L-glutaminase activity of soils

Abstract

l-Glutaminase [ l-glutamine amidohydrolasc, 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 NH + 4 released by l-glutaminase activity when soil is exposed to l-glutamine, Tris(hydroxymethyl)aminomethane (THAM) buffer and toluene at 37°C for 2 h. The NH + 4-N released is determined by treatment of the soil sample with 2.5 m KCl containing a l-glutaminase inhibitor (Ag 2SO 4) and steam distillation of an aliquot of the resulting soil suspension. The optimum pH for NH + 4-N released by l-glutaminase activity in soils was 10. l-Glutaminase was saturated with 50 m m 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 5m m NH + 4 or l-glutamic acid. The optimal temperature for soil l-glutaminase activity was at 50°C and denaturation began at 55°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) m m and the V max values ranged from 43 to 854 μg NH + 4-N released g −1 of soil 2h −1. The Q 10 values ranged from 1.19 to 1.85 (av. = 1.49). Among the various treatments that affected l-glutaminase activity in soils, autoclaving (121°C, 1h), formaldehyde (1ml 5 g −1 soil), dimethytsulfoxide (1 ml 5g −1 soil) and NaF (5m m) 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 Ca 2+ and Mg 2+ (5m m) activated l-glutaminase activity in soils by an average of 4 and 12%, respectively.