Biotechnology and Bioprocess Engineering

, Volume 24, Issue 2, pp 326–336 | Cite as

Preparation of High-purity 1,3-Diacylglycerol Using Performance-enhanced Lipase Immobilized on Nanosized Magnetite Particles

  • Jiong-feng Zhao
  • Tao-Wang
  • Jian-ping Lin
  • Li-rong Yang
  • Mian-Bin WuEmail author
Research Paper


Early research on the nutritional value of 1,3-diacylglycerols (1,3-DAGs) has resulted in a significant interest in their synthesis. 1,3-DAGs can be produced chemically and biologically. In this work, a regioselective lipase from Rhizopus oryzae was efficiently immobilized on nanosized magnetite particles (NSM) in an oriented way, resulting in significant enhancement of activity. The specific hydrolytic and esterification activities of the immobilized enzyme were 1,660% and 260% of those of the free enzyme, respectively. The immobilized enzyme was then used to catalyze the esterification of oleic acid with glycerol in a solvent-free system for preparation of 1,3-DAG in a 1 L reactor. The catalytic process was studied in detail, the final concentration of 1,3-DAG reached >76% under the optimal condition when the molar ratio of oleic acid to glycerol was 2.8:1. The regioselectivities of free and immobilized enzyme were both >97%. The immobilized enzyme was reused for 55 cycles with only ∼30% activity loss at 30°C. The purity of 1,3-DAG was up to ∼95% (w/w) after a simple purification step with the recovery ratio ∼85%. This is the first report of efficient 1,3-DAG purification by neutralization without acyl migration.


Rhizopus oryzae lipase nanosized magnetite particles Fe3O4 immobilization esterification 1,3-diacylglycerols 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.



The authors thank the National Natural Science Foundation of China (Grant No. 21376215), National Basic Research Program of China (973, 2011CB710803), the National High-Tech Research and Development Program of China (863, 2012AA022302), and the National Natural Science Foundation cultivation project of Jining medical university (Grant no.JYP201704) for financial support. The authors declare no conflicts of interest.

Supplementary material

12257_2018_458_MOESM1_ESM.pdf (270 kb)
Supplementary material, approximately 270 KB.


  1. 1.
    Morita, O. and M. G. Soni (2009) Safety assessment of diacylglycerol oil as an edible oil: a review of the published literature. Food Chem. Toxicol. 47: 9.CrossRefGoogle Scholar
  2. 2.
    Taguchi, H., T. Nagao, H. Watanabe, K. Onizawa, N. Matsuo, I. Tokimitsu, and H. Itakura (2001) Energy value and digestibility of dietary oil containing mainly 1,3-diacylglycerol are similar to those of triacylglycerol. Lipids 36: 379–382.CrossRefGoogle Scholar
  3. 3.
    Flickinger, B. D. and N. Matsuo (2003) Nutritional characteristics of DAG oil. Lipids 38: 129.CrossRefGoogle Scholar
  4. 4.
    Hu, T.-G., J.-H. Cheng, B.-B. Zhang, W.-Y. Lou, and M.-H. Zong (2015) Immobilization of Alkaline protease on amino-functionalized magnetic nanoparticles and its efficient use for preparation of oat polypeptides. Ind. Eng. Chem. Res. 54: 4689–4698.CrossRefGoogle Scholar
  5. 5.
    Ferreira, M. L. and G. M. Tonetto (2017) What is the importance of structured triglycerides and diglycerides? pp. 1–16. Enzymatic Synthesis of Structured Triglycerides. SpringerBriefs in Molecular Science. Springer, Cham.CrossRefGoogle Scholar
  6. 6.
    Singh, D., P. Patidar, A. Ganesh, and S. Mahajani (2013) Esterification of oleic acid with glycerol in the presence of supported zinc oxide as catalyst. Ind. Eng. Chem. Res. 52: 14776–14786.CrossRefGoogle Scholar
  7. 7.
    Garcia, H. S., R. Baeza-Jimenez, K. Miranda, and C. Otero (2013) Lipase-catalyzed glycerolysis of fish oil to obtain diacylglycerols. Grasas Aceites 64: 237–242.CrossRefGoogle Scholar
  8. 8.
    Cheong, L.-Z., C.-P. Tan, K. Long, M. S. Affandi Yusoff, N. Arifin, S.-K. Lo, and O.-M. Lai (2007) Production of a diacylglycerol-enriched palm olein using lipase-catalyzed partial hydrolysis: Optimization using response surface methodology. Food Chem. 105: 1614–1622.CrossRefGoogle Scholar
  9. 9.
    Guo, Z. and Y. Sun (2007) Solvent-free production of 1,3-diglyceride of CLA: Strategy consideration and protocol design. Food Chem. 100: 1076–1084.CrossRefGoogle Scholar
  10. 10.
    Yesiloglu, Y. and I. Kilic (2004) Lipase-catalyzed esterification of glycerol and oleic acid. J. Am. Oil Chem. Soc. 81: 281–284.CrossRefGoogle Scholar
  11. 11.
    Zhong, N., Z. Gui, L. Xu, J. Huang, K. Hu, Y. Gao, X. Zhang, Z. Xu, J. Su, and B. Li (2013) Solvent-free enzymatic synthesis of 1,3-Diacylglycerols by direct esterification of glycerol with saturated fatty acids. Lipids Health Dis. 12: 65.CrossRefGoogle Scholar
  12. 12.
    Kwon, S. J., J. J. Han, and J. S. Rhee (1995) Production and in situ separation of mono- or diacylglycerol catalyzed by lipases in n-hexane. Enzyme Microb. Tech. 17: 700–704.CrossRefGoogle Scholar
  13. 13.
    Wang, Z., W. Du, L. Dai, and D. Liu (2016) Study on Lipozyme TL IM-catalyzed esterification of oleic acid and glycerol for 1,3-diolein preparation. J. Mol. Catal. B-Enzym. 127: 11–17.CrossRefGoogle Scholar
  14. 14.
    Liu, M., J. Fu, Y. Teng, Z. Zhang, N. Zhang, and Y. Wang (2016) Fast production of diacylglycerol in a solvent free system via lipase catalyzed esterification using a bubble column reactor. J. Am. Oil Chem. Soc. 93: 637–648.CrossRefGoogle Scholar
  15. 15.
    Liu, N., Y. Wang, Q. Zhao, Q. Zhang, and M. Zhao (2011) Fast synthesis of 1,3-DAG by Lecitase® Ultra-catalyzed esterification in solvent-free system. Eur. J. Lipid Sci. Technol. 113: 973–979.CrossRefGoogle Scholar
  16. 16.
    Duan, Z.-Q., W. Du, and D.-H. Liu (2013) Improved synthesis of 1,3-diolein by Novozym 435-mediated esterification of monoolein with oleic acid. J. Mol. Catal. B-Enzym. 89: 1–5.CrossRefGoogle Scholar
  17. 17.
    Meng, X., G. Xu, Q. L. Zhou, J. P. Wu, and L. R. Yang (2014) Highly efficient solvent-free synthesis of 1,3-diacylglycerols by lipase immobilised on nano-sized magnetite particles. Food Chem. 143: 319–324.CrossRefGoogle Scholar
  18. 18.
    Tecelao, C., I. Rivera, G. Sandoval, and S. Ferreira-Dias (2012) Carica papaya latex: A low-cost biocatalyst for human milk fat substitutes production. Eur. J. Lipid Sci. Technol. 114: 266–276.CrossRefGoogle Scholar
  19. 19.
    Esteban, L., M. J. Jiménez, E. Hita, P. A. González, L. Martín, and A. Robles (2011) Production of structured triacylglycerols rich in palmitic acid at sn-2 position and oleic acid at sn-1,3 positions as human milk fat substitutes by enzymatic acidolysis. Biochem. Eng. J. 54: 62–69.CrossRefGoogle Scholar
  20. 20.
    Nagao, T., Y. Shimada, A. Sugihara, A. Murata, S. Komemushi, and Y. Tominaga (2001) Use of thermostable Fusarium heterosporum lipase for production of structured lipid containing oleic and palmitic acids in organic solvent-free system. J. Am. Oil Chem. Soc.78: 167–172.CrossRefGoogle Scholar
  21. 21.
    Marszałł, M. P., and T. Siódmiak (2012) Immobilization of Candida rugosa lipase onto magnetic beads for kinetic resolution of (R,S)-ibuprofen. Catal. Commun. 24: 80–84.CrossRefGoogle Scholar
  22. 22.
    Kharrat, N., Y. B. Ali, S. Marzouk, Y.-T. Gargouri, and M. Karra-Châabouni (2011) Immobilization of Rhizopus oryzae lipase on silica aerogels by adsorption: Comparison with the free enzyme. Process. Biochem. 46: 1083–1089.CrossRefGoogle Scholar
  23. 23.
    Adlercreutz, P. (2013) Immobilisation and application of lipases in organic media. Chem. Soc. Rev. 42: 6406–6436.CrossRefGoogle Scholar
  24. 24.
    Pashangeh, K., M. Akhond, H. R. Karbalaei-Heidari, and G. Absalan (2017) Biochemical characterization and stability assessment of Rhizopus oryzae lipase covalently immobilized on amino-functionalized magnetic nanoparticles. Int. J. Biol. Macromol. 105: 300–307.CrossRefGoogle Scholar
  25. 25.
    Tecelão, C., M. Guillén, F. Valero, and S. Ferreira-Dias (2012) Immobilized heterologous Rhizopus oryzae lipase: A feasible biocatalyst for the production of human milk fat substitutes. Biochem. Eng. J. 67: 104–110.CrossRefGoogle Scholar
  26. 26.
    Zhu, J. and G. Sun (2012) Lipase immobilization on glutaraldehyde-activated nanofibrous membranes for improved enzyme stabilities and activities. React. Funct. Polym. 72: 839–845.CrossRefGoogle Scholar
  27. 27.
    Zhou, W. J., L. Fang, Z. Fan, B. Albela, L. Bonneviot, F. De Campo, M. Pera-Titus, and J. M. Clacens (2014) Tunable catalysts for solvent-free biphasic systems: pickering interfacial catalysts over amphiphilic silica nanoparticles. J. Am. Chem. Soc. 136: 4869–4872.CrossRefGoogle Scholar
  28. 28.
    Cipolatti, E. P., M. J. A. Silva, M. Klein, V. Feddern, M. M. C. Feltes, J. V. Oliveira, J. L. Ninow, and D. de Oliveira (2014) Current status and trends in enzymatic nanoimmobilization. J. Mol. Catal. B-Enzym. 99: 56–67.CrossRefGoogle Scholar
  29. 29.
    Qian, S., C. Wang, H. Wang, F. Yu, C. Zhang, and H. Yu (2015) Synthesis and characterization of surface-functionalized paramagnetic nanoparticles and their application to immobilization of a-acetolactate decarboxylase. Process. Biochem. 50: 1388–1393.CrossRefGoogle Scholar
  30. 30.
    Xie, W. and N. Ma (2009) Immobilized lipase on Fe3 O4 nanoparticles as biocatalyst for biodiesel production. Energ. Fuel. 23: 1347–1353.CrossRefGoogle Scholar
  31. 31.
    Chen, L., Z. Xu, H. Dai, and S. Zhang (2010) Facile synthesis and magnetic properties of monodisperse Fe3O4/silica nano-composite microspheres with embedded structures via a direct solution-based route. J. Alloy Compd. 497: 221–227.CrossRefGoogle Scholar
  32. 32.
    Tang, J., Matt Myers, K. A. B. And, and L. E. Brus (2003) Magnetite Fe3 O4 nanocrystals: spectroscopic observation of aqueous oxidation kinetics. J. Phys. Chem. B. 107: 7501–7506.CrossRefGoogle Scholar
  33. 33.
    Feitknecht, W. and K. J. Gallagher (1970) mechanisms for the oxidation of Fe3O4. Nature 228: 548–549.CrossRefGoogle Scholar
  34. 34.
    Yamaura, M., R. L. Camilo, L. C. Sampaio, M. A. Macêdo, M. Nakamura, and H. E. Toma (2004) Preparation and characterization of (3-aminopropyl)triethoxysilane-coated magnetite nanoparticles. J. Magn. Magn. Mater. 279: 210–217.CrossRefGoogle Scholar
  35. 35.
    Liu, Y., Y. Li, X.-M. Li, and T. He (2013) Kinetics of (3-aminopropyl)triethoxylsilane (APTES) silanization of superparamagnetic iron oxide nanoparticles. Langmuir 29: 15275–15282.CrossRefGoogle Scholar
  36. 36.
    Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.CrossRefGoogle Scholar
  37. 37.
    Pencreac’h, G. and J. C. Baratti (1996) Hydrolysis of pnitrophenyl palmitate in n-heptane by the Pseudomonas cepacia lipase: A simple test for the determination of lipase activity in organic media. Enzyme Microb. Tech. 18: 417–422.CrossRefGoogle Scholar
  38. 38.
    Liu, T., Y. Zhao, X. Wang, X. Li, and Y. Yan (2013) A novel oriented immobilized lipase on magnetic nanoparticles in reverse micelles system and its application in the enrichment of polyunsaturated fatty acids. Bioresource Technol. 132: 99–102.CrossRefGoogle Scholar
  39. 39.
    Mine, Y., K. Fukunaga, K. Itoh, M. Yoshimoto, K. Nakao, and Y. Sugimura (2003) Enhanced enzyme activity and enantio-selectivity of lipases in organic solvents by crown ethers and cyclodextrins. J. Biosci. Bioeng. 95: 441–447.CrossRefGoogle Scholar
  40. 40.
    Kartal, F. (2016) Enhanced esterification activity through interfacial activation and cross-linked immobilization mechanism of Rhizopus oryzae lipase in a nonaqueous medium. Biotechnol. Prog. 32: 899–904.CrossRefGoogle Scholar
  41. 41.
    Luna, C., C. Verdugo, E. D. Sancho, D. Luna, J. Calero, A. Posadillo, F. M. Bautista, and A. A. Romero (2014) Biocatalytic behaviour of immobilized Rhizopus oryzae lipase in the 1,3-selective ethanolysis of sunflower oil to obtain a biofuel similar to biodiesel. Molecules 19: 11419–11439.CrossRefGoogle Scholar
  42. 42.
    Ashjari, M., M. Mohammadi, and R. Badri (2015) Chemical amination of Rhizopus oryzae lipase for multipoint covalent immobilization on epoxy-functionalized supports: modulation of stability and selectivity. J. Mol. Catal. B-Enzym. 115: 128–134.CrossRefGoogle Scholar
  43. 43.
    Kumar, V., F. Jahan, S. Raghuwanshi, R. V. Mahajan, and R. K. Saxena (2013) Immobilization of Rhizopus oryzae lipase on magnetic Fe3O4-chitosan beads and its potential in phenolic acids ester synthesis. Biotechnol. Bioproc. E. 18: 787–795.CrossRefGoogle Scholar
  44. 44.
    Ghamgui, H., N. Miled, M. Karra-chaabouni, and Y. Gargouri (2007) Immobilization studies and biochemical properties of free and immobilized Rhizopus oryzae lipase onto CaCO3: A comparative study. Biochem. Eng. J. 37: 34–41.CrossRefGoogle Scholar
  45. 45.
    Duan, Z. Q., W. Du, and D. H. Liu (2012) Rational synthesis of 1,3-diolein by enzymatic esterification. J. Biotechnol. 159: 44–49.CrossRefGoogle Scholar
  46. 46.
    Duan, Z. Q., X. L. Fang, Z. Y. Wang, Y. H. Bi, and H. Sun (2015) A sustainable process for 1,3-diolein synthesis catalyzed by immobilized lipase from Penicillium expansum. Acs. Sustain. Chem. Eng. 3: 2804–2808.CrossRefGoogle Scholar
  47. 47.
    Li, W., W. Du, Q. Li, T. Sun, and D. Liu (2010) Study on acylmigration kinetics of partial glycerides: dependence on temperature and water activity. J. Mol. Catal. B-Enzym. 63: 17–22.CrossRefGoogle Scholar
  48. 48.
    Eom, T.-K., C.-S. Kong, H.-G. Byun, W.-K. Jung, and S.-K. Kim (2010) Lipase catalytic synthesis of diacylglycerol from tuna oil and its anti-obesity effect in C57BL/6J mice. Process Biochem. 45: 738–743.CrossRefGoogle Scholar
  49. 49.
    Von der Haar, D., A. Stabler, R. Wichmann, and U. Schweiggert-Weisz (2015) Enzyme-assisted process for DAG synthesis in edible oils. Food Chem. 176: 263–270.CrossRefGoogle Scholar
  50. 50.
    Liu, N., Y. Wang, Q. Zhao, C. Cui, M. Fu, and M. Zhao (2012) Immobilisation of lecitase® ultra for production of diacylglycerols by glycerolysis of soybean oil. Food Chem. 134: 301–307.CrossRefGoogle Scholar
  51. 51.
    Zhao, Y., J. Liu, L. Deng, F. Wang, and T. Tan (2011) Optimization of Candida sp. 99–125 lipase catalyzed esterification for synthesis of monoglyceride and diglyceride in solvent-free system. J. Mol. Catal. B-Enzym. 72: 157–162.CrossRefGoogle Scholar
  52. 52.
    Wang, Y., M. Zhao, K. Song, L. Wang, X. Han, S. Tang, and Y. Wang (2010) Separation of diacylglycerols from enzymatically hydrolyzed soybean oil by molecular distillation. Sep. Purif. Technol. 75: 114–120.CrossRefGoogle Scholar

Copyright information

© The Korean Society for Biotechnology and Bioengineering and Springer 2019

Authors and Affiliations

  • Jiong-feng Zhao
    • 1
  • Tao-Wang
    • 2
  • Jian-ping Lin
    • 1
  • Li-rong Yang
    • 1
  • Mian-Bin Wu
    • 1
    Email author
  1. 1.Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of chemical and biological engineeringZhejiang UniversityHangzhouChina
  2. 2.School of biological scienceJining medical universityJiningChina

Personalised recommendations