Expression and purification of a recombinant enantioselective amidase

Doran, John P. and Duggan, Patrick and Masterson, Michael and Turner, Peter D. and O'Reilly, Catherine (2005) Expression and purification of a recombinant enantioselective amidase. Protein Expression and Purification, 40 (1). pp. 190-196. ISSN 1046-5928

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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.

Item Type: Article
Additional Information: Funding Information: This work has been funded under the Department of Education Technological Sector Research and Innovation initiative Strand III.
Uncontrolled Keywords: /dk/atira/pure/subjectarea/asjc/1300/1305
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Depositing User: Admin SSL
Date Deposited: 19 Oct 2022 23:03
Last Modified: 22 Jun 2023 19:35

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