Purification and characterization of R-stereospecific amidase from Brevibacterium epidermidis ZJB-07021

A R-stereospecific amidase was purified from Brevibacterium epidermidis ZJB-07021 and characterized in detail. The amidase was purified to homogeneity by three chromatographic steps for up to 328.9-fold with specific activity of 31.9 U mg(-1). The enzyme was a homodimer with a molecular mass of 94 kDa. It exhibited maximum activity at 40 °C and pH 7.5. The enzyme was strongly inactivated by serine protease inhibitor PMSF. The values of Km and Vmax for racemic 2,2-dimethylcyclopropane carboxamide (DMCPCA) were 4.58 mM and 35.03 μmol min(-1) mg(-1) protein, respectively. The amidase showed a broad substrate spectrum toward aliphatic, aromatic and heterocyclic amides, but could hardly hydrolyze the bulky side-chain-containing amides. Furthermore, kinetic resolution of racemic DMCPCA by the amidase afforded S-DMCPCA in 46.3% yield and 99% ee with an average E-value of 67. These unique properties of the amidase imply that it is a promising biocatalyst for the production of chiral amides and carboxylic acids.