Applied Biochemistry and Biotechnology

, Volume 160, Issue 4, pp 1017–1031 | Cite as

Biochemical Properties and Potential Applications of a Solvent-Stable Protease from the High-Yield Protease Producer Pseudomonas aeruginosa PT121

Article

Abstract

An organic solvent-stable protease from Pseudomonas aeruginosa PT121 was purified in a single step with 55% recovery by hydrophobic interaction chromatography on a Phenyl Sepharose High Performance matrix. The purified protease was homogenous on SDS-PAGE and had an estimated molecular mass of 33 kDa. The optimal pH and temperature conditions for enzyme activity were 8.0 and 60°C, respectively. The enzyme was classified as a metalloprotease based on its strong inhibition by EDTA and 1,10-phenanthroline and exhibited good stability across a broad pH range (6.0–11.0). The protease was quite stable in the presence of various water-miscible organic solvents. This is a unique property of the protease which makes it an ideal choice for application in aqueous-organic phase organic synthesis including peptides synthesis. The synthetic activity of the protease was tested using N-carbobenzoxy-l-asparagine (Z-Asp) and l-phenylalaninamide (Phe-NH2) as substrate in the presence of various water-miscible organic solvents for aspartame precursor synthesis. The highest yield was obtained in the presence of 50% DMSO (91%). The synthesis rate in the presence of DMSO was also much higher than the rates in the other tested organic solvents, and the initial rates of Z-Asp-Phe-NH2 synthesis in mixtures of various water-miscible organic solvents, with the exception of ethanol, correlated with the yields of Z-Asp-Phe-NH2. Furthermore, the PT121 protease was able to use various carboxyl components (Z-AA) and Phe-NH2 as substrates to catalyze the syntheses of the dipeptides, indicating that this protease has a broad specificity for carboxylic acid residue.

Keywords

Purification Characterization Organic solvent-stable protease Enzymatic synthesis 

References

  1. 1.
    Gupta, R., Beg, Q., & Lorenz, P. (2002). Applied Microbiology and Biotechnology, 59, 15–32. doi:10.1007/s00253-002-0975-y.CrossRefGoogle Scholar
  2. 2.
    Bordusa, F. (2002). Chemical Reviews, 102, 4817–4868. doi:10.1021/cr010164d.CrossRefGoogle Scholar
  3. 3.
    Kumar, D., & Bhalla, T. C. (2005). Applied Microbiology and Biotechnology, 68, 726–736. doi:10.1007/s00253-005-0094-7.CrossRefGoogle Scholar
  4. 4.
    Ogino, H., & Ishikawa, H. (2001). Journal of Bioscience and Bioengineering, 91, 109–116. doi:10.1263/jbb.91.109.CrossRefGoogle Scholar
  5. 5.
    Sardessai, Y. N., & Bhosle, S. (2004). Biotechnology Progress, 20, 655–660. doi:10.1021/bp0200595.CrossRefGoogle Scholar
  6. 6.
    Heipieper, H. J., Neumann, G., Cornelissen, S., & Meinhardt, F. (2007). Applied Microbiology and Biotechnology, 74, 961–973. doi:10.1007/s00253-006-0833-4.CrossRefGoogle Scholar
  7. 7.
    Rahman, R., Mahamad, S., Salleh, A. B., & Basri, M. (2007). Journal of Industrial Microbiology & Biotechnology, 34, 509–517. doi:10.1007/s10295-007-0222-8.CrossRefGoogle Scholar
  8. 8.
    Doddapaneni, K. K., Tatineni, R., Vellanki, R. N., Rachcha, S., Anabrolu, N., Narakuti, V., et al. (2007). Microbiological Research, . doi:10.1016/j.micres.2007.04.005.Google Scholar
  9. 9.
    Ghorbel, B., Sellami-Kamoun, A., & Nasri, M. (2003). Enzyme and Microbial Technology, 32, 513–518. doi:10.1016/S0141-0229(03)00004-8.CrossRefGoogle Scholar
  10. 10.
    Li, S., He, B. F., Bai, Z. Z., & Ouyang, P. K. (2008). Journal of Molecular Catalysis B, Enzymatic, 56, 85–88. doi:10.1016/j.molcatb.2008.08.001.CrossRefGoogle Scholar
  11. 11.
    Rahman, R., Geok, L. P., Basri, M., & Salleh, A. B. (2006). Enzyme and Microbial Technology, 39, 1484–1491. doi:10.1016/j.enzmictec.2006.03.038.CrossRefGoogle Scholar
  12. 12.
    Ogino, H., Watanabe, F., Yamada, M., Nakagawa, S., Hirose, T., Noguchi, A., et al. (1999). Journal of Bioscience and Bioengineering, 87, 61–68. doi:10.1016/S1389-1723(99)80009-7.CrossRefGoogle Scholar
  13. 13.
    Gupta, A., & Khare, S. K. (2006). Bioresource Technology, 97, 1788–1793. doi:10.1016/j.biortech.2005.09.006.CrossRefGoogle Scholar
  14. 14.
    Ogino, H., Yamada, M., Watanabe, F., Ichinose, H., Yasuda, M., & Ishikawa, H. (1999). Journal of Bioscience and Bioengineering, 88, 513–518. doi:10.1016/S1389-1723(00)87668-9.CrossRefGoogle Scholar
  15. 15.
    Sareen, R., Bornscheuer, U. T., & Mishra, P. (2004). Journal of Molecular Catalysis B, Enzymatic, 32, 1–5. doi:10.1016/j.molcatb.2004.09.006.CrossRefGoogle Scholar
  16. 16.
    Sareen, R., & Mishra, P. (2008). Applied Microbiology and Biotechnology, 79, 399–405. doi:10.1007/s00253-008-1429-y.CrossRefGoogle Scholar
  17. 17.
    Tang, X. Y., Pan, Y., Li, S., & He, B. F. (2008). Bioresource Technology, 99, 7388–7392. doi:10.1016/j.biortech.2008.01.030.CrossRefGoogle Scholar
  18. 18.
    Shimogaki, H., Takeuchi, K., Nishino, T., Ohdera, M., Kudo, T., Ohba, K., et al. (1991). Agricultural and Biological Chemistry, 55, 2251–2258.Google Scholar
  19. 19.
    Bradford, M. M. (1976). Analytical Biochemistry, 72, 248–254. doi:10.1016/0003-2697(76)90527-3.CrossRefGoogle Scholar
  20. 20.
    Laemmli, U. K. (1970). Nature, 227, 680. doi:10.1038/227680a0.CrossRefGoogle Scholar
  21. 21.
    Gupta, A., Roy, I., Patel, R. K., Singh, S. P., Khare, S. K., & Gupta, M. N. (2005). Journal of Chromatography A, 1075, 103–108. doi:10.1016/j.chroma.2005.03.127.CrossRefGoogle Scholar
  22. 22.
    Wang, S. L., & Yeh, P. Y. (2006). Process Biochemistry, 41, 1545–1552. doi:10.1016/j.procbio.2006.02.018.CrossRefGoogle Scholar
  23. 23.
    Gupta, A., Roy, I., Khare, S. K., & Gupta, M. N. (2005). Journal of Chromatography A, 1069, 155–161. doi:10.1016/j.chroma.2005.01.080.CrossRefGoogle Scholar
  24. 24.
    Nicas, T. I., & Iglewski, B. H. (1985). Canadian Journal of Microbiology, 31, 387–392.CrossRefGoogle Scholar
  25. 25.
    Umeki, S. (1989). Journal of Medical Microbiology, 28, 109–112.CrossRefGoogle Scholar
  26. 26.
    Schokker, E. P., & van Boekel, M. A. J. S. (1997). International Dairy Journal, 7, 165–171. doi:10.1016/S0958-6946(97)00008-3.CrossRefGoogle Scholar
  27. 27.
    Wang, S. L., Kao, T. Y., Wang, C. L., Yen, Y. H., Chern, M. K., & Chen, Y. H. (2006). Enzyme and Microbial Technology, 39, 724–731. doi:10.1016/j.enzmictec.2005.12.007.CrossRefGoogle Scholar
  28. 28.
    Sierecka, J. K. (1998). The International Journal of Biochemistry & Cell Biology, 30, 579–595. doi:10.1016/S1357-2725(98)00007-7.CrossRefGoogle Scholar
  29. 29.
    Maurer, K. H. (2004). Current Opinion in Biotechnology, 15, 330–334. doi:10.1016/j.copbio.2004.06.005.CrossRefGoogle Scholar
  30. 30.
    Zaks, A., & Klibanov, A. M. (1988). The Journal of Biological Chemistry, 263, 8017–8021.Google Scholar
  31. 31.
    Ogino, H., Nakagawa, S., Shinya, K., Muto, T., Fujimura, N., Yasuda, M., et al. (2000). Journal of Bioscience and Bioengineering, 89, 451–457. doi:10.1016/S1389-1723(00)89095-7.CrossRefGoogle Scholar
  32. 32.
    Karbalaei-Heidari, H. R., Ziaee, A. A., & Amoozegar, M. A. (2007). Extremophiles, 11, 237–243. doi:10.1007/s00792-006-0031-4.CrossRefGoogle Scholar
  33. 33.
    Klibanov, A. M. (2001). Nature, 409, 241–246. doi:10.1038/35051719.CrossRefGoogle Scholar
  34. 34.
    Gupta, A., & Khare, S. K. (2007). Enzyme and Microbial Technology, 42, 11–16. doi:10.1016/j.enzmictec.2007.07.019.CrossRefGoogle Scholar
  35. 35.
    Zhou, Y. Y., Yang, T., Wang, N., Xu, L., Huang, Y. B., Wu, X. X., et al. (2003). Enzyme and Microbial Technology, 33, 55–61. doi:10.1016/S0141-0229(03)00095-4.CrossRefGoogle Scholar
  36. 36.
    Murakami, Y., Yoshida, T., Hayashi, S., & Hirata, A. (2002). Biotechnology and Bioengineering, 69, 57–65. doi:10.1002/(SICI)1097-0290(20000705)69:1<57::AID-BIT7>3.0.CO;2-J.CrossRefGoogle Scholar
  37. 37.
    Voyushina, T. L., Potetinova, J. V., Milgotina, E. I., & Stepanov, V. M. (1999). Bioorganic & Medicinal Chemistry, 7, 2953–2959. doi:10.1016/S0968-0896(99)00237-0.CrossRefGoogle Scholar
  38. 38.
    Toledano, S., Williams, R. J., Jayawarna, V., & Ulijn, R. V. (2006). Journal of the American Chemical Society, 128, 1070–1071. doi:10.1021/ja056549l.CrossRefGoogle Scholar

Copyright information

© Humana Press 2009

Authors and Affiliations

  • Xiao-Yu Tang
    • 1
  • Bin Wu
    • 1
  • Han-Jie Ying
    • 1
  • Bing-Fang He
    • 1
    • 2
  1. 1.State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical EngineeringNanjing University of TechnologyNanjingChina
  2. 2.College of Pharmaceutical EngineeringNanjing University of TechnologyNanjingChina

Personalised recommendations