2019 Volume 65 Issue 3 Pages 137-144
An extracellular L-asparaginase was isolated and purified from Bacillus megaterium MG1 to apparent homogeneity. The purification procedure involved a combination of ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration techniques, resulting in a purification factor of 31.52 fold with a specific activity of 215 U mg-1. The molecular mass of the purified enzyme was approximately 47 kDa on SDS-PAGE and 185 kDa on native PAGE gel as well as in gel filtration column chromatography, revealing that the enzyme was a homotetramer. The Km and Vmax values of the purified enzyme were calculated to be 2.0 ⅹ 10-4 M and 1.198 mM s-1. Maximum enzyme activity was observed over a wide range of temperature and pH values with an optimum temperature of 37°C and pH 8.5. SDS and metal ions such as Fe2+, Cu2+, Mg2+, Co2+, Mn2+, and Ca2+ decreased the enzyme activity remarkably, whereas the addition of Na+ and K+ led to an increase in activity. The insensitivity of the protein in the presence of EDTA suggested that the enzyme might not essentially be a metalloprotein. Its marked stability and activity in organic solvents and reducing agents suggest that this asparaginase is highly suitable as a biotechnological tool with industrial applications.