l-Asparaginase activity of soils

Summary

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 NH ^sup+_inf4 produced by l-asparaginase activity when soil is incubated with buffered (0.1 M THAM, pH 10) l-asparagine solution and toluene at 30°C for 2 h. The procedure developed gives quantitative recovery of NH ^sup+_inf4 -N added to soils and does not cause chemical hydrolysis of l-asparagine. The optimum buffer pH for NH ^sup+_inf4 -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°C and denaturation began at 65°C. The Arrhenius equation plot for l-asparaginase activity in three selected soils was linear between 10 and 50°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 μg NH ^sup+_inf4 -N released g^-1 soil 2 h^-1. The temperature coefficients (Q ₁₀) for soil l-asparaginase activity ranged from 1.12 to 1.70 (average 1.39). Steam sterilization (121°C for 1 h), formaldehyde, and NaF decreased the activity but the presence of toluene increased the amount of NH ^sup+_inf4 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.