Skip to main content
SpringerLink
Log in

The Purification and Characterization of Lipases from Lasiodiplodia theobromae, and Their Immobilization and Use for Biodiesel Production from Coconut Oil

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

The coconut kernel-associated fungus, Lasiodiplodia theobromae VBE1, was grown on coconut cake with added coconut oil as lipase inducer under solid-state fermentation conditions. The extracellular-produced lipases were purified and resulted in two enzymes: lipase A (68,000 Da)—purified 25.41-fold, recovery of 47.1%—and lipase B (32,000 Da)—purified 18.47-fold, recovery of 8.2%. Both lipases showed optimal activity at pH 8.0 and 35 °C, were activated by Ca2+, exhibited highest specificity towards coconut oil and p-nitrophenyl palmitate, and were stable in iso-octane and hexane. Ethanol supported higher lipase activity than methanol, and n-butanol inactivated both lipases. Crude lipase immobilized by entrapment within 4% (w/v) calcium alginate beads was more stable than the crude-free lipase preparation within the range pH 2.5–10.0 and 20–80 °C. The immobilized lipase preparation was used to catalyze the transesterification/methanolysis of coconut oil to biodiesel (fatty acyl methyl esters (FAMEs)) and was quantified by gas chromatography. The principal FAMEs were laurate (46.1%), myristate (22.3%), palmitate (9.9%), and oleate (7.2%), with minor amounts of caprylate, caprate, and stearate also present. The FAME profile was comparatively similar to NaOH-mediated transesterified biodiesel from coconut oil, but distinctly different to petroleum-derived diesel. This study concluded that Lasiodiplodia theobromae VBE1 lipases have potential for biodiesel production from coconut oil.

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

Similar content being viewed by others

References

  1. Sharma, R., Chisti, Y., & Banergee, V. C. (2002). Production, purification, characterization and applications of lipases. Biotechnology Advances, 19, 627–662.

    Article  CAS  Google Scholar 

  2. Su, F., Peng, C., Li, G. L., Xu, L., & Yan, Y. J. (2016). Biodiesel production from woody oil catalyzed by Candida rugosa lipase in ionic liquid. Renewable Energy, 90, 329–335. https://doi.org/10.1016/j.renene.2016.01.029.

    Article  CAS  Google Scholar 

  3. Norjannah, B., Ong, H. C., Masjuki, H. H., Juan, J. C., & Chong, W. T. (2016). Enzymatic transesterification for biodiesel production: a comprehensive review. RSC Advances, 6(65), 60034–60055. https://doi.org/10.1039/C6RA08062F.

    Article  CAS  Google Scholar 

  4. Souza, L. T. A., Oliveira, J. S., Rodrigues, M. Q. R. B., Santos, V. L. D., Pessela, B. C., & Resende, R. R. (2015). Macaúba (Acrocomia aculeata) cake from biodiesel processing: a low-cost substrate to produce lipases from Moniliella spathulata R25L270 with potential application in the oleochemical industry. Microbial Cell Factories, 14(1), 87. https://doi.org/10.1186/s12934-015-0266-9.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Treichel, H., de Oliveira, D., Mazutti, M. A., Di Luccio, M., & Oliveira, J. V. (2010). A review on microbial lipases production. Food and Bioprocess Technology, 3(2), 182–196. https://doi.org/10.1007/s11947-009-0202-2.

    Article  CAS  Google Scholar 

  6. Di Luccio, M., Capra, F., Ribeiro, N. P., Vargas, G. D. L. P., Freire, D. M. G., Denise, M. G., & De Oliveira, D. (2004). Effect of temperature, moisture, and carbon supplementation on lipase production by solid-state fermentation of soy cake by Penicillium simplicissimum. Applied Biochemistry and Biotechnology, 113(1-3), 173–180. https://doi.org/10.1385/ABAB:113:1-3:173.

    Article  PubMed  Google Scholar 

  7. Ul-Haq, I., Idrees, S., & Rajoka, M. I. (2002). Production of lipases by Rhizopus oligosporous by solid-state fermentation. Process Biochemistry, 37(6), 637–641. https://doi.org/10.1016/S0032-9592(01)00252-7.

    Article  CAS  Google Scholar 

  8. Bogar, B., Szak’acs, G., Pandey, A., Abdulhameed, S., Linden, J. C., & Tengerdy, R. P. (2003). Production of phytase by Mucor racemosus in solid-state fermentation. Biotechnology Progress, 19(2), 312–319. https://doi.org/10.1021/bp020126v.

    Article  CAS  PubMed  Google Scholar 

  9. Mahadik, N. D., Puntambekar, U. S., Bastawde, K. B., Khire, J. M., & Gokhale, D. V. (2002). Production of acidic lipase by Aspergillus niger in solid state fermentation. Process Biochemistry, 38(5), 715–721. https://doi.org/10.1016/S0032-9592(02)00194-2.

    Article  CAS  Google Scholar 

  10. Dominguez, A., Costas, M., Longo, M. A., & Sanroman, A. (2003). A novel application of solid state culture: production of lipases by Yarrowia lipolytica. Biotechnology Letters, 25(15), 1225–1229. https://doi.org/10.1023/A:1025068205961.

    Article  CAS  PubMed  Google Scholar 

  11. Venkatesagowda, B., Ponugupaty, E., Barbosa, A. M., & Dekker, R. F. H. (2012). Diversity of plant oil seed-associated fungi isolated from seven oil-bearing seeds and their potential for the production of lipolytic enzymes. World Journal of Microbiology and Biotechnology, 28(1), 71–80. https://doi.org/10.1007/s11274-011-0793-4.

    Article  CAS  PubMed  Google Scholar 

  12. Castilho, L. R., Polato, C. M. S., Baruque, E. A., Sant’Anna Jr., G. L., & Freire, D. M. G. (2000). Economic analysis of lipase production by Penicillium restrictum in solid-state and submerged fermentations. Biochemical Engineering Journal, 4(3), 239–247. https://doi.org/10.1016/S1369-703X(99)00052-2.

    Article  CAS  Google Scholar 

  13. Venkatesagowda, B., Ponugupaty, E., Barbosa, A. M., & Dekker, R. F. H. (2015). Solid-state fermentation of coconut kernel-cake as substrate for the production of lipases by the coconut kernel-associated fungus Lasiodiplodia theobromae VBE-1. Annals of Microbiology, 65(1), 129–142. https://doi.org/10.1007/s13213-014-0844-9.

    Article  CAS  Google Scholar 

  14. Kamini, N. R., Mala, J. G. S., & Puvanakrishnan, P. (1998). Lipase production from Aspergillus niger by solid-state fermentation using gingelly oil cake. Process Biochemistry, 33(5), 505–511. https://doi.org/10.1016/S0032-9592(98)00005-3.

    Article  CAS  Google Scholar 

  15. Mahanta, N., Gupta, A., & Khare, S. K. (2008). Production of protease and lipase by solvent tolerant Pseudomonas aeruginosa PseA in solid state fermentation using Jatropha curcas seed cake as substrate. Bioresource Technology, 99(6), 1729–1735. https://doi.org/10.1016/j.biortech.2007.03.046.

    Article  CAS  PubMed  Google Scholar 

  16. Sethi, B. K., Rout, J. R., Das, R., Nanda, P. K., & Sahoo, S. L. (2013). Lipase production by Aspergillus terreus using mustard seed oil cake as a carbon source. Annals of Microbiology, 63(1), 241–252. https://doi.org/10.1007/s13213-012-0467-y.

    Article  CAS  Google Scholar 

  17. Balaji, V., & Ebenezer, P. (2008). Optimization of extracellular lipase production in Colletotrichum gloeosporioides by solid state fermentation. Indian Journal of Science and Technology, 1, 1–8.

    Article  Google Scholar 

  18. Babu, I. S., & Rao, G. H. (2007). Lipase production by Yarrowia lipolytica NCIM 3589 in solid state fermentation using mixed substrate. Research Journal of Microbiology, 5, 469–474.

    Google Scholar 

  19. Benjamin, S., & Pandey, A. (1998). Mixed-solid substrate fermentation. A novel process for enhanced lipase production by Candida rugosa. Acta Biotechnologica, 18(4), 315–324. https://doi.org/10.1002/abio.370180405.

    Article  CAS  Google Scholar 

  20. Edwinoliver, N. G., Thirunavukarasu, K., Naidu, R. B., Gowthaman, M. K., Nakajima Kambe, T., & Kamini, N. R. (2010). Scale up of a novel tri-substrate fermentation for enhanced production of Aspergillus niger lipase for tallow hydrolysis. Bioresource Technology, 101(17), 6791–6796. https://doi.org/10.1016/j.biortech.2010.03.091.

    Article  CAS  PubMed  Google Scholar 

  21. Gutarra, M. L. E., Godoy, M. G., Castilho, L. R., & Freire, D. M. G. (2007). Inoculum strategies for Penicillium simplicissimum lipase production by solid-state fermentation using a residue from the babassu oil industry. Journal of Chemical Technology and Biotechnology, 82(3), 313–318. https://doi.org/10.1002/jctb.1674.

    Article  CAS  Google Scholar 

  22. Uranga, C. C., Ghassemian, M., & Hernandez-Martínez, R. (2017). Novel proteins from proteomic analysis of the trunk disease fungus Lasiodiplodia theobromae (Botryosphaeriaceae). Biochimie Open, 4, 88–98. https://doi.org/10.1016/j.biopen.2017.03.001.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Kumar, A., & Kanwar, S. S. (2012). Lipase production in solid-state fermentation (SSF): recent developments and biotechnological applications. Dynamic Biochemistry, Process Biotechnology and Molecular Biology, 6, 13–27.

    Google Scholar 

  24. Ito, K., Gomi, K., & Kariyama, M. (2013). Rapid enzyme production and mycelial growth in solid-state fermentation using the non-airflow box. Journal of Bioscience and Bioengineering, 116(5), 585–590. https://doi.org/10.1016/j.jbiosc.2013.04.024.

    Article  CAS  PubMed  Google Scholar 

  25. Ban, K., Kaieda, M., Matsumoto, T., Kondo, A., & Fukuda, H. (2001). Whole cell biocatalyst for biodiesel fuel production utilizing Rhizopus oryzae cells immobilized within biomass support particles. Biochemical Engineering Journal, 8(1), 39–43. https://doi.org/10.1016/S1369-703X(00)00133-9.

    Article  CAS  PubMed  Google Scholar 

  26. Pizarro, P. A. V., & Park, E. Y. (2003). Lipase-catalyzed production of biodiesel fuel from vegetable oils contained in waste activated bleaching earth. Process Biochemistry, 38(7), 1077–1082. https://doi.org/10.1016/S0032-9592(02)00241-8.

    Article  CAS  Google Scholar 

  27. Nurfitri, I., Maniam, G. P., Hindryawati, N., Yusoff, M. M., & Ganesan, S. (2013). Potential of feedstock and catalysts from waste in biodiesel preparation: a review. Energy Conversion and Management, 74, 395–402. https://doi.org/10.1016/j.enconman.2013.04.042.

    Article  CAS  Google Scholar 

  28. Dias, J. M., Araújo, J. M., Costa, J. F., Alvim-Ferraz, M. C. M., & Almeida, M. F. (2013). Biodiesel production from raw castor oil. Energy, 53, 58–66. https://doi.org/10.1016/j.energy.2013.02.018.

    Article  CAS  Google Scholar 

  29. Patil, P. D., & Deng, S. (2009). Optimization of biodiesel production from edible and non-edible vegetable oils. Fuel, 88(7), 1302–1306. https://doi.org/10.1016/j.fuel.2009.01.016.

    Article  CAS  Google Scholar 

  30. Alptekin, E., & Canakci, M. (2010). Optimization of pretreatment reaction for methyl ester production from chicken fat. Fuel, 89(12), 4035–4039. https://doi.org/10.1016/j.fuel.2010.04.031.

    Article  CAS  Google Scholar 

  31. Borowitzka, M. A., & Moheimani, N. R. (Eds.). (2013). Algae for Biofuels and Energy, developments in applied phycology 5 (p. 288). Dordrecht: Springer Science + Business Media. https://doi.org/10.1007/978-94-007-5479-9.

    Book  Google Scholar 

  32. Liu, Y., Li, C., Wang, S., & Chen, W. (2014). Solid-supported microorganism of Burkholderia cenocepacia cultured via solid-state fermentation for biodiesel production: optimization and kinetics. Applied Energy, 113, 713–721. https://doi.org/10.1016/j.apenergy.2013.08.009.

    Article  CAS  Google Scholar 

  33. Christopher, L. P., Kumar, V., & Zambare, V. P. (2012). Enzymatic biodiesel: Challenges and opportunities. Applied Energy, 119, 497–520.

    Article  CAS  Google Scholar 

  34. Akoh, C. C., Chang, S. W., Lee, G. C., & Shaw, J. F. (2007). Enzymatic approach to biodiesel production. Journal of Agricultural and Food Chemistry, 55(22), 8995–9005. https://doi.org/10.1021/jf071724y.

    Article  CAS  PubMed  Google Scholar 

  35. Korman, T. P., Sahachartsiri, B., & Charbonneau, D. M. (2013). Dieselzymes: development of a stable and methanol tolerant lipase for biodiesel production by directed evolution. Biotechnology for Biofuels, 6(1), 70. https://doi.org/10.1186/1754-6834-6-70.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Yang, K. S., Sung, B. H., Park, M. K., Lee, J. H., Lim, K. J., Park, S. C., Kim, S. J., Kim, H. K., Sohn, J.-H., Kim, H. M., & Kim, S. C. (2015). Recombinant lipase engineered with amphipathic and coiled-coil peptides. ACS Catalysis, 5(9), 5016–5025. https://doi.org/10.1021/cs502079g.

    Article  CAS  Google Scholar 

  37. Hobden, R. (2014). Commercializing enzymatic biodiesel production. Biodiesel Magazine, 11, 34–37.

    Google Scholar 

  38. Sommer, P., Bormann, C., & Gotz, F. (1997). Genetic and biochemical characterization of a new extracellular lipase from Streptomyces cinnamomeus. Applied and Environmental Microbiology, 63(9), 3553–3560.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Analytical Biochemistry, 72(1-2), 248–254. https://doi.org/10.1016/0003-2697(76)90527-3.

    Article  CAS  PubMed  Google Scholar 

  40. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680–685. https://doi.org/10.1038/227680a0.

    Article  CAS  PubMed  Google Scholar 

  41. Blum, H., Beier, H., & Gross, H. J. (1987). Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis, 8(2), 93–99. https://doi.org/10.1002/elps.1150080203.

    Article  CAS  Google Scholar 

  42. Davis, B. J. (1964). Disc electrophoresis II method and application to human serum proteins. Annals of the New York Academy of Sciences, 121, 404–423.

    Article  CAS  PubMed  Google Scholar 

  43. Brush, T. S., Chapman, R., Kurzman, R., & Williams, D. P. (1999). Purification and characterization of extracellular lipase from Ophiostoma piliferum. Bioorganic & Medicinal Chemistry, 10, 2131–2138.

    Article  Google Scholar 

  44. Benjamin, S., & Pandey, A. (2001). Isolation and characterization of three distinct forms of lipases from Candida rugosa produced in solid state fermentation. Brazilian Archives of Biology and Technology, 44(2), 213–221. https://doi.org/10.1590/S1516-89132001000200016.

    Article  CAS  Google Scholar 

  45. Mateos Diaz, J. C., Rodriguez, J. A., Roussos, S., Cordova, J., Abousalham, A., Carriere, F., & Baratti, J. (2006). Lipase from the thermotolerant fungus Rhizopus homothallicus is more thermostable when produced using solid state fermentation than liquid fermentation procedures. Enzyme and Microbial Technology, 39(5), 1042–1050. https://doi.org/10.1016/j.enzmictec.2006.02.005.

    Article  CAS  Google Scholar 

  46. Lima, V. M. G., Krieger, N., Mitchell, D. A., & Fontana, J. D. (2004). Activity and stability of a crude lipase from Penicillium aurantiogriseum in aqueous media and organic solvents. Biochemical Engineering Journal, 18(1), 65–71. https://doi.org/10.1016/S1369-703X(03)00165-7.

    Article  CAS  Google Scholar 

  47. Saxena, R. K., Davidson, W. S., Sheoran, A., & Giri, B. (2003). Purification and characterization of an alkaline thermostable lipase from Aspergillus carneus. Process Biochemistry, 39(2), 239–247. https://doi.org/10.1016/S0032-9592(03)00068-2.

    Article  CAS  Google Scholar 

  48. Heerden, E. V., Litthaue, D., & Verger, R. (2002). Biochemical characterization and kinetic properties of a purified lipase from Aspergillus niger in bulk phase and monomolecular films. Enzyme and Microbial Technology, 30(7), 902–909. https://doi.org/10.1016/S0141-0229(02)00031-5.

    Article  Google Scholar 

  49. Shu, C.-H., Xu, C.-H., & Lin, G.-C. (2006). Purification and partial characterization of a lipase from Antrodia cinnamomea. Process Biochemistry, 41(3), 734–738. https://doi.org/10.1016/j.procbio.2005.09.007.

    Article  CAS  Google Scholar 

  50. Mehta, A., Saun, N. K., & Gupta, R. (2016). Purification and characterization of lipase from thermophilic Geobacillus sp. Current Biotechnology, 5(1), 81–89. https://doi.org/10.2174/2211550105666151222183051.

    Article  CAS  Google Scholar 

  51. da Silva, V. C. F., Contesini, F. J., & Carvalho, P. D. O. (2008). Characterization and catalytic activity of free and immobilized lipase from Aspergillus niger: a comparative study. Journal of the Brazilian Chemical Society, 19(8), 1468–1474. https://doi.org/10.1590/S0103-50532008000800005.

    Article  Google Scholar 

  52. Hiol, A., Jonzo, M. D., & Comeau, L. (1999). Production, purification and characterization of an extracellular lipase from Mucor hiemalis f. hiemalis. Enzyme and Microbial Technology, 25(1-2), 80–87. https://doi.org/10.1016/S0141-0229(99)00009-5.

    Article  CAS  Google Scholar 

  53. Abbas, H., Hiol, A., Deyris, V., & Comeau, L. (2002). Isolation and characterization of an extracellular lipase from Mucor sp. strain isolated from palm fruit. Enzyme and Microbial Technology, 31(7), 968–975. https://doi.org/10.1016/S0141-0229(02)00190-4.

    Article  CAS  Google Scholar 

  54. Benjamin, S., & Pandey, A. (2000). Isolation and characterization of three distinct forms of lipases from Candida rugosa produced in solid state fermentation. Brazilian Archives of Biology and Technology, 43(5), 453–460. https://doi.org/10.1590/S1516-89132000000500002.

    Article  CAS  Google Scholar 

  55. Sharma, S., & Kanwar, S. S. (2014). Organic solvent tolerant lipases and applications. The Scientific World Journal, article ID, 625258, 15.

    Google Scholar 

  56. Kumar, A., Dhar, K., Kanwar, S. S., & Arora, P. K. (2016). Lipase catalysis in organic solvents: advantages and applications. Biological Procedures Online, 18(1), 2. https://doi.org/10.1186/s12575-016-0033-2.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  57. Ribeiro, B. D., de Castro, A. M., Coelho, M. A. Z., & Freire, D. M. G. (2011). Production and use of lipases in bioenergy: a review from the feedstocks to biodiesel production. Enzyme Research, 2011; ID 615803, 16.

    Google Scholar 

  58. Nakpong, P., & Wootthikanokkhan, S. (2010). High free fatty acid coconut oil as a potential feedstock for biodiesel production in Thailand. Renewable Energy, 35(8), 1682–1687. https://doi.org/10.1016/j.renene.2009.12.004.

    Article  CAS  Google Scholar 

  59. Tupufia, S. C., Jeon, Y. J., Marquis, C., Adesina, A. A., & Rogers, P. L. (2013). Enzymatic conversion of coconut oil for biodiesel production. Fuel Processing Technology, 106, 721–726. https://doi.org/10.1016/j.fuproc.2012.10.007.

    Article  CAS  Google Scholar 

  60. Winayanuwattikun, P., Kaewpiboon, C., Piriyakananon, K., Tantong, S., Thakernkarnkit, W., Chulalaksananukul, W., & Yongvanich, T. (2008). Potential plant oil feedstock for lipase-catalyzed biodiesel production in Thailand. Biomass and Bioenergy, 32(12), 1279–1286. https://doi.org/10.1016/j.biombioe.2008.03.006.

    Article  CAS  Google Scholar 

  61. Lafont, J. J., Espitia, A. A., & Sodre, J. R. (2015). Potential vegetable oils sources for biodiesel production: cashew, coconut and cotton. Material Renewable Sustainable Energy, March, 4(1), 1. https://doi.org/10.1007/s40243-014-0041-6.

    Article  Google Scholar 

  62. Giakoumis, E. G. (2013). A statistical investigation of biodiesel physical and chemical properties, and their correlation with the degree of unsaturation. Renewable Energy, 50, 858–878. https://doi.org/10.1016/j.renene.2012.07.040.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors acknowledge the Department of Chemistry, University of Madras and Indian Institute of Technology (IIT) Madras, and Central Leather Research Institute (CLRI), Adyar, Chennai, Tamil Nadu, India, for analysis of the biodiesel samples from coconut oil and commercial diesel. Professor R.F.H. Dekker gratefully acknowledges Universidade Tecnológica Federal do Paraná, Londrina-PR (Brazil) for support during the writing of the paper.

Funding

The University Grants Commission—Major Research Project at the University of Madras, INDIA, is acknowledged for financial support.

Author information

Authors and Affiliations

  1. Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai, Tamil Nadu, 600 025, India

    Balaji Venkatesagowda & Ebenezer Ponugupaty

  2. Departamento de Química, Centro de Ciências Exatas, Universidade Estadual de Londrina, Londrina, PR, CEP 86057-970, Brazil

    Aneli M. Barbosa-Dekker

  3. Programa de Pós-Graduação em Engenharia Ambiental, Universidade Tecnológica Federal do Paraná, Câmpus Londrina, Londrina, PR, CEP 86036-370, Brazil

    Robert F. H. Dekker

Authors
  1. Balaji Venkatesagowda
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ebenezer Ponugupaty
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Aneli M. Barbosa-Dekker
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Robert F. H. Dekker
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Balaji Venkatesagowda or Robert F. H. Dekker.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Venkatesagowda, B., Ponugupaty, E., Barbosa-Dekker, A.M. et al. The Purification and Characterization of Lipases from Lasiodiplodia theobromae, and Their Immobilization and Use for Biodiesel Production from Coconut Oil. Appl Biochem Biotechnol 185, 619–640 (2018). https://doi.org/10.1007/s12010-017-2670-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-017-2670-6

Keywords

Search

Navigation