Enhanced Agar Well Diffusion Method for Screening Enzyme-Producing Microorganisms

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Abstract
Conventional agar-based direct-plating assays are widely used to screen microorganisms for enzyme production, but their applicability can be limited, particularly by interference from complex growth media. We developed an ammonium sulfate-assisted agar well diffusion method that separates microbial cultivation from extracellular enzyme detection. Enzymes recovered from culture supernatants are concentrated and tested on substrate-containing agar without a nutrient-rich growth medium. Commercial reference enzymes produced concentration-dependent reaction zones and calibration curves for quantitative analysis of isolate-derived activities. Application to fruit by-product isolates W46 and W47 demonstrated distinct enzyme activity profiles and the method’s practical utility. However, W46 lipase activity detected by direct plating was not recovered after precipitation, indicating enzyme-dependent recovery and the need to optimize precipitation conditions for specific targets. Overall, the method provides a practical screening alternative with reduced contamination risk when conventional direct plating is unsuitable or insufficiently informative.Separates cultivation from enzyme detection, reducing interference from complex growth media, preventing substrate-related growth inhibition, and facilitating visualization of substrate hydrolysis.Concentrates extracellular enzymes and increases enzyme–substrate contact, improving sensitivity to low levels of enzyme production.Enables quantitative expression of microbial enzyme activity as reference-enzyme equivalents.

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