title: 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.
authors.affiliation: State Key Laboratory of Microbial Metabolism and School of Life Science and Biotechnology, State Key Laboratory of Ocean Engineering, Shanghai Jiao Tong University, Dongchuan Road 800, Shanghai, 200240, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Fu
authors.forename: Xuegong
authors.initials: X
authors.lastname: Li
authors.forename: Xiang
authors.initials: X
authors.lastname: Xiao
authors.forename: Jun
authors.initials: J
authors.lastname: Xu
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D019711:Pyrococcus / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D019843:Archaeal Proteins; D000581:Amidohydrolases