Objective Cloning and secretory expression of an amidase fromKluyvera cryocrescensand characterization of its potential in preparation of chiral amino acids. Results An amidase belonging to the Ntn-hydrolase superfamily was identified fromKluyvera cryocrescensZJB-17005 (Kc-Ami). The maximum activity ofKc-Ami was observed at pH 8.5 and 55 degrees C. Remarkably,Kc-Ami showed an excellent enantioselectivity (99% ee) usingrac-4-(hydroxy(methyl)phosphoryl)-2-(2-phenylacetamido) butanoic acid as substrate.Kc-Ami remained stable at pH 7.0-9.0 and exhibited prominent thermostability with a half-life time of 59.1, 47.4 and 20.4 h at 50, 55 and 60 degrees C, respectively.Kc-Ami could be appllied to synthesize chiral amino acids and its derivatives with excellent enantioselectivity (> 99% ee). The synthesized chiral amino acids could contain short or long side chain, and further the side chain could be replaced with -OH, -COOH or benzene ring. Conclusions Kc-Ami exhibited remarkable thermostability and excellent enantioselectivity for synthesizing chiral amino acids and its derivatives. This specific characteristic provides great potential for industrial application in preparation of chiral amino acids and its derivatives.