilosis: molecular cloning and characterization of purified recombinant enzymes. Eur J Biochem 269:1734–1745CrossRefGoogle Scholar
  20. Ohama T, Suzuki T, Mori M, Osawa S, Ueda T, Watanabe K, Nakase T (1993) Non-universal decoding of the leucine codon CUG in several Candida species. Nucleic Acids Res 21:4039–4045Google Scholar
  21. Palomo JM, Fuentes M, Fernandez-Lorente G, Mateo C, Guisan JM, Fernandez-Lafuente R (2003) General trend of lipase to self-assemble giving bimolecular aggregates greatly modifies the enzyme functionality. Biomacromolecules 4:1–6CrossRefGoogle Scholar
  22. Pandey A, Benjamin S, Soocol CR, Nigam P, Krieger N, Soccol TV (1999) The realm of microbial lipases in biotechnology. Biotechnol Appl Biochem 29:119–131Google Scholar
  23. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  24. Saxena RK, Ghosh PK, Gupta R, Davidson WS, Bradoo S, Gulati R (1999) Microbial lipases: potential biocatalysts for the future industry. Curr Sci 77:101–115Google Scholar
  25. Song JK, Rhee JS (2000) Simultaneous enhancement of thermostability and catalytic activity of phospholipase A1 by evolutionary molecular engineering. Appl Environ Biotechnol 66:890–894Google Scholar
  26. Stehr F, Felk A, Gácser A, Kretschmar M, Mähnß B, Neuber K, Hube B, Schäfer W (2004) Expression analysis of the Candida albicans lipase gene family during experimental infections and in patient samples. FEMS Yeast Res 4:401–408CrossRefGoogle Scholar
  27. Tsuzuki W, Ue A, Nagao A (2003) Polar organic solvent added to an aqueous solution changes hydrolytic property of lipase. Biosci Biotechnol Biochem 67:1660–1666CrossRefGoogle Scholar
  28. Turner NA, Needs EC, Khan JA, Vulfson EN (2001) Analysis of conformational states of Candida rugosa lipase in solution: implications for mechanism of interfacial activation and separation of open and closed forms. Biotechnol Bioeng 72:108–118Google Scholar
  29. Vaysse L, Ly A, Moulin G, Dubreucq E (2002) Chain-length selectivity of various lipases during hydrolysis, esterification and alcoholysis in biphasic aqueous medium. Enzyme Microb Technol 31:648–655CrossRefGoogle Scholar
  30. Vernet T, Dignard D, Thomas DY (1987) A family of yeast expression vectors containing the phage f1 intergenic region. Gene 52:225–233CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Jean Louis Roustan
    • 1
  • Agustin Rascon Chu
    • 1
  • Guy Moulin
    • 1
  • Frédéric Bigey
    • 1
    Email author
  1. 1.Unité Mixte de Recherche Ingénierie de la Réaction Biologique, BioproductionsInstitut National de la Recherche Agronomique-Ecole Nationale Supérieure Agronomique de MontpellierMontpellier cedex 1France

Personalised recommendations