Sensitive gas spectroscopy method for real-time determination of urease activity via ammonia production†

Abstract

In this study we propose the use of a laser-based photoacoustic spectrometer as a new method to investigate the kinetics of ammonia (NH₃) emission from the hydrolysis of urea (H₂N–CO–NH₂) in the presence of urease. Experiments explored the effects of varying the amounts of water and urease on nitrogen (N) loss from urea aqueous solutions. A linear increase in N–NH₃ loss and the maximum emission rate (r_NH3) was observed with increasing certified urease concentrations. Specifically, the addition of 10 units of urease resulted in an N loss of 8.1%, with a sensitivity of approximately 0.8% per unit of urease, and reached 12.8 μmol min⁻¹ with a sensitivity of 1.1 μmol min⁻¹ per unit of urease for r_NH3. These relationships enabled the estimation of urease quantities in commercial soy flour extracts. Variations in water and urease proportions revealed that maximum NH₃ emissions occurred within the first 2–5 h, with the highest N–NH₃ loss value attaining (9 ± 1)% for samples containing 2.0 mL soy flour extracts and additional water. For urease concentration assays, N–NH₃ loss and r_NH3 were (14.4 ± 0.1)% and (25.3 ± 0.1) μmol min⁻¹, respectively, with 4.0 mL of soy flour extract. The results underscored the dominant influence of urease compared to that of water in urea hydrolysis. The PA spectrometer demonstrated sufficient sensitivity for detecting NH₃, rendering it a promising tool for studying urease activity in urea decomposition. Future work could explore this system under crop field conditions to elucidate the roles of urease and water in the cycling of nutrients within agroecosystems.