Expression and purification of a recombinant enantioselective amidase

John P. Doran, Patrick Duggan, Michael Masterson, Peter D. Turner, Catherine O'Reilly

Research output: Contribution to journalArticlepeer-review

19 Citations (Scopus)

Abstract

Microbacterium sp. AJ115 metabolises a wide range of nitriles using the two-step nitrile hydratase/amidase pathway. In this study, the amidase gene of Microbacterium sp. AJ115 has been inserted into the pCal-n-EK expression vector and expressed in Escherichia coli BL21(DE3)pLysS. The expressed protein is active in E. coli and expression of the amidase gene allows E. coli to grow on acetamide as sole carbon and/or nitrogen source. Expression of active amidase in E. coli was temperature dependent with high activity found when cultures were grown between 20 and 30°C but no activity at 37°C. On induction, the amidase represents 28% of the total soluble protein in E. coli. The expressed amidase has been purified in a single step from the crude lysate using the calmodulin-binding peptide (CBP) affinity tag. The Vmax and K m of the purified enzyme with acetamide (50 mM) were 4.4 μmol/min/mg protein and 4.5 mM, respectively. The temperature optimum was found to be 50°C. Purified enzyme demonstrated enantioselectivity with the ability to preferentially act on the S enantiomer of racemic (R,S)-2-phenylpropionamide. S-2-phenylpropionic acid is produced with an enantiomeric excess of >82% at 50% conversion of the parent amide.

Original languageEnglish
Pages (from-to)190-196
Number of pages7
JournalProtein Expression and Purification
Volume40
Issue number1
DOIs
Publication statusPublished - Mar 2005

Keywords

  • Amidase
  • Enantioselective
  • Microbacterium sp. AJ115
  • pCal-n-EK
  • Rhodococcus erythropolis AJ270

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