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Applied Microbiology and Biotechnology

, Volume 68, Issue 2, pp 189–197 | Cite as

Optimization of extracellular production of recombinant asparaginase in Escherichia coli in shake-flask and bioreactor

  • Amardeep Khushoo
  • Yogender Pal
  • K. J. MukherjeeEmail author
Biotechnological Products and Process Engineering

Abstract

Various host–vector combinations were tested to maximize the extracellular production of recombinant asparaginase in Escherichia coli. Expression of recombinant asparaginase fused to pelB leader sequence under the inducible T7lac promoter in BLR (DE3) host cells resulted in optimum extracellular production in shake-flasks. Fed-batch studies were carried out using this recombinant strain and an exponential feeding strategy was used to maintain a specific growth rate of 0.3 h−1. To check the effect of the time of induction on expression, cultures were induced with 1 mM isopropyl-β-D-thiogalactopyranoside at varying cell optical densities (OD600: 33, 60, 90, 135). Although the specific product formation rates declined with increasing OD of induction, a maximum volumetric activity of 8.7×105 units l−1, corresponding to ∼5.24 g l−1 of recombinant asparaginase, was obtained when induction was done at an OD600 of 90. The recombinant protein was purified directly from the culture medium, using a rapid two-step purification strategy, which resulted in a recovery of ∼70% and a specific activity of ∼80% of that of the native enzyme.

Keywords

Specific Growth Rate Asparaginase Extracellular Production Volumetric Activity Export Efficiency 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgements

This work was funded by the Department of Biotechnology, Government of India. Y.P. is the recipient of a senior research fellowship from CSIR, Government of India

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Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Amardeep Khushoo
    • 1
  • Yogender Pal
    • 1
  • K. J. Mukherjee
    • 1
    Email author
  1. 1.Centre for BiotechnologyJawaharlal Nehru UniversityNew DelhiIndia

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