Rapid screening of enzyme inhibitors using profiling of enzyme-metabolite assay by HPLC (PREMA-HPLC).

A number of isolates from different ecosystems were screened for their ability to inhibit tyrosinase resulting in the selection of isolate CFR 101, which showed an inhibition of 72%. The metabolites present in the crude extract of the selected isolate was profiled through high-performance liquid chromatography (HPLC) before the enzyme inhibition assay to reveal a 66% decrease in area of the peak at room temperature for 13.9 min, after the assay. Upon purification, this peak was identified as kojic acid, a known inhibitor of tyrosinase. This unique technique of combining a reaction assay mixture with HPLC profile wherein inhibitors can be rapidly pinpointed in crude extracts addresses the drawback of rapid chemical high-throughput screening (HTS) systems, which is limited to the chemical nature of metabolites without any evidence of their biological activities.