Skip to main content
SpringerLink
Log in

Production of MLM-Type Structured Lipids Catalyzed by Immobilized Heterologous Rhizopus oryzae Lipase

  • Original Paper
  • Published:
Journal of the American Oil Chemists' Society

Abstract

This work aims to produce triacylglycerols (TAG) containing a medium-chain fatty acid (M) at positions sn-1,3 and a long-chain fatty acid (L) at sn-2 position, i.e. TAG of MLM type, by acidolysis of virgin olive oil with caprylic (C8:0) or capric (C10:0) acids, catalyzed by 1,3-selective Rhizopus oryzae heterologous lipase (rROL) immobilized in Eupergit® C and modified sepiolite. This lipase was produced by the methylotrophic yeast Pichia pastoris. Reactions were performed at 25 and 40 °C, for 24 h, either in solvent-free or in n-hexane media, at a molar ratio 1:2 (olive oil:free fatty acid). Higher incorporations of C8:0 (21.6 mol%) and C10:0 (34.8 mol%) into the TAG were attained in solvent-free media, at 40 °C, when rROL immobilized in Eupergit® C was used. In organic media, at 40 °C, only 15.9 and 14.1 mol%, incorporation of C8:0 or C10:0 were, respectively observed. Lower incorporations were attained for both acids (3.4–7.0 mol%) when native ROL (nROL) in both supports and rROL in modified sepiolite were used. rROL in Eupergit® C maintained its activity during the first four or three 23-h batches, respectively when C8:0 (half-life time, t 1/2 = 159 h) or C10:0 (t 1/2 = 136 h) were used, decreasing thereafter following a time delay model.

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

Similar content being viewed by others

Abbreviations

FFA:

Free fatty acid(s)

L:

Long-chain fatty acid(s)

M:

Medium-chain fatty acid(s)

MAG:

Monoacylglycerol(s)

MLM:

Triacylglycerol(s) containing medium-chain fatty acid at sn-1 and sn-3 positions (M) and a long-chain fatty acid (L) at position sn-2

nROL:

Native ROL

rROL:

Heterologous ROL

ROL:

Rhizopus oryzae lipase

SL:

Structured lipid(s)

TAG:

Triacylglycerol(s)

t 1/2 :

Half-life time

References

  1. Osborn HT, Akoh CC (2002) Structured lipids–novel fat with medical, nutraceutical and food applications. Compr Rev Food Sci Saf 3:93–103

    Google Scholar 

  2. Low CT, Mohamad R, Tan CP, Long K, Ismail R, Lo SK, Lai OM (2007) Lipase-catalyzed production of medium-chain triacylglycerols from palm kernel oil distillate: optimization using response surface methodology. Eur J Lipid Sci Technol 10:107–119

    Article  Google Scholar 

  3. Sharma R, Chisti Y, Banerjee UC (2001) Production, purification, characterization, and applications of lipases. Biotechnol Adv 19(8):627–662

    Article  CAS  Google Scholar 

  4. Gunstone FD (2008) Extraction, refining, and modification processes in: oils and fats in the food industry. Wiley-Blackwell, UK

    Google Scholar 

  5. Kim I, Kim H, Lee K, Chung S, Ko S (2002) Lipase-catalyzed acidolysis of perilla oil with caprylic acid to produce structured lipids. J Am Oil Chem Soc 79:363–367

    Article  CAS  Google Scholar 

  6. Senanayake SPJ, Shahidi F (2002) Enzyme-catalyzed synthesis of structured lipids via acidolysis of seal (Phoca groenlandica) blubber oil with capric acid. Food Res Int 35:745–752

    Article  Google Scholar 

  7. Moreno PA, Medina AR, Rubio FC, Páez BC, Grima EM (2004) Production of structured lipids by acidolysis of an EPA-enriched fish oil and caprylic acid in a packed bed reactor: analysis of three different operation modes. Biotechnol Prog 20:1044–1052

    Article  Google Scholar 

  8. Kim BH, Akoh CC (2005) Modeling of lipase-catalyzed acidolysis of sesame oil and caprylic acid by response surface methodology: optimization of reaction conditions by considering both acyl incorporation and migration. J Agric Food Chem 53:8033–8037

    Article  CAS  Google Scholar 

  9. Kim BH, Akoh CC (2006) Characteristics of structured lipid prepared by lipase-catalyzed acidolysis of roasted sesame oil and caprylic acid in a bench-scale continuous packed bed reactor. J Agric Food Chem 54:5132–5141

    Article  CAS  Google Scholar 

  10. Turan S, Karabulut I, Vurak H (2006) Effects of reaction parameters on the incorporation of caprylic acid into soybean oil for production of structured lipids. J Food Lipids 13:306–317

    Article  CAS  Google Scholar 

  11. Feltes MMC, de Oliveira Pitol L, Gomes Correia JF, Grimaldi R, Block JM, Ninow JL (2009) Incorporation of medium chain fatty acids into fish oil by chemical and enzymatic interesterification. Grasas y Aceites 60:168–176

    Article  CAS  Google Scholar 

  12. Fomuso BL, Akoh CC (2002) Lipase-catalyzed acidolysis of olive oil and caprylic acid in a bench-scale packed bed bioreactor. Food Res Int 35:15–21

    Article  CAS  Google Scholar 

  13. Öztürk T, Ustun G, Aksoy H (2010) Production of medium-chain triacylglycerols from corn oil: optimization by response surface methodology. Bioresour Technol 101(19):7456–7461

    Article  Google Scholar 

  14. Jennings BH, Akoh CC (2000) Lipase-catalysed modification of rice bran oil to incorporate capric acid. J Agric Food Chem 48(9):4439–4443

    Article  CAS  Google Scholar 

  15. Houde A, Kademi A, Leblanc D (2004) Lipases and their industrial applications: an overview. Appl Biochem Biotechnol 118:155–170

    Article  CAS  Google Scholar 

  16. Hasan F, Shah AA, Hameed A (2005) Industrial applications of microbial lipases. Enzyme Microb Technol 39:235–251

    Article  Google Scholar 

  17. Caballero V, Bautista FM, Campelo JM, Luna D, Marinas JM, Romero AA, Hidalgo JM, Luque R, Macario A, Giodarno G (2009) Sustainable preparation of novel glycerol-free biofuel by using pig pancreatic lipase: partial 1,3-regiospecific alcoholysis of sunflower oil. Process Biochem 44:334–342

    Article  CAS  Google Scholar 

  18. Illanes A (1999) Stability of biocatalysts. Electron J Biotechnol 2:1–9

    Google Scholar 

  19. Hamam F, Shahidi F (2004) Synthesis of structured lipids via acidolysis of docosahexaenoic acid single cell oil (DHASCO) with capric acid. J Agric Food Chem 52:2900–2906

    Article  CAS  Google Scholar 

  20. Kim H, Hou CT, Lee K, Kim BH, Kim I (2010) Enzymatic synthesis of structured lipids using a novel cold-active lipase from Pichia lynferdii NRRL Y-7723. Food Chem 122:846–849

    Article  CAS  Google Scholar 

  21. Resina D, Serrano A, Valero F, Ferrer P (2004) Expression of a Rhizopus oryzae lipase in Pichia pastoris under control of the nitrogen source-regulated formaldehyde dehydrogenase promoter. J Biotechnol 109:103–113

    Article  CAS  Google Scholar 

  22. Cos O, Serrano A, Montesinos JL, Ferrer P, Cregg JM, Valero F (2005) Combined effect of methanol utilization (Mut) phenotype and gene dosage on recombinant protein production in Pichia pastoris fed-batch cultures. J Biotechnol 116(4):321–335

    Article  Google Scholar 

  23. Katchalski-Katzir E, Kraemer DM (2000) Eupergit® C, a carrier for immobilization of enzymes of industrial potential. J Mol Catal B Enzym 10:157–176

    Article  CAS  Google Scholar 

  24. Knezevic Z, Milosavic N, Bezbradica D, Jakovljevic Z, Prodanovic R (2006) Immobilization of lipase from Candida rugosa on Eupergit® C supports by covalent attachment. Biochem Eng J 30:269–278

    Article  CAS  Google Scholar 

  25. Bautista FM, Bravo C, Campelo JM, Garcia A, Luna D, Marinas JM (1998) Covalent immobilization of porcine pancreatic lipase on amorphous AlPO4 and other inorganic supports. J Chem Technol Biotechnol 72:249–254

    Article  CAS  Google Scholar 

  26. Bautista FM, Campelo JM, Garcia Jurado A, Luna D, Marinas JM (2001) Properties of a glucose oxidase covalently immobilized on amorphous AlPO4 support. J Mol Catal B Enzym 11:567–577

    Article  CAS  Google Scholar 

  27. Arnau C, Ramón R, Casas C, Valero F (2010) Optimization of the heterologous production of a Rhizopus oryzae lipase in Pichia pastoris system using mixed substrates on controlled fed-batch bioprocess. Enzyme Microb Technol 46:494–500

    Article  CAS  Google Scholar 

  28. Sayari A, Frikha F, Miled N, Mtibaa H, Ali YB, Verger R, Gargouri Y (2005) N-terminal peptide of Rhizopus oryzae lipase is important for its catalytic properties. FEBS Lett 579:976–982

    Article  CAS  Google Scholar 

  29. Bradford MM (1976) A rapid and sensitive for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  30. Xu X, Fomuso LB, Akoh CC (2000) Synthesis of structured triacylglycerols by lipase-catalyzed acidolysis in packed bed bioreactor. J Agric Food Chem 48(1):3–10

    Article  CAS  Google Scholar 

  31. Zhao H, Lu Z, Bie X, Lu F, Liu Z (2007) Lipase catalyzed acidolysis of lard with capric acid in organic solvent. J Food Eng 78:41–46

    Article  CAS  Google Scholar 

  32. Camacho Paez B, Robles Medina A, Camacho Rubio F, Estebán Cerdán L, Molina Grima E (2003) Kinetics of lipase-catalysed interesterification of triolein and caprylic acid to produce structured lipids. J Chem Technol Biotechnol 78:461–470

    Article  Google Scholar 

  33. Jennings BH, Akoh CC (2001) Lipase-catalyzed modification of fish oil to incorporate capric acid. Food Chem 72(3):273–278

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors are grateful to Fundação para a Ciência e a Tecnologia (FCT), Portugal, for a Ph.D. fellowship for Mrs. Patrícia Nunes (SFRH/BD/39280/2007). This study was supported by the: (i) Program CYTED, under the scope of the “Ibero-American Network for the Extraction and Enzymatic Transformation of Functional Ingredients and Nutraceuticals from Regional Plants and Agro-Residues (ENZNUT)” and (ii) by the project CTQ2007-60347 of the Spanish Ministry of Science and Innovation, and the (iii) 2009-SGR-281 and Reference Network in Biotechnology (XRB) (Generalitat de Catalunya).

Author information

Authors and Affiliations

  1. Instituto Superior de Engenharia, Universidade do Algarve, Campus da Penha, 8005-139, Faro, Portugal

    P. A. Nunes & P. Pires-Cabral

  2. Instituto Superior de Agronomia, CEER-Biosystems Engineering, Technical University of Lisbon, Tapada da Ajuda, 1349-017, Lisbon, Portugal

    P. A. Nunes, P. Pires-Cabral & S. Ferreira-Dias

  3. Departament d’Enginyeria Química, EE, Universitat Autònoma de Barcelona, Bellaterra, 08193, Barcelona, Spain

    M. Guillén & F. Valero

  4. Departamento de Química Orgánica, Universidad de Córdoba, Campus Universitario de Rabanales, Edificio Marie Curie, 14014, Córdoba, Spain

    D. Luna

Authors
  1. P. A. Nunes
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. P. Pires-Cabral
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. Guillén
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. F. Valero
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. D. Luna
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. S. Ferreira-Dias
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to S. Ferreira-Dias.

About this article

Cite this article

Nunes, P.A., Pires-Cabral, P., Guillén, M. et al. Production of MLM-Type Structured Lipids Catalyzed by Immobilized Heterologous Rhizopus oryzae Lipase. J Am Oil Chem Soc 88, 473–480 (2011). https://doi.org/10.1007/s11746-010-1702-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11746-010-1702-y

Keywords

Search

Navigation