Detection of microbial proteolytic activity by a cultivation plate assay in which different proteins adsorbed to a hydrophobic surface are used as substrates

Abstract

A screening technique for microbial proteases, the thin-layer enzyme assay cultivation technique, was developed. The inner surface of a polystyrene petri dish was coated with protein and then covered with a culture agar medium. The enzymes, produced during growth of the microorganisms, reach the protein-coated surface by diffusion in the agar. Degradation of the protein was visualized by condensation of water vapor on the surface after removal of the agar medium. The wettability of the enzyme-affected protein-coated polystyrene surface was decreased compared with the unaffected protein surface. Enzyme substrates used were fibrinogen, immunoglobulin G, egg albumin, human serum albumin, bovine serum albumin, hemoglobin, mucin, and gelatin. It was possible to use a variety of culture agar media, nonselective as well as selective, in the assay. The technique provides a sensitive, convenient, and inexpensive method for screening various microbial proteases. In addition, the technique can be used for screening proteolytic enzyme activity of specific microbial species in a mixed microbial sample as well as for studies of factors that influence the cultivation conditions for protease production and activity.