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- Title
Purification and characterization of a thermostable aliphatic amidase from the hyperthermophilic archaeon Pyrococcus yayanosii CH1.
- Authors
Fu, Ling; Li, Xuegong; Xiao, Xiang; Xu, Jun
- 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 (NaHPO-NaHPO) 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, 1 mM Al, and 1-10 mM Mg, but strongly inhibited by Zn, Cu, Ni, and Fe. 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.
- Subjects
AMIDASES; HEAT stability in proteins; HYDROTHERMAL vents; NITRILASES; BACTERIAL enzyme analysis; FORMAMIDE
- Publication
Extremophiles, 2014, Vol 18, Issue 2, p429
- ISSN
1431-0651
- Publication type
Article
- DOI
10.1007/s00792-014-0628-y