Arginase from Bacillus thuringiensis SK 20.001: Purification, characteristics, and implications for l-ornithine biosynthesis

Abstract

An l-ornithine high producing strain Bacillus thuringiensis SK20.001 was screened by our laboratory. An intracellular arginase used to biosynthesize l-ornithine from the strain was purified and characterized. The final specific arginase activity was 589.2units/mg, with 70.1 fold enrichment and 22.4% recovery. The molecular weight of the enzyme was approximately 33,000Da as evaluated by SDS-PAGE and 191,000Da as determined by gel filtration. The enzyme had an optimum pH of 10.0 and an optimum temperature of 40°C. It was stable from pH 8.0–12.0 and <50°C without Mn2+. The presence of Mn2+ and Ni2+ had strong effects on the enzyme activity, and Mn2+ significantly increased the thermal stability of the enzyme. The arginase was slightly inhibited by Ca2+, Fe2+ and Zn2+. Trp, Asp, Glu, Tyr, and Arg residues were directly involved in the arginase activity evaluated by chemical modifications. The Km and Vmax for l-arginine were estimated to be 15.6mM and 538.9μmol/min/mg. The biosynthesis yield of l-ornithine was 72.7g/L with the enzyme.