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Purification and biochemical characterization of a novel thermostable lipase from Aspergillus niger

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Lipids

Abstract

An extracellular 1,3-specific lipase with molecular weight of 35.5 kDa and an isoelectric point of 4.4 from Aspergillus niger has been purified 50-fold by pH precipitation followed by a series of chromatographic steps with an overall yield of 10%. The enzyme was homogeneous as judged by denaturing polyacrylamide gel electrophoresis and size-exclusion fast-performance liquid chromatography. It contained 2.8% sugar which was completely removed by endoglycosidase F treatment, and the deglycosylated enzyme retained full activity. The native lipase showed optimal activity between temperatures 35 and 55°C and pH 5.0 and 6.0. The amino acid composition and the N-terminal sequence were found to be different from lipases previously purified from A. niger. The enzyme was resistant to trypsin, chymotrypsin, endoprotease Glu-C, thrombin, and papain under native conditions but was susceptible to cleavage by the same proteases when heat-denatured.

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Abbreviations

BSA:

bovine serum albumin

DMSO:

dimethylsulfoxide

FPLC:

fast-performance liquid chromatography

HPLC:

high-performance liquid chromatography

PAS:

periodate Schiff's stain

PITC:

phenylisothio-cyanate

PMSF:

phenylmethylsulfonylfluoride

SDS-PAGE:

polyacrylamide gel electrophoresis in sodium dodecyl sulfate

TCA:

trichloroacetic acid

TLC:

thin-layer chromatography

References

  1. Sarda, L., and Desnuelle, P. (1958) Action de la Lipase Pancre-atique sur les Esters en Emulsion, Biochim. Biophys. Acta 30, 513–520.

    Article  PubMed  CAS  Google Scholar 

  2. Derewanda, Z.S., and Sharp, A.M. (1993) News from the Inter-face: the Molecular Structures of Triglyceride Lipases, TIBS 18, 20–25.

    Google Scholar 

  3. Derewanda, Z.S. (1995) A Twist in the Tale of Lipolytic Enzymes, Nat. Struct. Biol. 2, 347–349.

    Article  Google Scholar 

  4. Desnuelle, P. (1972) The Lipases, in The Enzymes (Boyer, P.D., ed.), pp. 575–616, Academic Press, New York.

    Google Scholar 

  5. Aires-Barros, M.R., Angela Taipa, M., and Cabral, J.M.S. (1994) Isolation and Purification of Lipases, in Lipases, Their Structure, Biochemistry, and Application (Woolley, P., and Peterson, S.B., eds.), pp. 243–270, Cambridge University Press, Cambridge.

    Google Scholar 

  6. Vulfson, E.N. (1994) Industrial Applications of Lipases, in Lipases, Their Structure, Biochemistry and Application (Woolley, P., and Peterson, S.B., eds.), pp. 271–288, Cambridge University Press, Cambridge.

    Google Scholar 

  7. Veeraragavan, K., Colpitts, T., and Gibbs, B.F. (1990) Purification and Characterization of Two Distinct Lipases from Geotrichum candidum, Biochim. Biophys. Acta 1044, 26–33.

    PubMed  CAS  Google Scholar 

  8. Nagaoka, K., and Yamada, Y. (1973) Purification of Mucor Lipases and Their Properties, Agric. Biol. Chem. 37, 2791–2796.

    CAS  Google Scholar 

  9. Fukumoto, J., Iwai, M., and Tsujisaka, Y. (1964) Purification and Crystallization of a Lipase Secreted by Aspergillus niger, J. Gen. Appl. Microbiol. 10, 257.

    Google Scholar 

  10. Tsujisaka, Y., Iwai, M., and Tominaga, Y. (1973) Purification, Crystallization, and Some Properties of Lipases from Geotrichum candidum, Agric. Biol. Chem. 37, 1457–1464.

    CAS  Google Scholar 

  11. Tomizuka, N., Ota, Y., and Yamada, K. (1966) Studies on Lipase from Candida cylindracea. Part 1 Purification and Properties, Agric. Biol. Chem. 30, 576–584.

    CAS  Google Scholar 

  12. Rubin, B. (1994) Grease Pit Chemistry Exposed, Nat. Struct. Biol. 1, 568–572.

    Article  PubMed  CAS  Google Scholar 

  13. Derewanda, U., Swenson, L., Green, R., Wei, Y., Dodson, G.G., Yamaguchi, S., Haas, M.J., and Derewanda, Z.S. (1994) An Unusual Buried Polar Cluster in a Family of Fungal Lipases, Nat. Struct. Biol. 1, 36–47.

    Article  Google Scholar 

  14. Tombs, M.P., and Blake, G.G. (1982) Stability and Inhibition of Aspergillus and Rhizopus Lipases, Biochim. Biophys. Acta 700, 81–89.

    PubMed  CAS  Google Scholar 

  15. Höfelmann, M. (1985) Isolation, Purification, and Characterization of Lipase Isozymes from a Technical Aspergillus niger Enzyme, J. Food Sci. 50, 1721–1725.

    Article  Google Scholar 

  16. Sugihara, A., Shimada, Y., and Tominaga, Y. (1988) Purification and Characterization of Aspergillus niger Lipase, Agric. Biol. Chem. 52, 1591–1592.

    CAS  Google Scholar 

  17. Torossian, K., and Bell, A.W. (1991) Purification and Characterization of an Acid-Resistant Lipase from Aspergillus niger, Biotechnol. Appl. Biochem. 13, 205–211.

    CAS  Google Scholar 

  18. Bernardi, G. (1971) Chromatography of Proteins on Hydroxyapatite, Methods Enzymol. 22, 325–339.

    Article  CAS  Google Scholar 

  19. Kwon, D.Y., and Rhee, J.S. (1986) A Simple and Rapid Colorimetric Method for Determination of Free Fatty Acids for Lipase Assay, J. Am. Oil Chem. Soc. 63, 89–92.

    CAS  Google Scholar 

  20. Kouker, G., and Jaeger, K. (1987) Specific and Sensitive Plate Assay for Bacterial Lipase, Appl. Environ. Microbiol. 53, 211–213.

    PubMed  CAS  Google Scholar 

  21. 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.

    Article  PubMed  CAS  Google Scholar 

  22. Laemmli, U.K. (1970) Cleavage of Structural Proteins During the Assembly of the Head of Bacteriophage T4, Nature 227, 680–685.

    Article  PubMed  CAS  Google Scholar 

  23. Butcher, L.A., and Tomkins, J.K. (1986) A Comparison of Silver Staining Methods for Detecting Proteins in Ultrathin Gels on Support Films After Isoelectric Focussing, Anal. Biochem. 148, 384–388.

    Article  Google Scholar 

  24. Konat, G., Offner, H., and Mellah, J. (1984) Improved Sensitivity for Detection and Quantitation of Glycoproteins on Polyacrylamide Gels, Experientia 40, 303.

    Article  CAS  Google Scholar 

  25. Thornton, D.J., Carlstedt, I., and Sheehan, J.K. (1994) Identification of Glycoproteins on Nitrocellulose Membranes and Gels, in Basic Protein and Peptide Protocols (Walker, J.M., ed.) pp. 119–128, Humana Press, New Jersey.

    Chapter  Google Scholar 

  26. Sztajer, H., Lunsdorf, H., Erdmann, H., Menge, U., and Schmid, R.D. (1992) Purification and Properties of Lipase from Penicillium simplissimum, Biochim. Biophys. Acta 1124, 253–261.

    PubMed  CAS  Google Scholar 

  27. Haas, M.J., Cichowicz, D.J., and Bailey, D.G. (1992) Purification and Characterization of an Extracellular Lipase from Rhizopus delemar, Lipids 27, 571–576.

    CAS  Google Scholar 

  28. Boel, E., Huge-Jensen, B., Christensen, M., Thim, L., and Fiil, N.P. (1988) Rhizomucor miehei Triglyceride Lipase Is Synthesized as a Precursor, Lipids 23, 701–706.

    PubMed  CAS  Google Scholar 

  29. Maley, F., Trimble, R.B., Tarentino, A.L., and Plummer, T.H., Jr. (1989) Characterization of Glycoproteins and Their Associated Oligosaccharides Through the Use of endoglycosidases, Anal. Biochem. 180, 195–204.

    Article  PubMed  CAS  Google Scholar 

  30. Klibanov, A.M. (1989) Enzymatic Catalysis in Anhydrous Organic Solvents, Trends. Biochem. Sci. 14, 141–144.

    Article  PubMed  CAS  Google Scholar 

  31. Bott, R., Shield, J.W., and Poulose, A.J. (1994) Protein Engineering of Lipases, in Lipases, Their Structure, Biochemistry and Application (Woolley, P., and Peterson, S.B., eds.), pp. 337–354, Cambridge University Press, Cambridge.

    Google Scholar 

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Author notes

  1. Variketta M. Haridasan Namboodiri

    Present address: Boston University School of Medicine, 700 Albany St., 02118-2526, Boston, MA

Authors and Affiliations

  1. Department of Biochemistry, Bose Institute, P-1/12 CIT Scheme VII M, 700054, Calcutta, India

    Variketta M. Haridasan Namboodiri & Rajagopal Chattopadhyaya

Authors
  1. Variketta M. Haridasan Namboodiri
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  2. Rajagopal Chattopadhyaya
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Correspondence to Rajagopal Chattopadhyaya.

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Haridasan Namboodiri, V.M., Chattopadhyaya, R. Purification and biochemical characterization of a novel thermostable lipase from Aspergillus niger . Lipids 35, 495–502 (2000). https://doi.org/10.1007/s11745-000-549-3

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  • DOI: https://doi.org/10.1007/s11745-000-549-3

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