Bio25388 Vol. 24 No. 1 (2025)

Vol. 24, No. 1 (2025), Bio25388 https://doi.org/10.24275/rmiq/Bio25388

Effect of temperature on the kinetics of the enzymatic esterification of n-3 polyunsaturated fatty acids and glycerol

Authors

C. Correa-Leyva, M. Pérez-Tello, A.R. Martin-Garcia1, H.S. García, J.A. Noriega-Rodríguez

Abstract

In this work, the effect of temperature on the kinetics of esterification of polyunsaturated fatty acids (PUFA) and glycerol catalyzed by Candida antarctica lipase was studied for the production of structured acylglycerols in a batch reactor. A concentrate of n-3 PUFA was prepared by chemical hydrolysis of Menhaden oil followed by urea precipitation. Experiments were conducted at 40, 50, and 60°C using the stoichiometric ratio of 3 mol of PUFA per mol of glycerol. At 60°C the overall esterification yield was 51.8%. The activation energy was estimated in 28.611 kJ mol-1. To describe the overall esterification kinetics, a Michaelis-Menten model with reversible effects was used. Esterification kinetics of the structured acylglycerols obeyed a kinetic model based on the ordered-sequential multi-product multi-substrate mechanism. The kinetic parameters of both models were obtained by a non-linear regression algorithm using the MATLAB® software (R2018b). The average coefficient of determination of the models was R_avg^2=0.93.

Keywords

acylglycerols, enzymatic esterification, kinetic parameters, n-3 PUFA.

References
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Prog. 29 (6), 1422–1429. https://doi.org/10.1002/btpr.1806 Bousquet, M. P., Willemot, R. M., Monsan, P. & Boures, E. (2000). Enzymatic Synthesis of α-Butylglucoside Linoleate in a Packed Bed Reactor for Future Pilot Scale-up. Biotechnol. Prog. 16 (4), 589–594. https://doi.org/10.1021/bp000043 Calder, P. C. (2013). Omega-3 polyunsaturated fatty acids and inflammatory processes: nutrition or pharmacology? Br. J. Clin. Pharmacol. 75 (3), 645–662. https://doi.org/10.1111/j.1365-2125.2012.04374.x Castejón, N. & Señoráns, F. J. (2019). Strategies for Enzymatic Synthesis of Omega-3 Structured Triacylglycerols from Camelina sativa oil enriched in EPA and DHA. Eur. J. Lipid Sci. Technol. 121, 1800412. https://doi.org/10.1002/ejlt.201800412 Castellanos T, L. & Rodriguez D, M. (2015). The effect of omega 3 in human health and considerations to its intake. Rev Chil. Nutr. 42 (1), 90–95. http://dx.doi.org/10.4067/S0717-75182015000100012 Cheirsilp, B., Kaewthong, W. & H-Kittikun, A. (2007). Kinetic study of glycerolysis of palm olein for monoacylglycerol production by immobilized lipase. Biochem. Eng. J. 35 (1), 71–80. https://doi.org/10.1016/j.bej.2006.12.024 Correa, C., Tejeda, A., Martin, A. R., García, H. S. & Noriega, J. A. (2017) Kinetics of the enzymatic esterification of n-3 polyunsaturated fatty acids to glycerol: multi-substrate multi-product ping-pong mechanism. Rev. Mex. Ing. Quim. 16 (3), 805-812. http://bit.ly/3lXH3Sp Du, B., Zielinski, D. C., Kavvas, E. S., Dräger, A., Tan, J., Zhang, Z., Ruggiero, K., Arzumanyan, G. & Palsson, B. O. (2016). Evaluation of rate law approximations in bottom-up kinetic models of metabolism. BMC Syst. Biol. 10 (1), 1-15. https://doi.org/10.1186/s12918-016-0283-2 González, J., Moreno, V. R. & del Monte, A. (2010). Lipases: enzymes having the potential for developing immobilised biocatalysts by interfacial adsorption. Rev. Colomb. Biotecnol. 12 (1), 113-140. http://bit.ly/3KtA3Hc Hallahan, B., Ryan, T., Hibbeln, J. R., Murray, I. T., Glynn, S., Ramsden, C. E., SanGiovanni J. P. & Davis, J. M. (2016). Efficacy of omega-3 highly unsaturated fatty acids in the treatment of depression. Br. J. Psychiatry. 209 (03), 192–201. https://doi.org/10.1192/bjp.bp.114.160242 He, Y. & Shahidi, F. (1997). Enzymatic esterification of θ-3 fatty acid concentrates from seal blubber oil with glycerol. J Am Oil Chem Soc. 74 (9), 1133–1136. https://doi.org/10.1007/s11746-997-0036-x Hernández, M. C., Morales P, J., Valenzuela B, R., Morales I, G. & Valenzuela B, A. (2016). Benefits of n-3 long-chain polyunsaturated fatty acids in non-alcoholic fatty liver disease. Rev Chil Nutr. 43 (2), 196–205. http://dx.doi.org/10.4067/S0717-75182016000200013 Jamie, A., Alshami, A., Maliabari, Z.O. & Ateih, M.A. (2017). Development and validation of a kinetic model for enzymatic hydrolysis using Candida rugosa lipase. J Bioprocess Biotech. 7 (1), 1-7. DOI: https://doi.org/10.4172/2155-9821.1000297 Khan, I., Hussain, M., Jiang, B., Zheng, L., Pan, Y., Hu, J., Khan, A., Ashraf, A. & Zou, X. (2023). Omega-3 long-chain polyunsaturated fatty acids: Metabolism and health implications. Progress in Lipid Research, 101255. https://doi.org/10.1016/j.plipres.2023.101255 Kołodziej, Ł., Czarny, P. L., Ziółkowska, S., Białek, K., Szemraj, J., Gałecki, P., Su, K. P. & Śliwiński, T. (2023). How fish consumption prevents the development of Major Depressive Disorder? A comprehensive review of the interplay between n-3 PUFAs, LTP and BDNF. Progress in Lipid Research, 101254. https://doi.org/10.1016/j.plipres.2023.101254 Mbatia, B., Mattiasson, B., Mulaa, F. & Adlercreutz, P. (2011). Strategies for the enzymatic enrichment of PUFA from fish oil. Eur. J. Lipid Sci. Technol. 113 (6), 717–723. https://doi.org/10.1002/ejlt.201000560 Noriega, J. A., Carrillo, E., Gamez, N., Medina, L. A., Baeza, R. & Garcia, H. S. (2013). Optimization of the lipase catalyzed production of structured acylglycerols with polyunsaturated fatty acids isolated from sardine oil. J. Food Res. 2 (6), 97. http://dx.doi.org/10.5539/jfr.v2n6p97 Pino, F. J. & Noriega, J. A. (2011). Análisis de Clases de Lípidos por Cromatografía de Capa Fina de Alta Resolución. Revista Invurnus. 6 (2), 38-43. Piñeiro, G., Lago, N. & Culebras, J. M. (2013). Role of omega-3 fatty acids in cardiovascular disease prevention. Review. Nutr Hosp. 28 (1), 1-5. https://doi.org/10.3305/nh.2013.28.1.6312 Rivero, F., Padial, M., Guadix, E. M. & Morales, R. (2020). Novozyme 435 and Lipozyme RM IM Preferably Esterify Polyunsaturated Fatty Acids at the sn‐2 Position. Eur. J. Lipid Sci. Technol. 122 (10), 2000115. https://doi.org/10.1002/ejlt.202000115 Schneider, C. A., Rasband, W. S. & Eliceiri, K. W., NIH Image to ImageJ: 25 years of image analysis. Nat. Methods. 2012, 9 (7), 671–675. doi: https://doi.org/10.1038/nmeth.2089 Shahidi, F. & Ambigaipalan, P. (2018). Omega-3 Polyunsaturated Fatty Acids and Their Health Benefits. Annu Rev Food Sci Technol. 9 (1), 345–381. https://doi.org/10.1146/annurev-food-111317-095850 Thuy, N. M., Giau, T. N., Tai, N. V. & Minh, V. Q. (2023). Drying kinetics and mathematical modeling of dried macaroni supplemented with Gac aril. Rev. Mex. Ing. Quim, 22 (3), 1-8. https://doi.org/10.24275/rmiq/Alim23103 Valle, P. A., Salazar, Y., Soto-Cruz, N. O., Páez-Lerma, J. B., Coria, L. N., Núñez-Guerrero, M. E., Rodriguez-Herrera R. & Herrera, L. D. (2024) Modelling and analysis on the ethanol production by the Torulaspora delbrueckii yeast. Rev. Mex. Ing. Quim. 23 (3), 1-10. https://doi.org/10.24275/rmiq/Sim24312 Von der Haar, D., Stäbler, A., Wichmann, R. & Schweiggert-Weisz, U. (2014). Enzymatic esterification of free fatty acids in vegetable oils utilizing different immobilized lipases. Biotechnol. Lett. 37 (1), 169–174. https://doi.org/10.1007/s10529-014-1668-1 Watanabe, Y., Yamauchi-Sato, Y., Nagao, T., Negishi, S., Terai, T., Kobayashi, T. & Shimada, Y. (2005). Production of MAG of CLA by esterification with dehydration at ordinary temperature using Penicillium camembertii lipase. J. Am. Oil Chem. Soc. 82 (9), 619–623. https://doi.org/10.1007/s11746-005-1119-9 Xie, D., Chen, Y., Yu, J., Yang, Z., Wang, X. & Wang, X. (2023). Progress in enrichment of n-3 polyunsaturated fatty acid: A review. Critical Reviews in Food Science and Nutrition, 63 (32), 11310-11326. https://doi.org/10.1080/10408398.2022.2086852 Zhang, Y., Di, X., Wang, W., Song, M., Yu, Q., Wang, Z., Yuan, Z., Chen, X., Xu, H. & Guo, Y. (2020). Kinetic study of lipase-catalyzed esterification of furoic acid to methyl-2-furoate. Biochem. Eng. J. 161, 107587. https://doi.org/10.1016/j.bej.2020.107587 Zhao, Y., Liu, J., Deng, L., Wang, F. & Tan, T. (2011). Optimization of Candida sp. 99-125 lipase catalyzed esterification for synthesis of monoglyceride and diglyceride in solvent-free system. J. Mol. Catal. 72 (3-4), 157-162. https://doi.org/10.1016/j.molcatb.2011.05.014 Zhu, Y., Feng, Y., Wang, J., Yuan, Z., Miao, Y., Miao, T., Gao, B. & Zhang, L. (2024). Selective Esterification Design of Lipases for TAG Synthesis Based on the Unique Structure of Curved DHA. ACS Food Science & Technology, 4 (7), 1722-1730. https://doi.org/10.1021/acsfoodscitech.4c00174

References

Albalate-Ramírez, A., Alcalá-Rodríguez, M. M., Miramontes-Martínez, L. R., Estrada-Baltazar, A., Galván-Arzola, U., López-Hernández, B. N., Morones-Ramírez J. R. & Rivas-García, P. (2023). The Importance of Substrate Formulation on the Hydrolysis Process in Anaerobic Digestion: A Numerical and Experimental Study. Rev. Mex. Ing. Quim. 22 (2), 1-10. https://doi.org/10.24275/rmiq/Bio239
Baeza, R., López, L. X. & García, H. S. (2014). Biocatalytic modification of food lipids: reactions and applications. Rev. Mex. Ing. Quim. 2014, 13 (1), 29-47. http://bit.ly/3XSCS7J

Bisswanger, H. (2017). Enzyme Kinetics: Principles and Methods, John Wiley & Sons, Germany, pp. 80-82.

Bornadel, A., Åkerman, C. O., Adlercreutz, P., Hatti-Kaul, R. & Borg, N. (2013). Kinetic modeling of lipase-catalyzed esterification reaction between oleic acid and trimethylolpropane: A simplified model for multi-substrate multi-product ping-pong mechanisms. Biotechnol. Prog. 29 (6), 1422–1429. https://doi.org/10.1002/btpr.1806

Bousquet, M. P., Willemot, R. M., Monsan, P. & Boures, E. (2000). Enzymatic Synthesis of α-Butylglucoside Linoleate in a Packed Bed Reactor for Future Pilot Scale-up. Biotechnol. Prog. 16 (4), 589–594. https://doi.org/10.1021/bp000043

Calder, P. C. (2013). Omega-3 polyunsaturated fatty acids and inflammatory processes: nutrition or pharmacology? Br. J. Clin. Pharmacol. 75 (3), 645–662. https://doi.org/10.1111/j.1365-2125.2012.04374.x

Castejón, N. & Señoráns, F. J. (2019). Strategies for Enzymatic Synthesis of Omega-3 Structured Triacylglycerols from Camelina sativa oil enriched in EPA and DHA. Eur. J. Lipid Sci. Technol. 121, 1800412. https://doi.org/10.1002/ejlt.201800412

Castellanos T, L. & Rodriguez D, M. (2015). The effect of omega 3 in human health and considerations to its intake. Rev Chil. Nutr. 42 (1), 90–95. http://dx.doi.org/10.4067/S0717-75182015000100012
Cheirsilp, B., Kaewthong, W. & H-Kittikun, A. (2007). Kinetic study of glycerolysis of palm olein for monoacylglycerol production by immobilized lipase. Biochem. Eng. J. 35 (1), 71–80. https://doi.org/10.1016/j.bej.2006.12.024

Correa, C., Tejeda, A., Martin, A. R., García, H. S. & Noriega, J. A. (2017) Kinetics of the enzymatic esterification of n-3 polyunsaturated fatty acids to glycerol: multi-substrate multi-product ping-pong mechanism. Rev. Mex. Ing. Quim. 16 (3), 805-812. http://bit.ly/3lXH3Sp

Du, B., Zielinski, D. C., Kavvas, E. S., Dräger, A., Tan, J., Zhang, Z., Ruggiero, K., Arzumanyan, G. & Palsson, B. O. (2016). Evaluation of rate law approximations in bottom-up kinetic models of metabolism. BMC Syst. Biol. 10 (1), 1-15. https://doi.org/10.1186/s12918-016-0283-2

González, J., Moreno, V. R. & del Monte, A. (2010). Lipases: enzymes having the potential for developing immobilised biocatalysts by interfacial adsorption. Rev. Colomb. Biotecnol. 12 (1), 113-140. http://bit.ly/3KtA3Hc

Hallahan, B., Ryan, T., Hibbeln, J. R., Murray, I. T., Glynn, S., Ramsden, C. E., SanGiovanni J. P. & Davis, J. M. (2016). Efficacy of omega-3 highly unsaturated fatty acids in the treatment of depression. Br. J. Psychiatry. 209 (03), 192–201. https://doi.org/10.1192/bjp.bp.114.160242

He, Y. & Shahidi, F. (1997). Enzymatic esterification of θ-3 fatty acid concentrates from seal blubber oil with glycerol. J Am Oil Chem Soc. 74 (9), 1133–1136. https://doi.org/10.1007/s11746-997-0036-x

Hernández, M. C., Morales P, J., Valenzuela B, R., Morales I, G. & Valenzuela B, A. (2016). Benefits of n-3 long-chain polyunsaturated fatty acids in non-alcoholic fatty liver disease. Rev Chil Nutr. 43 (2), 196–205. http://dx.doi.org/10.4067/S0717-75182016000200013
Jamie, A., Alshami, A., Maliabari, Z.O. & Ateih, M.A. (2017). Development and validation of a kinetic model for enzymatic hydrolysis using Candida rugosa lipase. J Bioprocess Biotech. 7 (1), 1-7. DOI: https://doi.org/10.4172/2155-9821.1000297
Khan, I., Hussain, M., Jiang, B., Zheng, L., Pan, Y., Hu, J., Khan, A., Ashraf, A. & Zou, X. (2023). Omega-3 long-chain polyunsaturated fatty acids: Metabolism and health implications. Progress in Lipid Research, 101255. https://doi.org/10.1016/j.plipres.2023.101255

Kołodziej, Ł., Czarny, P. L., Ziółkowska, S., Białek, K., Szemraj, J., Gałecki, P., Su, K. P. & Śliwiński, T. (2023). How fish consumption prevents the development of Major Depressive Disorder? A comprehensive review of the interplay between n-3 PUFAs, LTP and BDNF. Progress in Lipid Research, 101254. https://doi.org/10.1016/j.plipres.2023.101254

Mbatia, B., Mattiasson, B., Mulaa, F. & Adlercreutz, P. (2011). Strategies for the enzymatic enrichment of PUFA from fish oil. Eur. J. Lipid Sci. Technol. 113 (6), 717–723. https://doi.org/10.1002/ejlt.201000560

Noriega, J. A., Carrillo, E., Gamez, N., Medina, L. A., Baeza, R. & Garcia, H. S. (2013). Optimization of the lipase catalyzed production of structured acylglycerols with polyunsaturated fatty acids isolated from sardine oil. J. Food Res. 2 (6), 97. http://dx.doi.org/10.5539/jfr.v2n6p97

Pino, F. J. & Noriega, J. A. (2011). Análisis de Clases de Lípidos por Cromatografía de Capa Fina de Alta Resolución. Revista Invurnus. 6 (2), 38-43.

Piñeiro, G., Lago, N. & Culebras, J. M. (2013). Role of omega-3 fatty acids in cardiovascular disease prevention. Review. Nutr Hosp. 28 (1), 1-5. https://doi.org/10.3305/nh.2013.28.1.6312
Rivero, F., Padial, M., Guadix, E. M. & Morales, R. (2020). Novozyme 435 and Lipozyme RM IM Preferably Esterify Polyunsaturated Fatty Acids at the sn‐2 Position. Eur. J. Lipid Sci. Technol. 122 (10), 2000115. https://doi.org/10.1002/ejlt.202000115

Schneider, C. A., Rasband, W. S. & Eliceiri, K. W., NIH Image to ImageJ: 25 years of image analysis. Nat. Methods. 2012, 9 (7), 671–675. doi: https://doi.org/10.1038/nmeth.2089

Shahidi, F. & Ambigaipalan, P. (2018). Omega-3 Polyunsaturated Fatty Acids and Their Health Benefits. Annu Rev Food Sci Technol. 9 (1), 345–381. https://doi.org/10.1146/annurev-food-111317-095850
Thuy, N. M., Giau, T. N., Tai, N. V. & Minh, V. Q. (2023). Drying kinetics and mathematical modeling of dried macaroni supplemented with Gac aril. Rev. Mex. Ing. Quim, 22 (3), 1-8. https://doi.org/10.24275/rmiq/Alim23103

Valle, P. A., Salazar, Y., Soto-Cruz, N. O., Páez-Lerma, J. B., Coria, L. N., Núñez-Guerrero, M. E., Rodriguez-Herrera R. & Herrera, L. D. (2024) Modelling and analysis on the ethanol production by the Torulaspora delbrueckii yeast. Rev. Mex. Ing. Quim. 23 (3), 1-10. https://doi.org/10.24275/rmiq/Sim24312

Von der Haar, D., Stäbler, A., Wichmann, R. & Schweiggert-Weisz, U. (2014). Enzymatic esterification of free fatty acids in vegetable oils utilizing different immobilized lipases. Biotechnol. Lett. 37 (1), 169–174. https://doi.org/10.1007/s10529-014-1668-1

Watanabe, Y., Yamauchi-Sato, Y., Nagao, T., Negishi, S., Terai, T., Kobayashi, T. & Shimada, Y. (2005). Production of MAG of CLA by esterification with dehydration at ordinary temperature using Penicillium camembertii lipase. J. Am. Oil Chem. Soc. 82 (9), 619–623. https://doi.org/10.1007/s11746-005-1119-9

Xie, D., Chen, Y., Yu, J., Yang, Z., Wang, X. & Wang, X. (2023). Progress in enrichment of n-3 polyunsaturated fatty acid: A review. Critical Reviews in Food Science and Nutrition, 63 (32), 11310-11326. https://doi.org/10.1080/10408398.2022.2086852

Zhang, Y., Di, X., Wang, W., Song, M., Yu, Q., Wang, Z., Yuan, Z., Chen, X., Xu, H. & Guo, Y. (2020). Kinetic study of lipase-catalyzed esterification of furoic acid to methyl-2-furoate. Biochem. Eng. J. 161, 107587. https://doi.org/10.1016/j.bej.2020.107587

Zhao, Y., Liu, J., Deng, L., Wang, F. & Tan, T. (2011). Optimization of Candida sp. 99-125 lipase catalyzed esterification for synthesis of monoglyceride and diglyceride in solvent-free system. J. Mol. Catal. 72 (3-4), 157-162. https://doi.org/10.1016/j.molcatb.2011.05.014

Zhu, Y., Feng, Y., Wang, J., Yuan, Z., Miao, Y., Miao, T., Gao, B. & Zhang, L. (2024). Selective Esterification Design of Lipases for TAG Synthesis Based on the Unique Structure of Curved DHA. ACS Food Science & Technology, 4 (7), 1722-1730. https://doi.org/10.1021/acsfoodscitech.4c00174