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Extremophiles

, Volume 18, Issue 2, pp 429–440 | Cite as

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

  • Ling Fu
  • Xuegong Li
  • Xiang Xiao
  • Jun Xu
Original Paper

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 Ca2+, 1 mM Al3+, and 1–10 mM Mg2+, but strongly inhibited by Zn2+, Cu2+, Ni2+, and Fe3+. 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.

Keywords

Amidase Nitrilase superfamily Pyrococcus yayanosii Hyperthermophilic archaeon Thermostability 

Notes

Acknowledgments

The project was supported by National High-tech R&D Program (863 Program, Grant 2012AA092103) and the State Key Laboratory of Ocean Engineering of China (Grant GKZD010045). We thank Geng Wu and Guangyu Yang for experimental assistance.

Supplementary material

792_2014_628_MOESM1_ESM.doc (1.3 mb)
Fig. S1 Relative activity of AmiE and E119D. Ammonia was detected when acetamide was incubated with AmiE and E119D mutant for 10 min at 85° C in triplicate repeats (DOC 1306 kb)

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Copyright information

© Springer Japan 2014

Authors and Affiliations

  1. 1.State Key Laboratory of Microbial Metabolism and School of Life Science and Biotechnology, State Key Laboratory of Ocean EngineeringShanghai Jiao Tong UniversityShanghaiChina

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