L-ASPARAGINASE ACTIVITY OF SOILS

A simple, precise, and sensitive method to assay L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) activity in soils is described. This method use steam distillation to determine the NH4+ produced by L-asparaginase activity when soil is incubated with buffered (0.1 M THAM, pH 10) L-asparagine solution and toluene at 30-degrees-C for 2 h. The procedure developed gives quantitative recovery of NH4+-N added to soils and does not cause chemical hydrolysis of L-asparagine. The optimum buffer pH for NH4+-N released by L-asparaginase activity in soils was 10. This enzyme was saturated with 50 mM L-asparagine, and the reaction rate essentially followed zero-order kinetics. The D-isomer of asparagine was also hydrolyzed in soils, but at only 16% of the activity of the L-isomer at a saturating concentration of the substrate. The optimal temperature for the soil L-asparaginase reaction occurred at 60-degrees-C and denaturation began at 65-degrees-C. The Arrhenius equation plot for L-asparaginase activity in three selected soils was linear between 10 and 50-degrees-C. The activation energy values of this enzyme ranged from 20.2 to 34.1 (average 26.6) kJ mol-1. Application of three linear transformations of the Michaelis-Menten equation showed that the K(m) values of L-asparaginase in nine soils ranged from 2.6 to 10.0 (average 6.1) mM and the V(max) values ranged from 9 to 131-mu-g NH4+-N released g-1 soil 2 h-1. The temperature coefficients (Q10) for soil L-asparaginase activity ranged from 1.12 to 1.70 (average 1.39). Steam sterilization (121-degrees-C for 1 h), formaldehyde, and NaF decreased the activity but the presence of toluene increased the amount of NH4+ released. Treatment of soils with dimethylsulfoxide completely destroyed L-asparaginase activity. The use of sulfhydryl reagents indicated that a free sulfhydryl moiety was required to maintain the active enzyme. L-Asparaginase activity in soils was increased by 13 to 18% in the presence of THAM buffer prepared to contain 5 mM Ca2+ and Mg2+, respectively.