Purification of L-Glutaminase from Bacillus sp. B12 and study its properties

Abstract

L-glutaminase is an enzyme that catalyse the conversion L-glutamine to glutamic acid and ammonia. The important application of the L-glutaminase is a chemotherapy agent. In this study a novel strain, Bacillus B12. that isolated from Diyala soil was explored for production of extracellular L-glutaminase. The enzyme has been purified 23.9-fold from cell-free extract with 45.4 recovery ( specific activity 76.6 U/mg protein). Enzyme has molecular weight of 199 kDa, and pH 8 and stable in pH range 6-9.5. Temperature optimum is 40 ?C and completely stable between 25-45 ?C. The kinetics studies revealed that the km and Vmax of purified L-glutaminase were estimated in 0.4mmol/L and 0.133mmol/min, respectively. The result showed that, the enzyme was active when incubated with 10 mM of Mn2+, Mg2+,Ca+2 and Na+. Whereas, K +, Co2+, and Ni2+ show no effect. However, Hg2+, Cu2+,Fe2+ and Zn2+ decreased L-glutaminase activity . The 10mM of DEAE, PMSF, and sodium azide did not show a clear effect against the enzyme activity. Higher decrease in enzyme activity was observed by using Cysteine and 2-Mercaptoethanol ( 30 and 25%, respectively).