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In Silico and Experimental Characterization of Chimeric Bacillus thermocatenulatus Lipase with the Complete Conserved Pentapeptide of Candida rugosa Lipase

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Abstract

Lipases are one of the highest value commercial enzymes as they have broad applications in detergent, food, pharmaceutical, and dairy industries. To provide chimeric Bacillus thermocatenulatus lipase (BTL2), the completely conserved pentapeptide (112Ala-His-Ser-Gln-Gly116) was replaced with similar sequences (207Gly-Glu-Ser-Ala-Gly211) of Candida rugosa lipase (CLR) at the nucleophilic elbow region. For this purpose, three mutations including A112G, H113E, and Q115A were inserted in the conserved pentapeptide sequence of btl2 gene. Based on the crystal structures of 2W22, the best structure of opened form of the chimeric lipases were garnered using the MODELLER v9.10 software. The native and chimeric lipases were docked to a set of ligands, and a trial version of Molegro Virtual Docker (MVD) software was used to obtain the energy values. Docking results confirmed chimeric lipase to be better than the native lipase. Following the in silico study, cloning experiments were conducted and expression of native and chimeric btl2 gene in Pichia pastoris was performed. The native and chimeric lipases were purified, and the effect of these mutations on characteristics of chimeric lipase studied and then compared with those of native lipase. Chimeric lipase exhibited 1.6-fold higher activity than the native lipase at 55 °C. The highest percentage of both lipases activity was observed at 60 °C and pH of 8.0. The ion Ca2+ slightly inhibited the activity of both lipases, whereas the organic solvent enhanced the lipase stability of chimeric lipase as compared with the native lipase. According to the results, the presence of two glycine residues at the conserved pentapeptide region of this chimeric lipase (112 Gly-Glu-Ser-Ala-Gly 116) may increase the flexibility of the nucleophilic elbow region and affect the enzyme activity level.

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References

  1. Widmann, M., Juhl, P. B., & Pleiss, J. (2010). BMC Genomics, 11, 123.

    Article  Google Scholar 

  2. Jaeger, K. E., & Reetz, M. T. (1998). Trends in Biotechnology, 16, 396–403.

    Article  CAS  Google Scholar 

  3. Schrag, J. D., & Cygler, M. (1997). Methods in Enzymology, 284, 85–107.

    Article  CAS  Google Scholar 

  4. Messaoudi, A., Belguith, H., & Ben Hamida, J. (1998). Biochemistry Journal, 322, 75–80.

    Google Scholar 

  5. Kim, H. K., Park, S. Y., Lee, J. K., & Oh, T. K. (1998). Bioscience, Biotechnology, and Biochemistry, 62, 66–71.

    Article  CAS  Google Scholar 

  6. Eggert, T., Pouderoyen, G. V., Dijkstra, B. W., & Jaeger, K. E. (2001). FEBS Letters, 502, 89–92.

    Article  CAS  Google Scholar 

  7. Eggert, T., van Pouderoyen, G., Pencreac’h, G., Douchet, I., Verger, R., Bauke, W., Dijkstra, B. W., & Jaeger, K.-E. (2002). Colloids and Surfaces. B, Biointerfaces, 26, 37–46.

    Article  CAS  Google Scholar 

  8. Schlieben, N. H., Niefind, K., & Schomburg, D. (2004). Protein Expression and Purification, 34, 103–110.

    Article  CAS  Google Scholar 

  9. Jaeger, K. E., Dijkstra, B. W., & Hertz, M. T. (1999). Annual Review of Microbiology, 53, 315–351.

    Article  CAS  Google Scholar 

  10. Ollis, D. L., Shea, E., Cygler, M., Dijkstra, B., Frolow, F., Franken, S. M., Harel, M., Remington, S. J., Silman, I., Schrag, J., Sussman, J. L., Verschueren, K. H. G., & Goldman, A. (1992). Protein Engineering, 5, 197–211.

    Article  CAS  Google Scholar 

  11. Lee, L. C., Chen, Y. T., Yen, C. C., Chiang, T. C., Tang, S. J., Lee, G. C., & Shaw, J. F. (2007). Journal of Agricultural and Food Chemistry, 55, 5103–5108.

    Article  CAS  Google Scholar 

  12. Lotti, M., Tramontano, A., Longhi, S., Fusetti, F., Brocca, S., Pizzi, E., & Alberghina, L. (1994). Protein Engineering, 7, 531–535.

    Article  CAS  Google Scholar 

  13. Jeong, S. T., Kim, H. K., Kim, S. J., & Chi, S. W. (2002). Journal of Biological Chemistry, 277, 17041–17047.

    Article  CAS  Google Scholar 

  14. Altschul, S. F., Madden, T. L., Schäffer, A. A., Zheng, J., Zhang, Z., Miller, W., & Lipman, D. J. (1997). Nucleic Acids Research, 25, 3389–3402.

    Article  CAS  Google Scholar 

  15. Eswar, N., Marti-Renom, M.A., Webb, B., Madhusudhan, M., Eramian, S.D., Shen, M., Pieper, U., & Sali, A. (2006). Current Protocols in Bioinformatics. New York: Wiley, Supplement 15, pp. 5.6.1–5.6.30.

  16. Laskowski, R. A., MacArthur, M. W., Moss, D. S., & Thornton, J. M. (1993). Journal of Applied Crystallography, 26, 283–291.

    Article  CAS  Google Scholar 

  17. Colovos, C., & Yeates, T. O. (1993). Protein Science, 2, 1511–1519.

    Article  CAS  Google Scholar 

  18. Benkert, P., Tosatto, S. C. E., & Schomburg, D. (2008). Proteins: Structure, Function, and Bioinformatics, 71, 261–277.

    Article  CAS  Google Scholar 

  19. Wiederstein, M., & Sippl, M. J. (2007). Nucleic Acids Research, 35, W407–W410.

    Article  Google Scholar 

  20. Humphrey, W., Dalke, A., & Schulten, K. (1996). Journal of Molecular Graphics, 14, 33–38.

    Article  CAS  Google Scholar 

  21. Thomsen, R., & Christensen, M. H. (2006). Journal of Medicinal Chemistry, 49, 3315–3321.

    Article  CAS  Google Scholar 

  22. Karkhane, A. A., Yakhchali, B., Rastgar Jazii, F., & Bambai, B. (2009). Journal of Molecular Catalysis B: Enzymatic, 61, 162–167.

    Article  CAS  Google Scholar 

  23. Quyen, D. T., Schmidt-Dannert, C., & Schmid, R. D. (2003). Protein Expression and Purification, 28, 102–111.

    Article  CAS  Google Scholar 

  24. Carrasco-Lopez, C., Godoy, C., De Las Rivas, B., Fernandez-Lorente, G., Palomo, J. M., Guisan, J. M., Fernandez-Lafuente, R., Martinez-Ripoll, M., & Hermoso, J. A. (2009). Journal of Biological Chemistry, 284, 4365–4372.

    Article  CAS  Google Scholar 

  25. Bolton, E., Wang, Y., Thiessen, P. A., & Bryant, S. H. (2008). Chapter 12 in Annual Reports in Computational Chemistry, Volume 4. Washington: American Chemical Society.

    Google Scholar 

  26. Abdul Rahman, M. B., Md Tajudin, S., Hussein, M. Z., Abdul Rahman, R. N. Z., Salleh, A. B., & Basri, M. (2005). Applied Clay Science, 29, 111–116.

    Article  CAS  Google Scholar 

  27. Yin, S., Ding, F., & Dokholyan, N. V. (2007). Nature Methods, 4, 466–467.

    Article  CAS  Google Scholar 

  28. Schmidt-Dannert, C., Rua, M. L., Atomi, H., & Schmid, R. D. (1996). Biochimica et Biophysica Acta, 1301, 105–114.

    Article  Google Scholar 

  29. Salameh, M. A., & Wiegel, J. (2010). The Open Biochemistry Journal, 4, 22–28.

    Article  CAS  Google Scholar 

  30. Joseph, D., Yunge, L., Miroslaw, C., Dietmar, L., Tanja, B., Hans-Juergen, H., Rolf, S., et al. (1997). Structure, 5, 187–202.

    Article  Google Scholar 

  31. Rahman, R. N. Z. A., Baharum, S. N., Basri, M., & Salleh, A. B. (2005). Analytical Biochemistry, 341, 267–274.

    Article  CAS  Google Scholar 

  32. Yan, B. X., & Sun, Y. Q. (1997). Journal of Biological Chemistry, 272, 3190–3194.

    Article  CAS  Google Scholar 

  33. Tsou, C. L. (1986). Trends in Biochemical Sciences, 111, 427–429.

    Article  Google Scholar 

  34. Tsou, C. L. (1993). Science, 262, 380–381.

    Article  CAS  Google Scholar 

Download references

Acknowledgment

This study has been supported by the Iran National Science Foundation (INSF), research project no. 88002118, for which the authors are very grateful.

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Authors and Affiliations

  1. Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), P.O. Box 14965/161, Tehran, Iran

    Mostafa Hosseini, Ali Asghar Karkhane, Bagher Yakhchali, Mehdi Shamsara, Saeed Aminzadeh, Dena Morshedi, Kamahldin Haghbeen, Ibrahim Torktaz, Esmat Karimi & Zahra Safari

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  1. Mostafa Hosseini
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  2. Ali Asghar Karkhane
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  3. Bagher Yakhchali
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  4. Mehdi Shamsara
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Correspondence to Ali Asghar Karkhane.

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Hosseini, M., Karkhane, A.A., Yakhchali, B. et al. In Silico and Experimental Characterization of Chimeric Bacillus thermocatenulatus Lipase with the Complete Conserved Pentapeptide of Candida rugosa Lipase. Appl Biochem Biotechnol 169, 773–785 (2013). https://doi.org/10.1007/s12010-012-0014-0

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