Enzyme-Catalyzed Production of FAME by Hydroesterification of Soybean Oil Using the Novel Soluble Lipase NS 40116

  • Daniela V. Rosset
  • João H. C. Wancura
  • Gustavo A. Ugalde
  • J. Vladimir Oliveira
  • Marcus V. TresEmail author
  • Raquel C. Kuhn
  • Sérgio L. Jahn


The performance of lipase NS 40116, a novel and promising soluble enzyme obtained from modified Thermomyces lanuginosus microorganism, was investigated in the production of biodiesel (fatty acid methyl esters—FAME) by hydroesterification. In order to investigate the potential of the biocatalyst in its soluble form, this work reports the effect of water content and enzyme load, as well as the recovery and reuse of the biocatalyst. A FAME yield of 94.30% after 12 h was achieved at 35 °C by combining 0.50 wt% of lipase, 15 wt% of water, and a methanol:oil molar ratio of 4.5:1. The analysis of the time course reaction suggests that the triacylglycerides (TAGs) are hydrolyzed by the enzyme in a first step, generating free fatty acids (FFAs), followed by the esterification of these FFAs into FAME. In relation to the reusability assays, the lipase kept approximately 90% of its catalytic activity after five cycles of reuse. In this context, the findings of this study demonstrate that lipase NS 40116 can efficiently catalyze hydroesterification reactions under mild conditions, arising as a competitive alternative for biodiesel synthesis.


FAME NS 40116 lipase Biodiesel production Hydroesterification Lipase reuse 



Acid value


Fourier transform infrared


Fatty acid methyl esters


Free fatty acids


Potassium hydroxide




Funding Information

This work received financial support from the National Council of Technological and Scientific Development (CNPq) and the Research Support Foundation of the State of Rio Grande do Sul (FAPERGS) throughout this research, as well the Coordination for the Improvement of Higher Education Personnel (CAPES) for scholarships.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. 1.
    Christopher, L. P., Hemanathan, K., & Zambare, V. P. (2014). Enzymatic biodiesel: challenges and opportunities. Applied Energy, 119(15), 497–520.CrossRefGoogle Scholar
  2. 2.
    Cesarini, S., Diaz, P., & Nielsen, P. M. (2013). Exploring a new, soluble lipase for FAMEs production in water-containing systems using crude soybean oil as a feedstock. Process Biochemistry, 48(3), 484–487.CrossRefGoogle Scholar
  3. 3.
    Ferreira-Leitão, V., Cammarota, M., Gonçalves Aguieiras, E., Vasconcelos de Sá, L., Fernandez-Lafuente, R., & Freire, D. (2017). The protagonism of biocatalysis in green chemistry and its environmental benefits. Catalysts, 7(1), 9.CrossRefGoogle Scholar
  4. 4.
    Trentin, C. M., Scherer, R. P., Dalla Rosa, C., Treichel, H., Oliveira, D., & Oliveira, J. V. (2014). Continuous lipase-catalyzed esterification of soybean fatty acids under ultrasound irradiation. Bioprocess and Biosystems Engineering, 37(5), 841–847.CrossRefGoogle Scholar
  5. 5.
    Remonatto, D., Santin, C. M. T., de Oliveira, D., Di Luccio, M., & de Oliveira, J. V. (2016). FAME production from waste oils through commercial soluble lipase Eversa® catalysis. Industrial Biotechnology, 12(4), 254–262.CrossRefGoogle Scholar
  6. 6.
    Ognjanovic, N., Bezbradica, D., & Knezevic-Jugovic, Z. (2009). Enzymatic conversion of sunflower oil to biodiesel in a solvent-free system: process optimization and the immobilized system stability. Bioresource Technology, 100(21), 5146–5154.CrossRefGoogle Scholar
  7. 7.
    Baskar, G., & Aiswarya, R. (2016). Trends in catalytic production of biodiesel from various feedstocks. Renewable and Sustainable Energy Reviews, 57, 496–504.CrossRefGoogle Scholar
  8. 8.
    Zeng, L., He, Y., Jiao, L., Li, K., & Yan, Y. (2017). Preparation of biodiesel with liquid synergetic lipases from rapeseed oil deodorizer distillate. Applied Biochemistry and Biotechnology, 183(3), 778–791.CrossRefGoogle Scholar
  9. 9.
    Hama, S., & Kondo, A. (2013). Enzymatic biodiesel production: an overview of potential feedstocks and process development. Bioresource Technology, 135, 386–395.CrossRefGoogle Scholar
  10. 10.
    Brady, D., & Jordaan, J. (2009). Advances in enzyme immobilisation. Biotechnology Letters, 31(11), 1639–1650.CrossRefGoogle Scholar
  11. 11.
    Manoel, E. A., dos Santos, J. C. S., Freire, D. M. G., Rueda, N., & Fernandez-Lafuente, R. (2015). Immobilization of lipases on hydrophobic supports involves the open form of the enzyme. Enzyme and Microbial Technology, 71, 53–57.CrossRefGoogle Scholar
  12. 12.
    Poppe, J. K., Matte, C. R., Fernandez-Lafuente, R., Rodrigues, R. C., & Ayub, M. A. Z. (2018). Transesterification of waste frying oil and soybean oil by combi-lipases under ultrasound-assisted reactions. Applied Biochemistry and Biotechnology, 186(3), 576–589.CrossRefGoogle Scholar
  13. 13.
    Aguieiras, E. C. G., Ribeiro, D. S., Couteiro, P. P., Bastos, C. M. B., de Queiroz, D. S., Parreira, J. M., & Langone, M. A. P. (2016). Investigation of the reuse of immobilized lipases in biodiesel synthesis: influence of different solvents in lipase activity. Applied Biochemistry and Biotechnology, 179(3), 485–496.CrossRefGoogle Scholar
  14. 14.
    Nordblad, M., Silva, V. T. L., Nielsen, P. M., & Woodley, J. M. (2014). Identification of critical parameters in liquid enzyme-catalyzed biodiesel production. Biotechnology and Bioengineering, 111(12), 2446–2453.CrossRefGoogle Scholar
  15. 15.
    Wancura, J. H. C., Rosset, D. V., Brondani, M., Mazutti, M. A., Oliveira, J. V., Tres, M. V., & Jahn, S. L. (2018). Soluble lipase-catalyzed synthesis of methyl esters using a blend of edible and nonedible raw materials. Bioprocess and Biosystems Engineering, 41(8), 1185–1193.CrossRefGoogle Scholar
  16. 16.
    Lotti, M., Pleiss, J., Valero, F., & Ferrer, P. (2015). Effects of methanol on lipases: molecular, kinetic and process issues in the production of biodiesel. Biotechnology Journal, 10(1), 22–30.CrossRefGoogle Scholar
  17. 17.
    Vieitez, I., da Silva, C., Alckmin, I., Borges, G. R., Corazza, F. C., Oliveira, J. V., Grompone, M. A., & Jachmanián, I. (2010). Continuous catalyst-free methanolysis and ethanolysis of soybean oil under supercritical alcohol/water mixtures. Renewable Energy, 35(9), 1976–1981.CrossRefGoogle Scholar
  18. 18.
    Pedersen, A. T., Nordblad, M., Nielsen, P. M., & Woodley, J. M. (2014). Batch production of FAEE-biodiesel using a liquid lipase formulation. Journal of Molecular Catalysis B: Enzymatic, 105, 89–94.CrossRefGoogle Scholar
  19. 19.
    Lam, M. K., Lee, K. T., & Mohamed, A. R. (2010). Homogeneous, heterogeneous and enzymatic catalysis for transesterification of high free fatty acid oil (waste cooking oil) to biodiesel: a review. Biotechnology Advances, 28(4), 500–518.CrossRefGoogle Scholar
  20. 20.
    Facin, B. R., Valério, A., Bresolin, D., Centenaro, G., de Oliveira, D., & Oliveira, J. V. (2018). Improving reuse cycles of Thermomyces lanuginosus lipase (NS-40116) by immobilization in flexible polyurethane. Biocatalysis and Biotransformation, 36(5), 372–380.CrossRefGoogle Scholar
  21. 21.
    Price, J., Nordblad, M., Martel, H. H., Chrabas, B., Wang, H., Nielsen, P. M., & Woodley, J. M. (2016). Scale-up of industrial biodiesel production to 40 m3 using a liquid lipase formulation. Biotechnology and Bioengineering, 113(8), 1719–1728.CrossRefGoogle Scholar
  22. 22.
    Li, N., Ding, M.-H. Z., & Ma, D. (2009). Unexpected reversal of the regioselectivity in Thermomyces lanuginosus lipase-catalyzed acylation of floxuridine. Biotechnology Letters, 31(8), 1241–1244.CrossRefGoogle Scholar
  23. 23.
    Wancura, J. H. C., Rosset, D. V., Ugalde, G. A., de Oliveira, J. V., Mazutti, M. A., Tres, M. V., & Jahn, S. L. (2018). Feeding strategies of methanol and lipase on Eversa® transform-mediated hydroesterification for FAME production. The Canadian Journal of Chemical Engineering.
  24. 24.
    Standard UNE-EN 14103 Fat and oil derivates—fatty acid methyl esters (FAME)—determination of ester and linolenic methyl ester contents 2011. Available from: Accessed Nov 10, 2018.
  25. 25.
    Rabelo, S. N., Ferraz, V. P., Oliveira, L. S., & Franca, A. S. (2015). FTIR analysis for quantification of fatty acid methyl esters in biodiesel produced by microwave-assisted transesterification. International Journal of Environmental Science and Development, 6(12), 964–969.CrossRefGoogle Scholar
  26. 26.
    Mahamuni, N. N., & Adewuyi, Y. G. (2009). Fourier transform infrared spectroscopy (FTIR) method to monitor soy biodiesel and soybean oil in transesterification reactions, petrodiesel-biodiesel blends, and blend adulteration with soy oil. Energy and Fuels, 23(7), 3773–3782.CrossRefGoogle Scholar
  27. 27.
    Siatis, N. G., Kimbaris, a. C., Pappas, C. S., Tarantilis, P. a., & Polissiou, M. G. (2006). Improvement of biodiesel production based on the application of ultrasound: monitoring of the procedure by FTIR spectroscopy. Journal of the American Oil Chemists’ Society, 83(1), 53–57.CrossRefGoogle Scholar
  28. 28.
    Soares, I. P., Rezende, T. F., Silva, R. C., Castro, E. V. R., & Fortes, I. C. P. (2008). Multivariate calibration by variable selection for blends of raw soybean oil/biodiesel from different sources using Fourier transform infrared spectroscopy (FTIR) spectra data. Energy and Fuels, 22(3), 2079–2083.CrossRefGoogle Scholar
  29. 29.
    Jaeger, K. E., & Reetz, M. T. (1998). Microbial lipases form versatile tools for biotechnology. Trends in Biotechnology, 16(9), 396–403.CrossRefGoogle Scholar
  30. 30.
    Chen, X., Du, W., & Liu, D. (2008). Effect of several factors on soluble lipase-mediated biodiesel preparation in the biphasic aqueous-oil systems. World Journal of Microbiology and Biotechnology, 24(10), 2097–2102.CrossRefGoogle Scholar
  31. 31.
    Lv, L., Dai, L., Du, W., & Liu, D. (2017). Effect of water on lipase NS81006-catalyzed alcoholysis for biodiesel production. Process Biochemistry, 58(February), 239–244.CrossRefGoogle Scholar
  32. 32.
    Nielsen, P. M., Rancke-Madsen, A., Holm, H. C., & Burton, R. (2016). Production of biodiesel using liquid lipase formulations. Journal of the American Oil Chemists’ Society, 93(7), 3–8.CrossRefGoogle Scholar
  33. 33.
    Firdaus, M. Y., Brask, J., Nielsen, P. M., Guo, Z., & Fedosov, S. (2016). Kinetic model of biodiesel production catalyzed by free liquid lipase from Thermomyces lanuginosus. Journal of Molecular Catalysis B: Enzymatic, 133, 55–64.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Chemical EngineeringFederal University of Santa MariaSanta MariaBrazil
  2. 2.Department of Crop ProtectionFederal University of Santa MariaSanta MariaBrazil
  3. 3.Department of Chemical and Food EngineeringFederal University of Santa CatarinaFlorianopolisBrazil
  4. 4.Laboratory of Agroindustrial Processes Engineering (LAPE)Federal University of Santa MariaCachoeira do SulBrazil

Personalised recommendations