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Purification, Immobilization and Characterization of Lipase Isoenzyme from Aspergillus niger with C8 Magnetic Particles

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DOI: 10.4236/abb.2014.57075    3,904 Downloads   4,983 Views   Citations

ABSTRACT

The purification of a lipase isoenzyme from an Aspergillus niger lipase A is reported in this manuscript. Purification was carried out in a simple adsorption step, in which the lipase was offered at low ionic strength to the commercially available C8 modified magnetic particles, MaKProt C8. When the isoenzyme was desorbed with a 0.2% solution of Triton X-100, the SDS-PAGE gel showed a single pure band with a molecular weight of 35 KDa. The purified fraction showed 66.75-fold purification compared with the crude extract. The pure fraction was characterized along with the crude extract and the lipase adsorbed on the MaKProt C8. The purified and the adsorbed lipase showed better activity for the tested substrates (p-nitrophenyl acetate, decanoate, myristate and palmitate) than the crude extract, the preferred substrates being myristate (26.7 μmol·min-1·mg-1) and decanoate (17.4 μmol·min-1·mg-1), respectively. The temperature and pH profiles showed no change for the three enzymes, the optimum temperature being 37°C and the best pH 7.0.

Conflicts of Interest

The authors declare no conflicts of interest.

Cite this paper

Hernández-García, S. , García-García, M. and García-Carmona, F. (2014) Purification, Immobilization and Characterization of Lipase Isoenzyme from Aspergillus niger with C8 Magnetic Particles. Advances in Bioscience and Biotechnology, 5, 633-641. doi: 10.4236/abb.2014.57075.

References

[1] Jaeger, K.E. and Reetz, M.T. (2000) Directed Evolution of Enantioselective Enzymes for Organic Chemistry. Current Opinion in Chemical Biology, 4, 68-73.
http://dx.doi.org/10.1016/S1367-5931(99)00054-X
[2] Koeller, K.M. and Wong, C.H. (2001) Enzymes for Chemical Synthesis. Nature, 409, 232-240.
http://dx.doi.org/10.1038/35051706
[3] Virto, M.D., Lascaray, J.M., Solozabal, R. and de Renobales, M. (1991) Enzymic Hydrolysis of Animal Fats in Organic Solvents at Temperatures below Their Melting Points. Journal of the American Oil Chemists Society, 68, 324-327.
http://dx.doi.org/10.1007/BF02657686
[4] Chen, Y.Z., Yang, C.T., Ching, C.B. and Xu, R. (2008) Immobilization of Lipases on Hydrophobilized Zirconia Nanoparticles: Highly Enantioselective and Reusable Biocatalysts. Langmuir, 24, 8877-8884.
http://dx.doi.org/10.1021/la801384c
[5] Pera, L.M., Romero, C.M., Baigori, M.D. and Castro, G.R. (2006) Catalytic Properties of Lipase Extracts from Aspergillus niger. Food Technology & Biotechnology, 44, 247-252.
[6] Palomo, J.M., Fernández-Lorente, G., Mateo, C., Fernández-Lafuente, R. and Guisan, J.M. (2002) Enzymatic Resolution of (±)-trans-4-(4’-Fluorophenyl)-6-Oxo-Piperidin-3-Ethyl Carboxylate, an Intermediate in the Synthesis of (-) Paroxetine. Tetrahedron: Asymmetry, 13, 2375-2381.
http://dx.doi.org/10.1016/S0957-4166(02)00645-6
[7] Wang, J., Meng, G., Tao, K., Feng, M., Zhao, X., Li, Z. and Lu, J.R. (2012) Immobilization of Lipases on Alkyl Silane Modified Magnetic Nanoparticles: Effect of Alkyl chain Length on Enzyme Activity. PloS one, 7, e43478.
http://dx.doi.org/10.1371/journal.pone.0043478
[8] Franzreb, M., Ebner, N. and Siemann-Herzberg, M. (2003) Magnettechnologie in der Bioproduktaufreinigung. Transkript, 9, 112-115.
[9] Tong, X.D., Xue, B. and Sun, Y. (2001) A Novel Magnetic Affinity Support for Protein Adsorption and Purification. Biotechnology Progress, 17, 134-139.
http://dx.doi.org/10.1021/bp000134g
[10] Holschuh, K. and Schwämmle, A. (2005) Preparative Purification of Antibodies with Protein A—An Alternative to Conventional Chromatography. Journal of Magnetism and Magnetic Materials, 293, 345-348.
http://dx.doi.org/10.1016/j.jmmm.2005.02.050
[11] Van Tilbeurgh, H., Egloff, M.P., Martinez, C., Rugani, N., Verger, R. and Cambillau, C. (1993) Interfacial Activation of the Lipase-Procolipase Complex by Mixed Micelles Revealed by X-Ray Crystallography. Nature, 362, 814-820.
http://dx.doi.org/10.1038/362814a0
[12] Brady, L., Brzozowski, A.M., Derewenda, Z.S., Dodson, E., Dodson, G., Tolley, S. and Menge, U. (1990) A Serine Protease Triad Forms the Catalytic Centre of a Triacylglycerol Lipase. Nature, 343, 767-770.
http://dx.doi.org/10.1038/343767a0
[13] Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M. and Klenk, D.C. (1985) Measurement of Protein Using Bicinchoninic Acid. Analytical biochemistry, 150, 76-85.
http://dx.doi.org/10.1016/0003-2697(85)90442-7
[14] Gupta, R., Rathi, P., Gupta, N. and Bradoo, S. (2003) Lipase Assays for Conventional and Molecular Screening: An Overview. Biotechnology and Applied Biochemistry, 37, 63-71.
http://dx.doi.org/10.1042/BA20020059
[15] Höfelmann, M., Hartmann, J., Zink, A. and Schreier, P. (1985) Isolation, Purification, and Characterization of Lipase Isoenzymes from a Technical Aspergillus niger Enzyme. Journal of Food Science, 50, 1721-1725.
http://dx.doi.org/10.1111/j.1365-2621.1985.tb10574.x
[16] Namboodiri, V.M.H. and Chattopadhyaya, R. (2000) Purification and Biochemical Characterization of a Novel Thermostable Lipase from Aspergillus niger. Lipids, 35, 495-502.
http://dx.doi.org/10.1007/s11745-000-549-3
[17] Chang, S.W., Lee, G.C. and Shaw, J.F. (2006) Efficient Production of Active Recombinant Candida Rugosa LIP3 Lipase in Pichiapastoris and Biochemical Characterization of the Purified Enzyme. Journal of Agricultural and Food Chemistry, 54, 5831-5838.
http://dx.doi.org/10.1021/jf060835e
[18] Shangguan, J.J., Liu, Y.Q., Wang, F.J., Zhao, J., Fan, L.Q., Li, S.X. and Xu, J.H. (2011) Expression and Characterization of a Novel Lipase from Aspergillus fumigatus with High Specific Activity. Applied Biochemistry and Biotechnology, 165, 949-962.
http://dx.doi.org/10.1007/s12010-011-9311-2
[19] Karadzic, I., Masui, A., Zivkovic, L.I. and Fujiwara, N. (2006) Purification and Characterization of an Alkaline Lipase from Pseudomonas aeruginosa Isolated from Putrid Mineral Cutting Oil as Component of Metalworking Fluid. Journal of Bioscience and Bioengineering, 102, 82-89.
http://dx.doi.org/10.1263/jbb.102.82
[20] Quyen, D.T., Giang Le, T.T., Nguyen, T.T., Oh, T.K. and Lee, J.K. (2005) High-Level Heterologous Expression and Properties of a Novel Lipase from Ralstonia sp. M1. Protein Expression and Purification, 39, 97-106.
http://dx.doi.org/10.1016/j.pep.2004.10.001
[21] Van Heerden, E., Litthauer, D. and Verger, R. (2002) Biochemical Characterisation and Kinetic Properties of a Purified Lipase from Aspergillus niger in Bulk Phase and Monomolecular Films. Enzyme and Microbial Technology, 30, 902-909.
http://dx.doi.org/10.1016/S0141-0229(02)00031-5
[22] Romero, C.M., Baigori, M.D. and Pera, L.M. (2007) Catalytic Properties of Mycelium-Bound Lipases from Aspergillus niger MYA 135. Applied Microbiology and Biotechnology, 76, 861-866.
http://dx.doi.org/10.1007/s00253-007-1067-9

  
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