Characterization of a recombinant arginine deiminase from Halothermothrix orenii and its application in citrulline production.

In recent years, arginine deiminase (ADI, EC 3.5.3.6) has attracted much attention as a biocatalyst that produces the functional amino acid l‐citrulline from l‐arginine and also as an anticancer enzyme. Here, we identified and characterized a putative ADI from the thermophilic bacterium Halothermothrix orenii. The H. orenii ADI (H‐ADI) protein was expressed in Escherichia coli BL21(DE3) with a specific activity of 91.8 U/mg protein at 55°C and pH 6.5. The enzyme remained at 74% relative activity after incubation at 45°C for 180 min, only 25% at 50°C. The melting temperature was 56°C. H‐ADI is not a metal‐requiring enzyme; Ni2+ slightly improved the catalytic activity. The Km and Vmax for l‐arginine were 55.5 mM and 156.8 μmol/min/mg protein, respectively. Moreover, three residues (Arg183, Arg237, and His273) were key to the formation of l‐citrulline, as analyzed by alanine‐scanning mutagenesis. Finally, the enzymatic synthesis of l‐citrulline was carried out at 50°C with a conversion ratio reaching 99.03%. Together, these findings show that H‐ADI is a promising biocatalyst for the production of l‐citrulline. [ABSTRACT FROM AUTHOR]