A rapid UV-assisted OPA Fluorogenic Micro-spot assay for semi-quantitative screening of arginase activity in crude...

Rapid screening of arginase-producing microorganisms is challenging because traditional assays often need enzyme purification, large reagent volumes, spectrophotometric equipment, or labour-intensive procedures, limiting high-throughput microbial bioprospecting. We developed a compact UV-assisted O-phthalaldehyde (OPA) fluorogenic micro-spot assay for rapid semi-quantitative detection of arginase activity in crude marine bacterial extracts. After converting L-Arginine to L-Ornithine enzymatically, the reaction mixtures were derivatized with OPA and β-mercaptoethanol, producing cyan fluorescence visible under 365 nm UV light in a micro-spot format on a parafilm surface. Fluorescence was quantified digitally in ImageJ by calculating corrected total fluorescence (CTF), thereby eliminating the need for specialised fluorescence instrumentation. Out of 83 marine bacterial isolates, 17 showed clear fluorescence signals indicating arginase activity, while negative controls had minimal background fluorescence. Validation with a conventional ninhydrin-based ornithine assay showed a strong correlation (R² = 0.9392) between fluorescence and ornithine levels, confirming the method's semi-quantitative reliability. While it is primarily a rapid screening tool rather than a precise analytical method, its compatibility with crude extracts, low reagent use, fast workflow, and minimal equipment needs make it ideal for high-throughput microbial enzyme screening and marine bioprospecting.