Purification and characterization of a thermostable aliphatic amidase from the hyperthermophilic archaeon Pyrococcus yayanosii CH1

Ling Fu, Xuegong Li, Xiang Xiao, Jun Xu

Amidases catalyze the hydrolysis of amides to free carboxylic acids and ammonia. Hyperthermophilic archaea are a natural reservoir of various types of thermostable enzymes. Here, we report the purification and characterization of an amidase from Pyrococcus yayanosii CH1, the first representative of a strict-piezophilic hyperthermophilic archaeon that originated from a deep-sea hydrothermal vent. An open reading frame that encoded a putative member of the nitrilase protein superfamily was identified. We cloned and overexpressed amiE in Escherichia coli C41 (DE3). The purified AmiE enzyme displayed maximal activity at 85 °C and pH 6.0 (NaH2PO4-Na2HPO4) with acetamide as the substrate and showed activity over the pH range of 4-8 and the temperature range of 4-95 °C. AmiE is a dimer and active on many aliphatic amide substrates, such as formamide, acetamide, hexanamide, acrylamide, and L-glutamine. Enzyme activity was induced by 1 mM Ca(2+), 1 mM Al(3+), and 1-10 mM Mg(2+), but strongly inhibited by Zn(2+), Cu(2+), Ni(2+), and Fe(3+). The presence of acetone and ethanol significantly decreased the enzymatic activity. Neither 5% methanol nor 5% isopropanol had any significant effect on AmiE activity (99 and 96% retained, respectively). AmiE displayed amidase activity although it showed high sequence homology (78% identity) with the known nitrilase from Pyrococcus abyssi. AmiE is the most characterized archaeal thermostable amidase in the nitrilase superfamily. The thermostability and pH-stability of AmiE will attract further studies on its potential industrial applications.