title: Expression and purification of a recombinant enantioselective amidase.
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 degrees C but no activity at 37 degrees 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 V(max) and K(m) of the purified enzyme with acetamide (50 mM) were 4.4 micromol/min/mg protein and 4.5mM, respectively. The temperature optimum was found to be 50 degrees 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.
authors.affiliation: Department of Chemical and Life Sciences, Waterford Institute of Technology, Cork Road, Waterford, Ireland.
authors.forename: John P
authors.initials: JP
authors.lastname: Doran
authors.forename: Patrick
authors.initials: P
authors.lastname: Duggan
authors.forename: Michael
authors.initials: M
authors.lastname: Masterson
authors.forename: Peter D
authors.initials: PD
authors.lastname: Turner
authors.forename: Catherine
authors.initials: C
authors.lastname: O'Reilly
mesh_terms: D000192:Actinomycetales / Q000201:enzymology / Q000235:genetics; D000581:Amidohydrolases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013237:Stereoisomerism
chemical_list: D011993:Recombinant Fusion Proteins; D000581:Amidohydrolases; C019665:amidase