Microplate diffusion assay for screening of beta-glucanase-producing microorganisms

Area of Science:

• Biotechnology
• Enzymology
• Microbiology

Background:

• Screening fungal strains for enzyme production is crucial for industrial applications.
• Existing methods for detecting beta-1,4-endoglucanase activity can be time-consuming and affected by sugar compounds.

Purpose of the Study:

• To develop a rapid, semi-quantitative assay for screening fungal strains overexpressing extracellular beta-1,4-endoglucanase.
• To enable efficient enzyme activity screening in the presence of high sugar concentrations.

Main Methods:

• Utilized a 96-well microplate format with an azurine dye covalently cross-linked (AZCL) chromogenic substrate (AZCL-hydroxyethyl-beta-1,4-endoglucanase).
• Measured enzyme activity by the release of blue dye, quantified via absorbance at 590 nm against a standard curve.
• Compared results with the Ostazin Brilliant Red-hydroxyethyl cellulose assay.

Main Results:

• The AZCL-based microplate assay demonstrated rapid and effective screening of beta-1,4-glucanase activity in fungal supernatants.
• Results correlated well with established Ostazin Brilliant Red-based assays.
• The method is adaptable for screening other glucanase activities (e.g., beta-1,3-glucanase) using different AZCL substrates.

Conclusions:

• A novel, efficient microplate assay facilitates rapid screening of fungal beta-1,4-endoglucanase activity.
• This method is suitable for high-throughput screening in biotechnological and industrial settings.
• The assay's adaptability allows for the detection of various enzyme activities.