Applied Biochemistry and Biotechnology

, Volume 177, Issue 1, pp 36–47 | Cite as

Protein-Coated Microcrystals from Candida rugosa Lipase: Its Immobilization, Characterization, and Application in Resolution of Racemic Ibuprofen

  • Shuangshuang Huang
  • Xiang Li
  • Li XuEmail author
  • Caixia Ke
  • Rui Zhang
  • Yunjun YanEmail author


In this study, an economical heterogeneous biocatalyst, protein-coated microcrystals (PCMCs), was prepared from a commercial Candida rugosa lipase (CRL) and used for catalyzing esterification of (R, S)-ibuprofen enantiomers with isooctanol in isooctane. The main variables controlling the process (precipitating solvents, pH, saturated K2SO4 solution, and water content) were optimized via single-factorial experiments. Under optimum conditions, the enantiomeric excess of active S(+)-ibuprofen and total conversion rate were 97.34 and 49.83 %, respectively, and the corresponding enzyme (PCMC-CRL) activity attained 387.29 μmol/min/g protein, a 5.78-fold enhancement over the free lipase powder. Additionally, the thermostability, organic-solvent tolerance, and operational stability of PCMC-CRL were greatly improved as compared to the free enzyme. Fourier transform infrared (FTIR) spectroscopy was employed to reveal the correlation between conformation and enzyme activity enhancement. Moreover, the PCMC-CRL retained most of its original activity following use in more than 15 successive batches, suggesting that it exhibits adequate operational stability. These results indicate that PCMC-CRL is of great potential use in industrial applications.


Protein-coated micro-crystals (PCMCs) Enantioselective esterification Ibuprofen Lipase 



This work is financially supported by the National Natural Science Foundation of China (nos. 31170078, 31070089, and J1103514), the National High Technology Research and Development Program of China (nos. 2011AA02A204 and 2014AA093510), the Innovation Foundation of Shenzhen Government (no. JCYJ20120831111657864), the Innovation Foundation of HUST (nos. 2011TS100, 2014QN119, and 2014NY007), and the Fundamental Research Funds for the Central Universities HUST (no. 2172012SHYJ004). Many thanks to the Analytical and Testing Center of HUST for their valuable assistance with SEM and FT-IR measurements.


  1. 1.
    Schmid, R. D., & Verger, R. (1998). Lipases: interfacial enzymes with attractive applications. Angewandte Chemie International Edition, 37(12), 1608–1633.CrossRefGoogle Scholar
  2. 2.
    Singh, A. K., & Mukhopadhyay, M. (2012). Overview of fungal lipase: a review. Applied Biochemistry and Biotechnology, 166(2), 486–520.CrossRefGoogle Scholar
  3. 3.
    Xie, Y. C., Liu, H. Z., & Chen, J. Y. (1998). Candida rugosa lipase catalyzed esterification of racemic ibuprofen with butanol: racemization of R-ibuprofen and chemical hydrolysis of S-ester formed. Biotechnology Letters, 20(5), 455–458.CrossRefGoogle Scholar
  4. 4.
    Contesini, F. J., & de Oliveira Carvalho, P. (2006). Esterification of (RS)-Ibuprofen by native and commercial lipases in a two-phase system containing ionic liquids. Tetrahedron: Asymmetry, 17(14), 2069–2073.CrossRefGoogle Scholar
  5. 5.
    Santos, J. C., Mijone, P. D., Nunes, G. F., Perez, V. H., & de Castro, H. F. (2008). Covalent attachment of Candida rugosa lipase on chemically modified hybrid matrix of polysiloxane-polyvinyl alcohol with different activating compounds. Colloids and surfaces B, Biointerfaces, 61(2), 229–236.CrossRefGoogle Scholar
  6. 6.
    Adlercreutz, P. (2013). Immobilisation and application of lipases in organic media. Chemical Society Reviews, 42(15), 6406–6436.CrossRefGoogle Scholar
  7. 7.
    Kreiner, M., Parker, M. C., & Moore, B. D. (2001). Enzyme-coated micro-crystals: a 1-step method for high activity biocatalyst preparation. Chemical Communications, 12, 1096–1097.CrossRefGoogle Scholar
  8. 8.
    Shah, S., Sharma, A., & Gupta, M. N. (2008). Cross-linked protein-coated microcrystals as biocatalysts in non-aqueous solvents. Biocatalysis and Biotransformation, 26(4), 266–271.CrossRefGoogle Scholar
  9. 9.
    Kumari, V., Shah, S., & Gupta, M. N. (2007). Preparation of biodiesel by lipase- catalyzed transesterification of high free fatty acid containing oil from Madhuca indica. Energy & Fuels, 21(1), 368–372.CrossRefGoogle Scholar
  10. 10.
    Shah, S., & Gupta, M. N. (2007). Kinetic resolution of (+/−)-1-phenylethanol in [Bmim][PF6] using high activity preparations of lipases. Bioorganic & Medicinal Chemistry Letters, 17(4), 921–924.CrossRefGoogle Scholar
  11. 11.
    Kreiner, M., Amorim Fernandes, J. F., O’Farrell, N., Halling, P. J., & Parker, M. C. (2005). Stability of protein-coated microcrystals in organic solvents. Journal of Molecular Catalysis B: Enzymatic, 33(3-6), 65–72.CrossRefGoogle Scholar
  12. 12.
    Berglund, P. (2001). Controlling lipase enantioselectivity for organic synthesis. Biomolecular Engineering, 18(1), 13–22.CrossRefGoogle Scholar
  13. 13.
    Dominguez de Maria, P., Sanchez-Montero, J. M., Sinisterra, J. V., & Alcantara, A. R. (2006). Understanding Candida rugosa lipases: an overview. Biotechnology Advances, 24(2), 180–196.CrossRefGoogle Scholar
  14. 14.
    Li, X., Huang, S., Xu, L., & Yan, Y. (2013). Conformation and catalytic properties studies of Candida rugosa Lip7 via enantioselective esterification of Ibuprofen in organic solvents and ionic liquids. The Scientific World Journal, 2013, 1–7.Google Scholar
  15. 15.
    Colton, I. J., Ahmed, S. N., & Kazlauskas, R. J. (1995). A 2-propanol treatment increases the enantioselectivity of Candida rugosa lipase toward esters of chiral carboxylic acids. The Journal of Organic Chemistry, 60(1), 212–217.CrossRefGoogle Scholar
  16. 16.
    Yang, Z., Niu, X., Fang, X., Chen, G., Zhang, H., Yue, H., Wang, L., Zhao, D., & Wang, Z. (2013). Enantioselective esterification of Ibuprofen under microwave irradiation. Molecules, 18(5), 5472–5481.CrossRefGoogle Scholar
  17. 17.
    Tutar, H., Yilmaz, E., Pehlivan, E., & Yilmaz, M. (2009). Immobilization of Candida rugosa lipase on sporopollenin from Lycopodium clavatum. International Journal of Biological Macromolecules, 45(3), 315–320.CrossRefGoogle Scholar
  18. 18.
    Yu, H., Wu, J., & Ching, C. B. (2004). Enhanced activity and enantioselectivity ofCandida rugosa lipase immobilized on macroporous adsorptive resins for ibuprofen resolution. Biotechnology Letters, 26(8), 629–633.CrossRefGoogle Scholar
  19. 19.
    Hart, F. D., & Huskisson, E. (1984). Non-steroidal anti-inflammatory drugs. Current status and rational therapeutic use. Drugs, 27(3), 232–255.CrossRefGoogle Scholar
  20. 20.
    Liu, Y., Wang, F., & Tan, T. (2009). Effects of alcohol and solvent on the performance of lipase from Candida sp. in enantioselective esterification of racemic ibuprofen. Journal of Molecular Catalysis B: Enzymatic, 56(2), 126–130.CrossRefGoogle Scholar
  21. 21.
    Chen, J. C., & Tsai, S. W. (2000). Enantioselective synthesis of (S)Ibuprofen ester prodrug in cyclohexane by Candida rugosa lipase immobilized on accurel MP1000. Biotechnology Progress, 16(6), 986–992.CrossRefGoogle Scholar
  22. 22.
    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), 248–254.CrossRefGoogle Scholar
  23. 23.
    Yang, C., Wang, F., Lan, D., Whiteley, C., Yang, B., & Wang, Y. (2012). Effects of organic solvents on activity and conformation of recombinant Candida antarctica lipase A produced by Pichia pastoris. Process Biochemistry, 47(3), 533–537.CrossRefGoogle Scholar
  24. 24.
    Raita, M., Champreda, V., & Laosiripojana, N. (2010). Biocatalytic ethanolysis of palm oil for biodiesel production using microcrystalline lipase in tert-butanol system. Process Biochemistry, 45(6), 829–834.CrossRefGoogle Scholar
  25. 25.
    Costantino, H. R., Griebenow, K., Langer, R., & Klibanov, A. M. (1997). On the pH memory of lyophilized compounds containing protein functional groups. Biotechnology and Bioengineering, 53(3), 345–348.CrossRefGoogle Scholar
  26. 26.
    Yu, H. W., Chen, H., Wang, X., Yang, Y. Y., & Ching, C. B. (2006). Cross-linked enzyme aggregates (CLEAs) with controlled particles: application to Candida rugosa lipase. Journal of Molecular Catalysis B: Enzymatic, 43(1-4), 124–127.CrossRefGoogle Scholar
  27. 27.
    Yu, W. H., Fang, M., Tong, D. S., Shao, P., Xu, T. N., & Zhou, C. H. (2013). Immobilization of Candida rugosa lipase on hexagonal mesoporous silicas and selective esterification in nonaqueous medium. Biochemical Engineering Journal, 70, 97–105.CrossRefGoogle Scholar
  28. 28.
    Raita, M., Laothanachareon, T., Champreda, V., & Laosiripojana, N. (2011). Biocatalytic esterification of palm oil fatty acids for biodiesel production using glycine-based cross-linked protein coated microcrystalline lipase. Journal of Molecular Catalysis B: Enzymatic, 73(1-4), 74–79.CrossRefGoogle Scholar
  29. 29.
    Kreiner, M., & Parker, M. C. (2005). Protein-coated microcrystals for use in organic solvents: application to oxidoreductases. Biotechnology Letters, 27(20), 1571–1577.CrossRefGoogle Scholar
  30. 30.
    Moore, B. D., Partridge, J., Bradley, L. & Vos, J. (2008). Precipitation stabilising compositions. A61K 9/14 (2006.01).Google Scholar
  31. 31.
    Zheng, J., Xu, L., Liu, Y., Zhang, X., & Yan, Y. (2012). Lipase-coated K2SO4 micro-crystals: preparation, characterization, and application in biodiesel production using various oil feedstocks. Bioresource Technology, 110, 224–231.CrossRefGoogle Scholar
  32. 32.
    Zhu, K., Jutila, A., Tuominen, E. K., Patkar, S. A., Svendsen, A., & Kinnunen, P. K. (2001). Impact of the tryptophan residues of Humicola lanuginosa lipase on its thermal stability. Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology, 1547(2), 329–338.CrossRefGoogle Scholar
  33. 33.
    Bai, S., Guo, Z., Liu, W., & Sun, Y. (2006). Resolution of (±)-menthol by immobilized Candida rugosa lipaseon superparamagnetic nanoparticles. Food Chemistry, 96(1), 1–7.CrossRefGoogle Scholar
  34. 34.
    Chen, D., Peng, C., Zhang, H., & Yan, Y. (2013). Assessment of activities and conformation of lipases treated with sub- and supercritical carbon dioxide. Applied Biochemistry and Biotechnology, 169(7), 2189–2201.CrossRefGoogle Scholar
  35. 35.
    Liu, Y., Chen, D., & Yan, Y. (2013). Effect of ionic liquids, organic solvents and supercritical CO2 pretreatment on the conformation and catalytic properties of Candida rugosa lipase. Journal of Molecular Catalysis B: Enzymatic, 90, 123–127.CrossRefGoogle Scholar
  36. 36.
    Pavlidis, I. V., Gournis, D., Papadopoulos, G. K., & Stamatis, H. (2009). Lipases in water-in-ionic liquid microemulsions: structural and activity studies. Journal of Molecular Catalysis B: Enzymatic, 60(1-2), 50–56.CrossRefGoogle Scholar
  37. 37.
    Sheldon, R. A., Lau, R. M., Sorgedrager, M. J., van Rantwijk, F., & Seddon, K. R. (2002). Biocatalysis in ionic liquids. Green Chemistry, 4(2), 147–151.CrossRefGoogle Scholar
  38. 38.
    Solanki, K., Gupta, M. N., & Halling, P. J. (2012). Examining structure-activity correlations of some high activity enzyme preparations for low water media. Bioresource Technology, 115, 147–151.CrossRefGoogle Scholar
  39. 39.
    Gaur, R., Gupta, G. N., Vamsikrishnan, M., & Khare, S. K. (2008). Protein-coated microcrystals of Pseudomonas aeruginosa PseA lipase. Applied Biochemistry and Biotechnology, 151(2-3), 160–166.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Key Laboratory of Molecular Biophysics, The Ministry of Education, College of Life Science and TechnologyHuazhong University of Science and TechnologyWuhanChina

Personalised recommendations