Cloning, purification and properties of a hyperthermophilic esterase from archaeon Aeropyrum pernix K1

Abstract The gene APE1547 of the aerobic thermophilic Aeropyrum pernix K1 encoding 582 amino acid residues was cloned into Escherichia coli . BL21 (DE3) by using vector pET11a with a T7 promoter. An alignment of similarity analysis of APE1547 with protein sequences from A. pernix K1 databank revealed that it showed a lipase motif and low homology with the known thermophilic esterases. However, it had a high degree homology with several acyl amino acid-releasing enzymes. After purified by ion exchange chromatography and gel filtration chromatography, the recombinant protein showed both esterase activity and acylamino acid-releasing enzyme (AARE) activities. The optimum of temperature and pH of the esterase activity are 90 °C and 8.0, respectively. The recombinant protein showed the hydrolytic activity for a wide range of substrates, such as p -nitrophenyl alkanoate esters of varying alkyl chain lengths, pNA-labelled amino acid and peptide. The highest activity was observed for the substrate p -nitrophenyl caprylate. The recombinant enzyme was extremely stable and protein concentration-dependent. Its half-life at 90 °C was over 160 h. at the concentration of 2.14 mg/ml, which renders this new esterase very attractive for biotechnological applications.