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pH-Induced Molten Globule State of Rhizopus niveus Lipase is More Resistant Against Thermal and Chemical Denaturation Than Its Native State

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Abstract

Here, we have characterized four pH-dependent states: alkaline state, “B” (pH 9.0), native state, “N” (pH 7.4), acid-induced state, “A” (pH 2.2) and molten globule state, “MG” (pH 1.8) of Rhizopus niveus lipase (RNL) by CD, tryptophanyl fluorescence, ANS binding, DLS, and enzyme activity assay. This “MG” state lacks catalytic activity and tertiary structure but it has native-like significant secondary structure. The “R h” of all the four states of RNL obtained from DLS study suggests that the molecular compactness of the protein increases as the pH of solution decreases. Kinetic analysis of RNL shows that it has maximum catalytic efficiency at state “B” which is 15-fold higher than state “N.” The CD and tryptophanyl fluorescence studies of RNL on GuHCl and temperature-induced unfolding reveal that the “MG” state is more stable than the other states. The DSC endotherms of RNL obtained at pH 9.0, 7.4, and 2.2 were with two transitions, while at pH 1.8 it showed only a single transition.

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Abbreviations

ANS:

1-Anilino-8-napthalene sulfonate

C m :

Midpoint concentration

DSC:

Differential scanning calorimetry

GuHCl:

Guanidine hydrochloride

∆H :

Enthalpy

MG:

Molten globule

T m :

Midpoint temperature

RNL:

Rhizopus niveus lipase

DLS:

Dynamic light scattering

References

  1. Porter-Peden, L., Kamper, S. G., Wal, M. V., Blankespoor, R., & Sinniah, K. (2008). Estimating kinetic and thermodynamic parameters from single molecule enzyme-inhibitor interactions. Langmuir, 24, 11556–11561.

    Article  PubMed  CAS  Google Scholar 

  2. Povarova, O. I., Kuznetsova, I. M., & Turoverov, K. K. (2010). Differences in the pathways of proteins unfolding induced by urea and guanidine hydrochloride: Molten globule state and aggregates. PLoS ONE, 5, e15035.

    Article  PubMed  CAS  Google Scholar 

  3. Siddiqui, K. S., & Cavicchioli, R. (2005). Improved thermal stability and activity in the cold-adapted lipase B from Candida antarctica following chemical modification with oxidized polysaccharides. Extremophiles, 9, 471–476.

    Article  PubMed  CAS  Google Scholar 

  4. Akhtar, M. S., Ahmad, A., & Bhakuni, V. (2002). Guanidinium chloride- and urea-induced unfolding of the dimeric enzyme glucose oxidase. Biochemistry, 19, 3819–3827.

    Article  Google Scholar 

  5. Anfinsen, C. B., & Scheraga, H. A. (1975). Experimental and theoretical aspects of protein folding. Advances in Protein Chemistry, 29, 205–300.

    Article  PubMed  CAS  Google Scholar 

  6. Privalov, P. L. (1996). Intermediate states in protein folding. Journal of Molecular Biology, 258, 707–725.

    Article  PubMed  CAS  Google Scholar 

  7. Ptitsyn, O. B. (1995). Molten globule and protein folding. Advances in Protein Chemistry, 47, 83–87.

    Article  PubMed  CAS  Google Scholar 

  8. Brenner, S. (1988). The molecular evolution of genes and proteins: A tale of two serines. Nature, 334, 528–530.

    Article  PubMed  CAS  Google Scholar 

  9. Ollis, D. L., Cheah, E., Cygler, M., Dijkstra, B., Frolow, F., Franken, S. M., et al. (1992). The alpha/beta hydrolase fold. Protein Engineering Design and Selection, 5, 197–211.

    Article  CAS  Google Scholar 

  10. Jaeger, K. E., & Eggert, T. (2002). Lipases for biotechnology. Current Opinion in Biotechnology, 13, 390–397.

    Article  PubMed  CAS  Google Scholar 

  11. Jaeger, K. E., Dijkstra, B. W., & Reetz, M. T. (1999). Bacterial biocatalysts: Molecular biology, three-dimensional structures and biotechnological applications of lipases. Annual Review of Microbiology, 53, 315–351.

    Article  PubMed  CAS  Google Scholar 

  12. Weber, N., Klein, E., Vosmann, K., & Mukherjee, K. D. (2004). Mono-thioesters and di-thioesters by lipase-catalyzed reactions of alpha, omega alkanedithiols with palmitic acid or its methyl ester. Applied Microbiology and Biotechnology, 64, 800–805.

    Article  PubMed  CAS  Google Scholar 

  13. Gutiérrez, A., del Río, J. C., & Martínez, A. T. (2009). Microbial and enzymatic control of pitch in the pulp and paper industry. Applied Microbiology and Biotechnology, 82, 1005–1018.

    Article  PubMed  Google Scholar 

  14. Zandonella, G., Haalck, L., Spener, F., Paltauf, F., & Hermetter, A. (1997). New fluorescent glycerolipids for a dual wavelength assay of lipase activity and stereoselectivity. Journal of Molecular Catalysis B: Enzymatic, 3, 127–130.

    Article  CAS  Google Scholar 

  15. Theil, F. (1995). Lipase-supported synthesis of biologically active compounds. Chemical Reviews, 95, 2203–2227.

    Article  CAS  Google Scholar 

  16. Chen, Y. H., Yang, J. T., & Martinez, H. (1972). Determination of the secondary structures of proteins by circular dichroism and optical rotatory dispersion. Biochemistry, 11, 4120–4131.

    Article  PubMed  CAS  Google Scholar 

  17. Pace, C. N., & Shaw, K. L. (2000). Linear extrapolation method of analyzing solvent denaturation curves. Proteins, 4, 1–7.

    Article  PubMed  Google Scholar 

  18. Aune, K. C., Salahuddin, A., Zarlengo, M. H., & Tanford, C. (1967). Evidence for residual structure in acid- and heat-denatured proteins. Journal of Biological Chemistry, 242, 4486–4489.

    PubMed  CAS  Google Scholar 

  19. Wong, K. P., & Hamlin, L. M. (1974). Acid denaturation of bovine carbonic anhydrase B. Biochemistry, 13, 2678–2683.

    Article  PubMed  CAS  Google Scholar 

  20. Eftink, M. R. (1994). The use of fluorescence methods to monitor unfolding transitions in proteins. Biophysical Journal, 66, 482–501.

    Article  PubMed  CAS  Google Scholar 

  21. Gasymov, O. K., & Glasgow, B. J. (2007). ANS fluorescence: Potential to augment the identification of the external binding sites of proteins. Biochimica et Biophysica Acta, 1774, 403–411.

    PubMed  CAS  Google Scholar 

  22. Varshney, A., Ahmad, B., Rabbani, G., Kumar, V., Yadav, S., & Khan, R. H. (2010). Acid-induced unfolding of didecameric keyhole limpet hemocyanin: Detection and characterizations of decameric and tetrameric intermediate states. Amino Acids, 39, 899–910.

    Article  PubMed  CAS  Google Scholar 

  23. Fatima, S., Mishra, A., Sen, P., & Khan, R. H. (2008). Characterization of fluoroalcohols-induced intermediates of Mucor miehei lipase at low pH. Protein Peptide Letters, 15, 346–355.

    Article  CAS  Google Scholar 

  24. Rabbani, G., Ahmad, E., Zaidi, N., & Khan, R. H. (2011). pH-dependent conformational transitions in conalbumin (ovotransferrin), a metalloproteinase from hen egg white. Cell Biochemistry and Biophysics, 61, 551–560.

    Article  PubMed  CAS  Google Scholar 

  25. Khodadadi, S., Pawlus, S., Roh, J. H., Garcia-Sakai, V., Mamontov, E., & Sokolov, A. P. (2008). The origin of the dynamic transition in proteins. Journal of Chemical Physics, 128, 195–206.

    Article  Google Scholar 

  26. Andreas, B., Tobias, R., Matthias, H., Winfried, H., Jochen, B., & Marion, A. S. (2005). pH optima in lipase-catalysed esterification. Biocatalysis and Biotransformation, 23, 307–314.

    Article  Google Scholar 

  27. Paiva, A. L., Balcão, V. M., & Malacta, F. X. (2000). Review: Kinetics and mechanisms of reactions catalyzed by immobilized lipases. Enzyme and Microbial Technology, 27, 187–204.

    Article  PubMed  CAS  Google Scholar 

  28. Ahmad, E., Fatima, S., Khan, M. M., & Khan, R. H. (2010). More stable structure of wheat germ lipase at low pH than its native state. Biochimie, 92, 885–893.

    Article  PubMed  CAS  Google Scholar 

  29. Khyami-Horani, H. (1996). Thermotolerant strain of Bacillus licheniformis producing lipase. World Journal of Microbiology and Biotechnology, 12, 399–401.

    Article  CAS  Google Scholar 

  30. Nagaoka, K., & Yamada, Y. (1967). Purification of Mucor lipases and their properties. Agricultural and Biological Chemistry, 31, 1357–1366.

    Article  Google Scholar 

  31. Schmidt-Dannert, C., Sztajer, H., Stocklein, W., Menge, U., & Schmid, R. D. (1994). Screening purification and properties of a thermophilic lipase from Bacillus thermocatenulatus. Biochimica et Biophysica Acta, 1214, 43–53.

    PubMed  CAS  Google Scholar 

  32. He, G. J., Zhang, A., Liu, W. F., Cheng, Y., & Yan, Y. B. (2009). Conformational stability and multistate unfolding of poly (A)-specific ribonuclease. FEBS J, 276, 2849–2860.

    Article  PubMed  CAS  Google Scholar 

  33. Biyani, K., Kahsai, M. A., Clark, A. T., Armstrong, T. L., & Edmondson, S. P. (2005). Solution structure, stability, and nucleic acid binding of the hyperthermophile protein Sso10b2. Biochemistry, 44, 14217–14230.

    Article  PubMed  CAS  Google Scholar 

  34. Robic, S., Guzman-Casado, M., Sanchez-Ruiz, J. M., & Marqusee, S. (2003). Role of residual structure in the unfolded state of a thermophilic protein. Proceedings of the National Academy of Sciences of the United States of America, 100, 11345–11349.

    Article  PubMed  CAS  Google Scholar 

  35. Makhatadze, G. I., & Privalov, P. L. (1992). Protein interactions with urea and guanidinium chloride. A calorimetric study. Journal of Molecular Biology, 226, 491–505.

    Article  PubMed  CAS  Google Scholar 

  36. Yamasaki, M., Yano, H., & Aoki, K. (1990). Differential scanning calorimetric studies on bovine serum albumin: I. Effects of pH and ionic strength. International Journal of Biological Macromolecules, 12, 263–268.

    Article  PubMed  CAS  Google Scholar 

  37. Lackowicz, J. (1999). Principals of fluorescence spectroscopy. New York: Kluwer Academic/Plenum Publishers.

    Google Scholar 

  38. Demaurex, N. (2002). pH homeostasis of cellular organelles. News in Physiological Sciences, 17, 1–5.

    PubMed  CAS  Google Scholar 

  39. Hanakam, F., Gerisch, G., Lotz, S., Alt, T., & Seelig, A. (1996). Binding of hisactophilin I and II to lipid membranes is controlled by a pH-dependent myristoyl-histidine switch. Biochemistry, 35, 11036–11044.

    Article  PubMed  CAS  Google Scholar 

  40. Goto, Y., & Fink, A. L. (1989). Conformational states of beta-lactamase: Molten-globule states at acidic and alkaline pH with high salt. Biochemistry, 28, 945–952.

    Article  PubMed  CAS  Google Scholar 

  41. Narimoto, T., Sakurai, K., Okamoto, A., Chatani, E., Hoshino, M., Hasegawa, K., et al. (2004). Conformational stability of amyloid fibrils of beta 2-microglobulin probed by guanidine-hydrochloride-induced unfolding. FEBS Letters, 576, 313–319.

    Article  PubMed  CAS  Google Scholar 

  42. Ren, M. Y., Bai, S., Zhang, D. H., & Sun, Y. (2008). pH memory of immobilized lipase for (±)-menthol resolution in ionic liquid. Journal of Agricultural and Food Chemistry, 56, 2388–2391.

    Article  PubMed  CAS  Google Scholar 

  43. Kohno, M., Funatsu, J., Mikami, B., Kugimiya, W., Matsuo, T., & Morita, Y. (1996). The crystal structure of lipase II from Rhizopus niveus at 2.2 Å resolution. Journal of Biochemistry, 120, 505–510.

    PubMed  CAS  Google Scholar 

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Acknowledgments

This study was supported by the University Grants Commission (UGC) New Delhi, funded project grant No. 32-543/2006 (SR). G. Rabbani acknowledged to Council of Scientific and Industrial Research (CSIR), New Delhi, India for financial assistance in the form of Senior Research Fellow (SRF).

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Authors and Affiliations

  1. Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, 202 002, India

    Gulam Rabbani, Ejaz Ahmad, Nida Zaidi, Sadaf Fatima & Rizwan Hasan Khan

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  1. Gulam Rabbani
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  2. Ejaz Ahmad
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Correspondence to Rizwan Hasan Khan.

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Rabbani, G., Ahmad, E., Zaidi, N. et al. pH-Induced Molten Globule State of Rhizopus niveus Lipase is More Resistant Against Thermal and Chemical Denaturation Than Its Native State. Cell Biochem Biophys 62, 487–499 (2012). https://doi.org/10.1007/s12013-011-9335-9

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