An improved method for simple and accurate colorimetric determination of l‐asparaginase enzyme activity using Nessler's reagent

Abstract

AbstractBACKGROUNDColorimetric determination of l‐asparaginase enzyme activity most commonly uses Nessler's reagent, but often yields unreliable results due to precipitation of tetraiodomercurate salts. Interference caused by some commonly used inorganic and organic compounds of biochemical buffers, reagents and microbiology fermentation media was determined. The stabilising effects of tartrate and polyvinyl alcohol upon the impact of these salts was next evaluated using the following two‐step assay in 96‐well microtitre plates: 160 μL of 50 mmol L−1 Tris–HCl pH 8.6, 32 μL of 100 mmol L−1 l‐asparagine, 16 μL analyte and 128 μL water were added to each well. After incubation at 37 °C for 1 h, 16 μL of 1.5 mol L−1 trichloroacetic acid was added to stop the reaction. A 35 μL volume was next transferred to a fresh well containing 35 μL Nessler's reagent, 35 μL stabiliser solution (4 mmol L−1 disodium tartrate and 10 mg L−1 polyvinyl alcohol) and 245 μL water. After incubation at room temperature for 10 min, absorbance was recorded at 436 nm and the enzyme activity calculated by extrapolation of a (NH4)2SO4 calibrated standard curve.RESULTSAddition of tartrate and polyvinyl alcohol proved to be effective in reducing interference caused by precipitation of salts, allowing determination of l‐asparaginase activity in a range from 0.050 to 0.796 IU mL−1, ideal for monitoring enzyme production in fermentations.CONCLUSIONA method is offered for rapid and reliable determination of l‐asparaginase activity, particularly in complex samples such as microbial fermentation broths where existing methodologies do not account for interferences that frequently yield overvalues. © 2020 Society of Chemical Industry