A simple, rapid and sensitive plate assay for detection of microbial hyaluronidase activity

Hyaluronidase (hyase) is a glycosidase enzyme that predominantly degrades hyaluronic acid (HA) having important applications in many biotechnological processes and therapeutics. Several assay methods have been proposed to screen hyase producing microorganisms; however, they rely on unique reagents and sophisticated instruments, which are expensive and could be unavailable in general laboratories. In the present studies, a rapid, simple, sensitive, highly reproducible, and cost-effective qualitative plate assay has been developed for the screening of hyase producing microorganisms. The routinely used plate assay method of Richman and Baer requires a special chemical cetylpyridinium chloride and long incubation period of 20 h; but still, the zones of clearance are not very clear and distinct. While, the present method requires an incubation period of only 1 h and the distinct zones of clearance appear with Gram's iodine within 1 min of time. This method does not require any special medium, unlike previously reported methods. Moreover, use of commonly available Gram's iodine makes this method suitable for many researchers. The results of the assay method were validated by TLC, zymographic analysis and determining the growth of isolates in minimal medium containing HA as a sole carbon source.