Spectrophotometric determination of l-asparagine by flow-injection analysis using l-asparaginase immobilized on an epoxy resin

A continuous flow procedure for the determination of L-asparagine based on its l-asparaginase-catalyzed conversion to l-aspartate and ammonia is proposed. The ammonia formed is measured via the indophenol blue generated in Berthelotʹs reaction. The enzyme l-asparaginase is covalently bound to an epoxy resin (VA-Epoxy Biosynth, Riedel-De Häen) packed in a glass column (50 × 3 mm i.d.). This enzyme reactor can be used for more than 5 months and maintains about 88% of its initial enzyme activity after 700 determinations. Under the proposed experimental conditions a linear calibration range is obtained for l-asparagine concentrations between 60 μM and 15 mM (Abs = −4.29 × 10−3 + 8.05 × 10−4[Asp]; r = 0.999). The proposed procedure has a detection limit (3σ) of 1.2 ppm (9 μM) and a relative standard deviation of 1.9% (12 determinations at 30 ppm). Among the 22 amino acids investigated, only l-histidine (≥ 960 ppm) and l-methionine (≥ 640 ppm) were found to interfere.