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 of the soil sample with 2.5 M KCl containing a L-glutaminase inhibitor (Ag2SO4) and steam distillation of 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 V(max) values ranged from 43 to 854-mu-g 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 enzyme. Both Ca2+ and Mg2+ (5 mM) activated L-glutaminase activity in soils by an average of 4 and 12%, respectively.