Microplate Diffusion Assay for Screening of β-Glucanase-Producing Microorganisms

Abstract

A method is described to screen fungal strains rapidly for overexpression of extracellular β-1,4-endoglucanase in the presence of high levels of sugar compounds. The semi-quantitative assay utilizes microplates in a 96-well format and an azurine dye covalently cross-linked (AZCL) chromogenic substrate. The digestion of AZCL-hydroxyethyl-β-1,4-endoglucanase results in the release of a blue dye directly proportional to the amount of enzyme activity present in the sample. Sample absorbance was read at 590 nm, and the enzyme activity was determined by reference to a standard curve. The results from the microplate diffusion assay were similar to the results derived from the Ostazin Brilliant Red-hydroxyethyl cellulose assay. The technique described allowed the rapid comparison and screening β-1,4-glucanase activity directly in spent fungal supernatant, from cultures grown in potato dextrose broth. The method could also be easily adapted for the screening of the presence of other activities such as β-1,3-glucanase activity by using either AZCL-β-glucan or AZCL-pachyman in place of the AZCL-hydroxyethyl-cellulose. This assay could be used to measure supernatant within an activity range of 0.1–2 U/mL.