A lysoplate assay for Escherichia coli cell wall-active enzymes

A benchtop assay based upon digestion of purified Escherichia coli peptidoglycan suspended in an agarose gel matrix is described. Enzymes for which these cell walls are substrates are applied to wells in the gel and diffuse into the gel. Activity is measured visually by the size of clear disks formed around the wells as the peptidoglycan is digested. Using this assay, it is possible to screen large numbers of cell wall-active enzymes for sensitivity to pH, ionic strength, denaturant, temperature, or other factors without interference from endogenous autolytic enzymes. Data are presented to show the limits of detection and linearity of the assay. For an assay time of 14 h, as little as 1 nmol per liter of bacteriophage T4 lysozyme and 200 nmol per liter of hen egg white lysozyme were detected. Longer assay times decrease these limits by as much as an order of magnitude. The salt dependence of T4 lysozyme and several of its temperature-sensitive mutants was also determined. Finally, an example of the use of the assay during lysozyme purification to determine active column fractions is presented.