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An effective method for extraction of glutaryl-7-aminocephalosporanic acid acylase from recombinant E. coli cells

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Abstract

In this study, several chemical treatment techniques commonly used for protein extraction were investigated for recovering glutaryl-7-aminocephalosporanic acid acylase (GLA) from recombinant E. coli cells. The best results were obtained by the combined use of cetyltrimethylammonium bromide (CTAB) and KCl. Subsequently, various extraction conditions, such as cation salt, concentrations of CTAB and KCl, extraction temperature, extraction time, and biomass, were optimized to further enhance the release yield and specific activity of GLA. Our results showed that 110% of GLA was released after treatment with 0.5% CTAB (w/v, %) and 0.3 M KCl at 10°C for 12 h, and its specific activity in this extracting solution was approximately 1.5 times higher as compared to that obtained by sonication. This extraction method could avoid the inactivation of GLA caused by drastic mechanical methods, and also enhance its specific activity for industrial extraction.

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References

  1. Kim, Y., K. H. Yoon, Y. Khang, S. Turley, and W. G. J. Hol (2000) The 2.0 A crystal structure of cephalosporin acylase. Structure 8: 1059–1068.

    Article  CAS  Google Scholar 

  2. Grçger, H., Y. Asano, U. T. Bornscheuer, and J. Ogawa (2012) Development of biocatalytic processes in Japan and Germany: From research synergies to industrial applications. Chem. Asian J. 7: 1138–1153.

    Article  Google Scholar 

  3. Sonawane, V. C. (2006) Enzymatic modifications of cephalosporins by cephalosporin acylase and other enzymes. Crit. Rev. Biotechnol. 26: 95–120.

    Article  CAS  Google Scholar 

  4. Li, Y., J. F. Chen, W. H. Jiang, X. Mao, G. P. Zhao, and E. D. Wang (1999) In vivo post-translational processing and subunit reconstitution of cephalosporin acylase from Pseudomonas sp 130. Eur. J. Biochem. 262: 713–719.

    Article  CAS  Google Scholar 

  5. Ma, X. Q., E. Z. Su, Y. Zhu, S. W. Deng, and D. Z. Wei (2013) High-level expression of glutaryl-7-aminocephalosporanic acid acylase from Pseudomonas diminuta NK703 in Escherichia coli by combined optimization strategies. J. Biotechnol. 168: 607–615.

    Article  CAS  Google Scholar 

  6. Volontè, F., F. Marinelli, L. Gastaldo, S. Sacchi, M. S. Pilone, L. Pollegioni, and G. Molla (2008) Optimization of glutaryl-7-aminocephalosporanic acid acylase expression in E. coli. Protein. Expr. Purif. 61: 131–137.

    Article  Google Scholar 

  7. Zhou, H., H. M. Yu, H. Luo, Y. Y. Shi, X. F. Ma, and Z. Y. Shen (2007) Inducible and constitutive expression of glutaryl-7-aminocephalosporanic acid acylase by fusion to maltose-binding protein. Enz. Microb. Technol. 40: 555–562.

    Article  CAS  Google Scholar 

  8. Kim, D. W. and K. H. Yoon (2001) Cloning and high expression of glutaryl 7-aminocephalosporanic acid acylase gene from Pseudomonas diminuta. Biotechnol. Lett. 23: 1067–1071.

    Article  CAS  Google Scholar 

  9. Li, Y., W. H. Jiang, Y. L. Yang, G. P. Zhao, and E. D. Wang (1998) Overproduction and purification of glutaryl-7-amino cephalosporanic acid acylase. Protein Expr. Purif. 12: 233–238.

    Article  CAS  Google Scholar 

  10. Zheng, Y. G., Y. Li, J. F. Chen, W. H. Jiang, G. P. Zhao, and E. D. Wang (2001) Two novel engineered bacteria for secretory expression of glutaryl7-amino-cephalosporanic acid acylase. Biotechnol. Lett. 23: 1781–1787.

    Article  CAS  Google Scholar 

  11. Novella, I. S., C. Fargues, and G. GréVillot (1994) Improvement of the extraction of penicillin acylase from Escherichia coli cells by a combined use of chemical methods. Biotechnol. Bioeng. 44: 379–382.

    Article  CAS  Google Scholar 

  12. Bansal-Mutalik, R. and V. G. Gaikar (2003) Cell permeabilization for extraction of penicillin acylase from Escherichia coli by reverse micellar solutions. Enz. Microb. Technol. 32: 14–26.

    Article  CAS  Google Scholar 

  13. Falconer, R. J., B. K. O’Neill, and A. P. J. Middelberg (1997) Chemical treatment of Escherichia coli: I. Extraction of intracellular protein from uninduced cells. Biotechnol. Bioeng. 53: 453–458.

    Article  CAS  Google Scholar 

  14. Tavares, F. and A. Sellstedt (2000) A simple, rapid and nondestructive procedure to extract cell wall-associated proteins from Frankia. J. Microbiol. Methods. 39: 171–178.

    Article  CAS  Google Scholar 

  15. Cheng, S. W., D. Z. Wei, and Q. X. Song (2006) Extraction penicillin G acylase from Alcaligenes faecalis in recombinant Escherichia coli with cetyl-trimethylammoniumbromide. Biochem. Eng. J. 32: 56–60.

    Article  CAS  Google Scholar 

  16. Wang, P. X., X. Y. Gong, E. Z. Su, J. L. Xie, and D. Z. Wei (2012) A facile pretreatment method for efficient immobilization of penicillin G acylase. Biochem. Eng. J. 56: 17–22.

    Article  Google Scholar 

  17. Sizmann, D., C. Keilmann, and A. Bock (1990) Primary structure requirements for the maturation in vivo of penicillin acylase from Escherichia coli ATCC 11105. Eur. J. Biochem. 192: 143–151.

    Article  CAS  Google Scholar 

  18. Kim, J. K., I. S Yang, H. J. Shin, K. J. Cho, E. K. Ryu, S. H. Kim, S. S Park, and K. H. Kim (2005) Insight into autoproteolytic activation from the structure of cephalosporin acylase: A protein with two proteolytic chemistries. PNAS. 103: 1732–1737.

    Article  Google Scholar 

  19. Kheirolomoom, A., M. Ardjmand, H. Fazelinia, and A. Zakeri (2001) Isolation of penicillin G acylase from Escherichia coli ATCC 11105 by physical and chemical treatments. Biochem. Eng. J. 8: 223–227.

    Article  CAS  Google Scholar 

  20. Balasingham, K., D. Warburton, P. Dunnill, and M. D. Lilly (1972) The isolation and kinetics of penicillin amidase from E. coli. Biochim. Biophys. Acta. (BBA)-Enzymol. 276: 250–256.

    Article  CAS  Google Scholar 

  21. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.

    Article  CAS  Google Scholar 

  22. Nagalakshmi, V. and J. S. Pai (1994) Permeabilization of Escherichia coli cells for enhanced penicillin acylase activity. Biotechnol. Tech. 8: 431–434.

    Article  CAS  Google Scholar 

  23. Felix, H. (1982) Permeabilized cells. Anal Biochem. 120: 211–234.

    Article  CAS  Google Scholar 

  24. Falconer, R. J., B. K. O’Neill, and A. P. J. Middelberg (1998) Chemical treatment of Escherichia coli: II. Direct extraction of recombinant protein from cytoplasmic inclusion bodies in intact cells. Biotechnol. Bioeng. 57: 381–386.

    Article  CAS  Google Scholar 

  25. Ignatova, Z., S. Stoeva, B. Galunsky, C. Hörnle, A. Nurk, E. Piotraschke, W. Voelter, and V. Kasche (1998) Proteolytic processing of penicillin amidase from Alcaligenes faecalis cloned in E. coli yields several active forms. Biotechnol. Lett. 20: 977–982.

    CAS  Google Scholar 

  26. Vaara, M. (1981) Increased outer membrane resistance to ethylenediaminetetraacetate and cations in novel lilip A mutants. J. Bacteriol. 148: 426–434.

    CAS  Google Scholar 

  27. Brass, J. M., W. Boos, and R. Hengge (1981) Reconstitution of maltose transport in malB mutants of Escherichia coli through calcium-induced disruptions of the outer membrane. J. Bacteriol. 146: 10–17.

    CAS  Google Scholar 

  28. Nikaido, H. and M. Vaara (1985) Molecular basis of bacterial outer membrane permeability. Microbiol. Rev. 49: 1–32.

    CAS  Google Scholar 

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Authors and Affiliations

  1. State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200-237, China

    Xiao-qiang Ma, Sen-wen Deng & Dong-zhi Wei

  2. Enzyme and Fermentation Technology Laboratory, College of Light Industry Science and Engineering, Nanjing Forestry University, Nanjing, 210-037, China

    Er-zheng Su

Authors
  1. Xiao-qiang Ma
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  2. Er-zheng Su
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  3. Sen-wen Deng
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  4. Dong-zhi Wei
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Correspondence to Er-zheng Su or Dong-zhi Wei.

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Ma, Xq., Su, Ez., Deng, Sw. et al. An effective method for extraction of glutaryl-7-aminocephalosporanic acid acylase from recombinant E. coli cells. Biotechnol Bioproc E 20, 718–724 (2015). https://doi.org/10.1007/s12257-013-0607-7

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  • DOI: https://doi.org/10.1007/s12257-013-0607-7

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