Purification and Characterization of Glutaminase from Bacillus subtilis GT Strain

Ex tracellular glutaminase of Bacillus subtilis GT strain was purified by ion-exchange, gel-filtration and hydrophobic chromatographies.After the purification, the specific activity was increased 259-fold (based on the enzyme in the culture filtrate) with 1. 5% recovery. Polyacrylamide gel electrophoresis of purified enzyme gave a single protein band.The molecular weight of the enzyme was estimated to be approximately 55, 000 by gel-filtration on Sephacryl S-200. The enzyme hydrolyzed only free L- and D-glutamine. The enzyme was most active at pH 6.0 and stable in a pH range of 5.0-8.0. This enzyme was stable up to 50°C, and the optimum temperature for enzyme reaction was 50°C. This enzyme manifested more than 85% of the original activity even in the presence of 25% NaCl at pH 5.5 Km value of the enzyme for L-glutamine was 0. 64 mM. The enzyme completely converted L-glutamine into L-glutamic acid, therefore, the enzyme seemed not to be inhibited by the reaction product, L-glutamic acid. Hg2+ ion strongly inhibited the enzyme activity.These results suggest that this glutaminase could widely be used in increasing the amount of Lglutamic acid in various fermentation seasonings such as miso, soy sauce, mirin and so on.