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

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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.

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References

  1. Gupta, R., Beg, Q., & Lorenz, P. (2002). Applied Microbiology and Biotechnology, 59, 15–32. doi:10.1007/s00253-002-0975-y.

    Article  CAS  Google Scholar 

  2. Bordusa, F. (2002). Chemical Reviews, 102, 4817–4868. doi:10.1021/cr010164d.

    Article  CAS  Google Scholar 

  3. Kumar, D., & Bhalla, T. C. (2005). Applied Microbiology and Biotechnology, 68, 726–736. doi:10.1007/s00253-005-0094-7.

    Article  CAS  Google Scholar 

  4. Ogino, H., & Ishikawa, H. (2001). Journal of Bioscience and Bioengineering, 91, 109–116. doi:10.1263/jbb.91.109.

    Article  CAS  Google Scholar 

  5. Sardessai, Y. N., & Bhosle, S. (2004). Biotechnology Progress, 20, 655–660. doi:10.1021/bp0200595.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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. Ghorbel, B., Sellami-Kamoun, A., & Nasri, M. (2003). Enzyme and Microbial Technology, 32, 513–518. doi:10.1016/S0141-0229(03)00004-8.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  13. Gupta, A., & Khare, S. K. (2006). Bioresource Technology, 97, 1788–1793. doi:10.1016/j.biortech.2005.09.006.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  16. Sareen, R., & Mishra, P. (2008). Applied Microbiology and Biotechnology, 79, 399–405. doi:10.1007/s00253-008-1429-y.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  18. Shimogaki, H., Takeuchi, K., Nishino, T., Ohdera, M., Kudo, T., Ohba, K., et al. (1991). Agricultural and Biological Chemistry, 55, 2251–2258.

    CAS  Google Scholar 

  19. Bradford, M. M. (1976). Analytical Biochemistry, 72, 248–254. doi:10.1016/0003-2697(76)90527-3.

    Article  CAS  Google Scholar 

  20. Laemmli, U. K. (1970). Nature, 227, 680. doi:10.1038/227680a0.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  22. Wang, S. L., & Yeh, P. Y. (2006). Process Biochemistry, 41, 1545–1552. doi:10.1016/j.procbio.2006.02.018.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  24. Nicas, T. I., & Iglewski, B. H. (1985). Canadian Journal of Microbiology, 31, 387–392.

    Article  CAS  Google Scholar 

  25. Umeki, S. (1989). Journal of Medical Microbiology, 28, 109–112.

    Article  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  28. Sierecka, J. K. (1998). The International Journal of Biochemistry & Cell Biology, 30, 579–595. doi:10.1016/S1357-2725(98)00007-7.

    Article  CAS  Google Scholar 

  29. Maurer, K. H. (2004). Current Opinion in Biotechnology, 15, 330–334. doi:10.1016/j.copbio.2004.06.005.

    Article  CAS  Google Scholar 

  30. Zaks, A., & Klibanov, A. M. (1988). The Journal of Biological Chemistry, 263, 8017–8021.

    CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  32. Karbalaei-Heidari, H. R., Ziaee, A. A., & Amoozegar, M. A. (2007). Extremophiles, 11, 237–243. doi:10.1007/s00792-006-0031-4.

    Article  CAS  Google Scholar 

  33. Klibanov, A. M. (2001). Nature, 409, 241–246. doi:10.1038/35051719.

    Article  CAS  Google Scholar 

  34. Gupta, A., & Khare, S. K. (2007). Enzyme and Microbial Technology, 42, 11–16. doi:10.1016/j.enzmictec.2007.07.019.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This study was supported by the National Basic Research Program of China (No. 2004CB719600) and the National High Technology Research and Development Program of China (No. 2006AA02Z202).

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Authors and Affiliations

  1. State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, 5 Xinmofan Road, Nanjing, 210009, China

    Xiao-Yu Tang, Bin Wu, Han-Jie Ying & Bing-Fang He

  2. College of Pharmaceutical Engineering, Nanjing University of Technology, 5 Xinmofan Road, Nanjing, 210009, Jiangsu, China

    Bing-Fang He

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  1. Xiao-Yu Tang
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  2. Bin Wu
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Correspondence to Bing-Fang He.

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Tang, XY., Wu, B., Ying, HJ. et al. Biochemical Properties and Potential Applications of a Solvent-Stable Protease from the High-Yield Protease Producer Pseudomonas aeruginosa PT121. Appl Biochem Biotechnol 160, 1017–1031 (2010). https://doi.org/10.1007/s12010-009-8665-1

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  • DOI: https://doi.org/10.1007/s12010-009-8665-1

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