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Secretory Expression, Purification, Characterization, and Application of an Aspergillus oryzae Prolyl Aminopeptidase in Bacillus subtilis

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Abstract

cDNA coding a prolyl aminopeptidase (PAP) was cloned from Aspergillus oryzae and over expressed in Bacillus subtilis with a 6×His tag in N-terminus. The recombinant prolyl aminopeptidase was secreted to extracellular by adding 2 mM CaCl2 and 5% D-sorbitol in TB medium; the enzyme activity in fermented supernatant increased from 7.2 to 41.5 U mL−1. It has been purified 4.3-fold through Ni-chelating affinity chromatography with a recovery of 47.3%. The purified enzyme is stable below 50 °C and within pH 6–11, and with the highest activity at pH 7.5 and 50 °C. Several kinds of salt can activate enzyme activity in a certain concentration and the relative activity was 127.02% even when the concentration of NaCl reached 4.36 M. It cleaved N-terminal Pro residues from many peptides but shown different hydrolysis rates for various Pro-X dipeptides or peptides which are of different lengths. It combined with alkaline protease and leucine aminopeptidase to hydrolyze casein, many free amino acid especially proline and small peptide of hydrolysate increased significantly.

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References

  1. Gonzales, T., & Robert-Baudouy, J. (1996). Bacterial aminopeptidases: properties and functions. FEMS Microbiology Reviews, 18, 319–344.

    Article  CAS  Google Scholar 

  2. Gao, X. X., Cui, W. J., Tian, Y. P., & Zhou, Z. M. (2013). Over-expression, secretion, biochemical characterisation, and structure analysis of Bacillus subtilis aminopeptidase. Journal of the Science of Food & Agriculture, 93, 2810–2815.

    Article  CAS  Google Scholar 

  3. Santos, K., & Medrano, F. J. (2007). Expression, purification, and characterization of an aminopeptidase (Xac2987) with broad specificity from Xanthomonas axonopodis pv. Citri. Protein Expression and Purification, 52, 117–122.

    Article  CAS  Google Scholar 

  4. Nicole, M., Holger, Z., & Martin, R. (2015). Prolyl-specifific peptidases for applications in food protein hydrolysis. Applied Microbiology and Biotechnology, 99, 7837–7846.

    Article  Google Scholar 

  5. Nandan, A., Pandey, A., & Nampoothiri, K. M. (2011). Proline-specific extracellular aminopeptidase purified from Streptomyces lavendulae. Applied biochemistry & biotechnology, 163, 994–1001.

    Article  CAS  Google Scholar 

  6. Polgar, L. (2005). The catalytic triad of serine peptidases. Cellular and Molecular Life Sciences, 62, 2167–2172.

    Article  Google Scholar 

  7. Li, M., Chen, C. A., Davies, D. R., & Chin, T. K. (2010). Induced-fit mechanism for prolyl endopeptidase. Journal of Biological Chemistry, 285, 21487–21495.

    Article  CAS  Google Scholar 

  8. Sarid, S., Berger, A., & Katchalski, E. (1959). Proline iminopeptidase. Journal of Biological Chemistry, 234, 1740–1746.

    CAS  Google Scholar 

  9. Matsushita-Morita, M., Furukawa, I., Suzuki, S., Yamagata, Y., Koide, Y., Ishida, H., Takeuchi, M., Kashiwagi, Y., & Kusumoto, K. I. (2010). Characterization of recombinant prolyl aminopeptidase from Aspergillus oryzae. Journal of Applied Microbiology, 109, 156–165.

    CAS  Google Scholar 

  10. Li, N., Wu, J. M., Zhang, L. F., Feng, H., et al. (2010). Characterization of a unique proline iminopeptidase from white-rot basidiomycetes Phanerochaete chrysosporium. Biochimie, 92, 779–788.

    Article  CAS  Google Scholar 

  11. Arima, J., Uesugi, Y., Iwabuchi, M., & Hatanaka, T. (2008). Streptomyces aminopeptidase P: biochemical characterization and insight into the roles of its N-terminal domain. Protein Engineering,Design & Selection, 21, 45–53.

    Article  CAS  Google Scholar 

  12. Murai, A., Tsujimoto, Y., Matsui, H., & Watanabe, K. (2004). An Aneurinibacillus sp. strain AM-1 produces a proline-specific aminopeptidase useful for collagen degradation. Journal of Applied Microbiology, 96, 810–818.

    Article  CAS  Google Scholar 

  13. Akioka, M., Nakano, H., Horikiri, A., Tsujimoto, Y., Matsui, H., Shimizu, T., Nakatsu, T., Kato, H., & Watanabe, K. (2006). Overexpression, purification, crystallization and preliminary X-ray crystallographic studies of a proline-specific aminopeptidase from Aneurinibacillus sp. strain AM-1. Acta Crystallographica Section F-Structural Biology and Crystallization Communications, 62, 1266–1268.

    Article  CAS  Google Scholar 

  14. Zhang, J., Kang, Z., Ling, Z., Cao, W., Liu, L., Wang, M., Du, G., & Chen, J. (2013). High-level extracellular production of alkaline polygalacturonate lyase in Bacillus subtilis with optimized regulatory elements. Bioresource Technology, 146, 543–548.

    Article  CAS  Google Scholar 

  15. Harwood, C. R., & Cranenburgh, R. (2008). Bacillus protein secretion: an unfolding story. Trends in Microbiology, 16, 73–79.

    Article  CAS  Google Scholar 

  16. Ploss, T. N., Reilman, E., Montefreeante, C. G., et al. (2016). Homogeneity and heterogeneity in amylase production by Bacillus subtilis under different growth conditions. Microbial Cell Factories, 15, 57.

    Article  Google Scholar 

  17. Barbe, V., Cruveiller, S., Kunst, F., et al. (2009). From a consortium sequence to a unified sequence: the Bacillus subtilis 168 reference genome a decade later. Microbiology-SGM., 155, 1758–1775.

    Article  CAS  Google Scholar 

  18. Kawabata, Y., Kimara, K., & Funane, K. (2012). Extracellular production of cycloisomaltooligosaccharide glucanotransferase and cyclodextran by a protease-deficient Bacillus subtilis host–vector system. Applied Microbiology and Biotechnology, 93, 1877–1884.

    Article  CAS  Google Scholar 

  19. Vavrova, L., Muchova, K., & Barak, I. (2010). Comparison of different Bacillus subtilis expression systems. Reserach Microbiology., 146, 791–797.

    Article  Google Scholar 

  20. Zweers, J. C., Barak, I., Becher, D., Driessen, A. J., Hecker, M., Kontinen, V. P., Saller, M. J., Vavrova, L., & Dijl, J. M. V. (2008). Towards the development of Bacillus subtilis as a cell factory for membrane proteins and protein complexes. Microbial Cell Factories, 7, 1–20.

    Article  Google Scholar 

  21. Dijl, J. M. V., & Hecker, M. (2013). Bacillus subtilis: from soil bacterium to super-secreting cell factory. Microbial Cell Factories, 12, 3–3.

    Article  Google Scholar 

  22. Liu, B., Zhang, J., Li, B., Liao, X. R., Du, G. C., & Chen, J. (2013). Expression and characterization of extreme alkaline, oxidation-resistant keratinase from Bacillus licheniformis in recombinant Bacillus subtilis WB600 expression system and its application in wool fiber processing. World Journal of Microbiology & Biotechnology, 29, 825–832.

    Article  Google Scholar 

  23. Dong, H. N., & Zhang, D. W. (2014). Current development in genetic engineering strategies of Bacillus species. Microbial Cell Factories, 13, 63.

    Article  Google Scholar 

  24. Ding, G. W., Zhou, N. D., & Tian, Y. P. (2014). Over-expression of a proline specific aminopeptidase from Aspergillus oryzae JN-412 and its application in collagen degradation. Applied Biochemistry & Biotechnology, 173, 1765–1777.

    Article  CAS  Google Scholar 

  25. Liu, J. M., Xin, X. J., Li, C. X., Xu, J. H., & Bao, J. (2012). Cloning of thermostable cellulase genes of Clostridium thermocellum and their secretive expression in Bacillus subtilis. Applied Biochemistry & Biotechnology, 166, 652–662.

    Article  CAS  Google Scholar 

  26. Atlan, D., Laloi, P., & Portalier, R. (1989). Isolation and characterization of aminopeptidase-deficient Lactobacillus bulgaricus mutants. Applied and Environmental Microbiology, 55, 1717–1723.

    CAS  Google Scholar 

  27. Fuke, Y., & Matsuoka, H. (1993). The purification and characterization of prolyl aminopeptidase from Penicillium camemberti. Journal of Dairy Science, 76, 2478–2484.

    Article  CAS  Google Scholar 

  28. Yu, Z., Wang, Q., Ma, Q., & Zhang, R. (2013). Secretory expression of lacticin Q fused with SUMO in Bacillus subtilis. Protein Expression & Purification, 89, 51–55.

    Article  CAS  Google Scholar 

  29. Rawlings, N. D., Barrett, A. J., & Bateman, A. (2010). MEROPS: the peptidase database. Nucleic Acids Research, 36, 320–325.

    Article  Google Scholar 

  30. Basten, D. E., Moers, A. P., Ooyen, A. J., & Schaap, P. J. (2005). Characterisation of Aspergillus niger prolyl aminopeptidase. Molecular Genetics & Genomics, 272, 673–679.

    Article  CAS  Google Scholar 

  31. Mahon, C. S., Goetz, D. H., Tuohy, M. G., et al. (2009). Characterization of a multimeric, eukaryotic prolyl aminopeptidase: an inducible and highly specific intracellular peptidase from the non-pathogenic fungus Talaromyces emersonii. Microbiology-SGM, 155, 3673–3682.

    Article  CAS  Google Scholar 

  32. Wang, Y., Liu, H., Wang, S., Li, H., & Xin, Q. (2015). Overexpressing of a novel wheat prolyl aminopeptidase gene enhances zinc stress tolerance in transgenic Arabidopsis thaliana. Plant Cell Tissue & Organ Culture, 121, 489–499.

    Article  CAS  Google Scholar 

  33. Uraji, M., Arima, J., Uesugi, Y., Iwabuchi, M., & Hatanaka, T. (2007). Effect of salt on the activity of Streptomyces prolyl aminopeptidase. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 1774, 1462–1469.

    Article  CAS  Google Scholar 

  34. Fukuchi, S., Yoshimune, K., Wakayama, M., Moriguchi, M., & Nishikawa, K. (2003). Unique amino acid composition of proteins in halophilic bacteria. Journal of Molecular Biology, 327, 347–357.

    Article  CAS  Google Scholar 

  35. Gao, X. X., Cui, W. J., Ding, N., Liu, Z. M., Tian, Y. P., & Zhou, Z. M. (2013). Structure-based approach to alter the substrate specificity of Bacillus subtilis aminopeptidase. Prion, 7, 328–334.

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by the National High Technology Research and Development Program of China (863 Program, 2011AA100905), the Fundamental Research Funds for the Central Universities (JUSRP51402A), and the Program for New Century Excellent Talents in University (NCET-12-0878).

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Authors and Affiliations

  1. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China

    Kai-Dao Wang, Ke-Hong Wang, Nan-Di Zhou & Ya-Ping Tian

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  1. Kai-Dao Wang
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  2. Ke-Hong Wang
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Correspondence to Ya-Ping Tian.

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Wang, KD., Wang, KH., Zhou, ND. et al. Secretory Expression, Purification, Characterization, and Application of an Aspergillus oryzae Prolyl Aminopeptidase in Bacillus subtilis . Appl Biochem Biotechnol 181, 1611–1623 (2017). https://doi.org/10.1007/s12010-016-2305-3

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  • DOI: https://doi.org/10.1007/s12010-016-2305-3

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