Purification and properties of an enantioselective and thermoactive amidase from the thermophilic actinomycete Pseudonocardia thermophila.
A constitutively expressed thermoactive amidase from the thermophilic actinomycete Pseudonocardia thermophila was purified to homogeneity by applying hydrophobic interaction, anion exchange and gel filtration chromatography, giving a yield of 26% and a specific activity of 19.5 units mg(-1). The purified enzyme has an estimated molecular mass of 108 kDa and an isoelectric point of 4.2. The amidase is active at a broad pH range (pH 4-9) and temperature range (40-80 degrees C) and has a half-life of 1.2 h at 70 degrees C. Inhibition of enzyme activity was observed in the presence of metal ions, such as Co(2+), Hg(2+), Cu(2+), Ni(2+), and thiol reagents. The amidase has a broad substrate spectrum, including aliphatic, aromatic and amino acid amides. The presence of a double bond or a methyl group near the carboxamide group of aliphatic and amino acid amides enhances the enzymatic activity. Among aromatic amides with substitutions at the o-, m-, or p-position, the p-substituted amides are the preferred substrates. The highest acyl transferase activity was detected with hexanoamide, isobutyramide and propionamide. The K(m) values for propionamide, methacrylamide, benzamide and 2-phenylpropionamide are 7.4, 9.2, 4.9 and 0.9 mM, respectively. The amidase is highly S-stereoselective for 2-phenylpropionamide; and the racemic amide was converted to the corresponding S-acid with an enantiomeric excess of >95% at 50% conversion of the substrate. In contrast, the D,L-tryptophanamide and D,L-methioninamide were converted to the corresponding D,L-acids at the same rate. This thermostable enzyme represents the first reported amidase from a thermophilic actinomycete.
Egorova
Ksenia
K
Institute of Technical Microbiology, Technical University Hamburg-Harburg, Kasernenstr. 12, 21073 Hamburg, Germany.
Trauthwein
Harald
H
Verseck
Stefan
S
Antranikian
Garabed
G
D039903:Actinobacteria / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D000581:Amidohydrolases / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D013379:Substrate Specificity; D013696:Temperature
D000581:Amidohydrolases