I'm working on marine bacterial isolates for asparaginase enzyme, the broth assay (using modified M9 medium, glutamine as substrate) for screening glutaminase activity (side activity of asparaginase enzyme) shows negative result (no color change of medium into pink) for some isolates but on nesslerization it is showing positive result, i.e. appearance of yellowish brown color, why it is so? Also, my control sample (uninoculated medium) is also showing yellow colour on adding nessler's reagent..moreover, I'm getting absorbance at different wavelengths each time I repeat the experiment (385, 488, 522) which is opposed to as mentioned in literature i.e., 425 or 450 nm..why it is happening?
Nesslerization removes ammonia from solution, and ammonia inhibits glutaminase: it probably inhibits asparaginase as well, so by removing it from the solution (via reaction with nessler's reagent) the asparaginase becomes more active, and thus you are able to observe a reaction with glutamine.
As for your color changes, my best guesses are either A) your instrument is failing, and unable to maintain the correct wavelength (easily checked via some stock solutions of known absorbance spectra), or B) the pH of your buffer is changing enough to affect the color of assorted species (easily checked with pH strips or meter).
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