As my aim is to study the anti-fungal activity by the bacteria, Is there any expertise member who can explain how to know a bio-active compound(may be protein, enzyme or other molecules) isolated from a bacteria is extracellular or intracellular in nature ?
The literature search could not helped me to find out the appropriate solution for this particular microorganism so I want to go for various experimental methods. So is there any proper step by step procedure for better confirmatory tests ????
There are various tests to conduct to confirm the chemical nature of your bio-active compound. First is to establish that the molecule has biological activity in the first place, then, against what? which in your case is fungi. Next is to establish the chemical nature of your agent which is what I think you desire. Preliminary biochemical tests for carbohydrates, proteins and lipids will guide you. You could then confirm this result using the 'Eye of the Organic Chemist'. We call it "Thin layer chromatography". HPLC will purify the compound for Fourier transform infrared spectrometry which establishes the possible functional groups present in the molecule to confirm or negate the results already hinted at. GC-MS is the ultimate in structural characterization albeit a number of hi-tech chemical procedures have emerged in recent times as options. Go through the article attached. If your question was about localization of metabolite, then you should understand that extracellular compounds will be released into the fermentation medium post synthesis but intracellular products will be confined within. You can do this: Assay the fermentation broth without cell separation and note your result. Centrifuge and filter-sterilize the resulting supernatant and re-run the assay. Note the difference. If greater than 75% of the activity is retained in the sterile broth, then the metabolite is extracellular, if not, then it is intracellular or cell-associated. The protocols for the later analysis are abundant in literature.
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