Novel Reference Method for Precise Determination of Tryptophanase Activity

Tryptophanase (TnaA; EC 4.1.99.1) is a pyridoxal 5’-phosphate-dependent enzyme that catalyzes the β-elimination of L-tryptophan into indole, pyruvate, and ammonia. It plays a key role in bacterial signaling and biofilm formation and is industrially relevant for L-tryptophan biosynthesis. Existing assay methods for TnaA activity measurement, including Kovács reagent colorimetry, HPLC, and NADH-coupled spectrophotometry, each carry limitations in cost, complexity, or analytical sensitivity that restrict their routine use. The current study aims to develop and validate a simple, cost-effective spectrophotometric method for quantifying TnaA activity using 2,4-dinitrophenylhydrazine (2,4-DNPH) as a chromogenic reagent, targeting the pyruvate product of the enzymatic reaction. The DNPH-TnaA assay uses a stable hydrazone complex formed between pyruvate and 2,4-DNPH, which is measurable at 432 nm. It was applied to E. coli lysates (ATCC 8739 and five clinical isolates). Performance was evaluated for linearity (5–500 µM), detection limits, intra- and inter-assay precision (CV%), spike recovery, selectivity against nine interference classes, and signal stability over 120 hours. Validation included comparisons with Kovács colorimetric and HPLC methods, using Passing–Bablok and Bland–Altman analyses. The limits of detection (LOD) and quantification (LOQ) were 2.25 U/L and 6.7 U/L, respectively. Intra-assay coefficient of variation (CV) was 1.19% (mean 270.83 ± 3.23 U/L), inter-assay CV was 2.65% (mean 273.55 ± 7.25 U/L), both acceptable. Selectivity errors were within ±3.33%. The colored hydrazone was stable at 25 °C for 48 hours. Passing–Bablok regression showed r = 0.99 with near-unity slopes and negligible intercepts, and Bland–Altman analysis indicated minimal bias. ANOVA found no significant differences among the methods at the 0.05 level. The DNPH-TnaA assay is a precise, accurate, and robust method for measuring tryptophanase activity, comparable to Kovács and HPLC methods. Its simplicity, low cost, and compatibility with common lab equipment make it ideal for routine diagnostics and research applications.