Purification and properties of an enantioselective and thermoactive amidase from the thermophilic actinomycete Pseudonocardia thermophila

Abstract

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.

Amidases: versatile enzymes in nature

“…Majority of amidase producing microorganisms exhibited optimum growth and production of enzyme around neutral pH (Pal and Samanta 1999;Ciskanik et al 1995) except amidases in Rhodococcus rhodochrous NHB-2 and Kluyveromyces thermotolerans MGBY 37 which have optimum pH 5.5-6.0 and 6.2, respectively (Bhalla et al 1997;Prasad et al 2005). Amidase from thermophiles are generally active over wide pH range (5-9.5) (Egorova et al 2004;Makhongela et al 2007). Most of the mesophiles produce amidases in temperature range 25-30°C (Banerjee et al 2002), while optimum production temperatures for amidase by thermophilic bacteria lies in range of 50-65°C (Baek et al 2003;Egorova et al 2004;Makhongela et al 2007).…”

Enantioselective hydrolysis of (R)-2, 2-dimethylcyclopropane carboxamide by immobilized cells of an R-amidase-producing bacterium, Delftia tsuruhatensis CCTCC M 205114, on an alginate capsule carrier

“…With the ability to hydrolyze amides to corresponding optical pure compounds with high enantioselectivity, mild reaction conditions and environmental compatibility, amidases (EC 3.5.1.4) have been intensively studied [2,4,7,8,10,12,13,16,17,21,22,26,30]. However, current research on amidase, such as screening, characterization and its heterogeneous expression, is concentrated on the laboratory level, and applications of amidase on a large scale are quite few.…”

A novel amidase from Brevibacterium epidermidis ZJB-07021: gene cloning, refolding and application in butyrylhydroxamic acid synthesis

“…They are widespread in both eukaryotic and prokaryotic organisms. Due to excellent regio-, chemo-and enantioselective properties, AS amidases are increasingly recognized as attractive tools for the preparation of optically active compounds [12,24,30,43]. Although some AS amidases have been cloned and overexpressed in E. coli [11,23,39,41], high-level expression of amidases often leads to formation of insoluble inclusion bodies (IBs) in the cytoplasm [18,25,37].…”