A One-Step Dual Chromogenic Plate Assay for Reliable Screening of Naringinase-Producing Microorganisms

Area of Science:

• Applied Microbiology
• Enzyme Technology
• Biotechnology

Background:

• Naringinase, crucial for citrus processing and bioactive compound production, requires both α-L-rhamnosidase and β-D-glucosidase activities.
• Existing screening methods often yield false positives, failing to distinguish complete naringinase producers from partial enzyme expressors.

Purpose of the Study:

• To develop a reliable, single-plate assay for accurately identifying true naringinase-producing microorganisms.
• To overcome the limitations of conventional screening methods that struggle with the dual-enzyme nature of naringinase.

Main Methods:

• A novel dual chromogenic assay using p-nitrophenyl-α-L-rhamnopyranoside and X-gal was developed.
• Simultaneous detection of α-L-rhamnosidase (yellow) and β-glycosidase-like (blue) activities on a single agar plate.
• Assay validation through spectrophotometric enzyme assays, thin-layer chromatography, and testing known bacterial strains.

Main Results:

• The assay successfully differentiated microbial enzymatic profiles using distinct color signals.
• Only isolates showing both yellow and blue signals achieved complete naringin hydrolysis to naringenin.
• A positive correlation was observed between chromogenic halo size and enzyme activity.

Conclusions:

• The developed assay provides a simple, reliable, and scalable method for high-throughput screening of naringinase producers.
• This advancement facilitates accurate identification in applied microbiology and biotechnology.
• The assay enables efficient discovery of microorganisms for industrial enzyme applications.