L- glutaminase activity of soils

L-glutaminase [L-glutamine amidohydrolase, EC 3.5.1.2] catalyzes the hydrolysis of L-glutamine to produce ammonia and L-glutamic acid. We have detected this enzyme in soils. A simple, precise, rapid and sensitive method to assay its activity is described, which involves determination of the NH4+ released by L-glutaminase activity when soil is exposed to L-glutamine, Tris(hydroxymethyl)aminomethane (THAM) buffer and toluene at 37 degrees C for 2 h. The NH4+ -N released is determined by treatment or the soil sample with 2.5 M KCl containing a L-glutaminase inhibitor (Ag2SO4) and steam distillation or an aliquot of the resulting soil suspension. The optimum pH for NH4+ -N released by L-glutaminase activity in soils was 10. L-glutaminase was saturated with 50 mM L-glutamine and the reaction essentially followed zero-order kinetics. The D-isomer of glutamine was hydrolyzed in soils at only 7% of the activity of the L-isomer at saturating concentrations of the substrate. The L-glutaminase reaction in soils was not inhibited by the presence of 5 mM NH4+ or L-glutamic acid. The optimal temperature for soil L-glutaminase activity was at 50 degrees C and denaturation began at 55 degrees C. The activation energy values of this enzyme, calculated from the Arrhenius plot, ranged from 20.3 to 39.9 (av.= 32.4) kJ mol(-1). Application of three linear transformations of the Michaelis-Menten equation showed that the K(m) values of L-glutaminase in nine soils ranged from 8.2 to 38.6 (av.= 21.7) mM and the Vmax values ranged from 43 to 854 micrograms NH4+ -N released g(-1) of soil 2 h(-1). The Q10 values ranged from 1.19 to 1.85 (av.= 1.49). Among the various treatments that affected L-glutaminase activity in soils, autoclaving (121 degrees C, 1h), formaldehyde (1 ml 5 g(-1) soil), dimethylsulfoxide (1 ml 5 g(-1) soil) and NaF (5 mM) reduced the activity by 92, 96, 78 and 14%, respectively. L-glutaminase activity was greater in toluene treated soils than in untreated soils. The use of sulfhydryl reagents indicated that a free sulfhydryl moiety was required to maintain the active enyzme. Both Ca2+ and Mg2+ (5 mM) activated L-glutaminase activity in soils by an average of 4 and 12%, respectively.