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

Abstract

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.