A microplate assay for extracellular hydrolase detection

This article presents a new qualitative method to detect enzyme activity replacing the conventional Agar-Petri dishes. This new method is a simple rapid and low-cost technique that uses 24-well microplates. The detection of hydrolases producing microorganisms in bioprospecting studies by qualitative methods is time consuming, costly and requires a large quantity of strains or enzymatic extracts. Tests with different substrate concentrations (0 to 20 g/L) in agar solution for the enzymatic hydrolysis analysis were performed to determine the best substrate concentrations in 24-well microplates. Other quantitative and analytical methods, such as enzymatic assays and thin layer chromatography, were performed to validate this new method and to compare the relationship between enzymatic activity and substrate degradation. Statistically relevant results were observed for amylase, endoglucanase and polygalacturonase enzymes, even when there was a low substrate concentration in agar, where the halo diameter was high. The results also indicated that the concentrations for efficient enzyme index measurements were 4 g/L carboxymethylcellulose for endoglucanase detection and 8 g/L for amylase and polygalacturonase assays. The results were presented according to the traditional methods for detection of enzymatic activity. This new method can be used as a general test for the detection of important industrial hydrolases. It is a faster and less costly alternative for screening microbial enzyme producing microorganisms and is useful for studying the production of microbial enzymes under different growing conditions.