Characterization of a thermostable arginase from Rummeliibacillus pycnus SK31.001

L-arginase from Rummeliibacillus pycnus SK31.001 is newly discovered. A 906 bp complete open reading frame, which encodes a 301 amino acid protein, was identified using degenerate PCR and inverse PCR techniques. The arginase was found to have a conserved active site with 6 amino acid residues binding to 2 manganese ions: D123, H125, D228, D230, H100 and D127. Bioinformatics analysis revealed that R. pycnus arginase is a hexamer with a subunit molecular mass of 33 kDa and whole molecular mass of 195 kDa. R. pycnus arginase is thermostable with an optimal temperature of 80 °C and maintains 85% of its initial activity after 24 h of incubation at 40 or 50 °C. An arginase activity assay showed that R. pycnus arginase has an optimum pH of 9.5 and a preference for Mn²⁺. Using arginine as the substrate, the Michaelis-Menten constant (K_m) and catalytic efficiency (k_cat/K_m) were measured to be 0.212 mM and 2970 mM⁻¹s⁻¹, respectively. The biosynthesis yield of L-ornithine by the purified enzyme was 144.4 g/L, and the molar yield was 95.2%.