The activity of phage lysins can be determined using several methods; turbidimetry or the determination of the change in concentration of some solubilised cell wall component are frequently used.
Turbimetry, where a standardised suspension of cells in buffer is mixed with a sample of lysin-containing material is widely used. The lysin causes lysis of the cell suspension and a reduction in optical density (OD). Enzyme activity can be calculated from the decrease in OD with time.
The following method has been found to give satisfactory results (Mullan and Crawford, 1985a).
Cells were suspended in 0.1 M-K phosphate buffer, pH 6.8, to give an OD at 450nm of 0.62-0.75. Depending on the lytic activity, 0.1-1.0 ml of lysin containing solution was added to 5ml of standardized cell suspension at 37°C. After mixing, OD readings were taken at 30 s intervals over a 2-6 min period. Absorption readings were plotted against time and only values on the linear portion of the graphs were used for calculation.
Since higher lytic activity is obtained with logarithmic compared with late stationary phase cells care is required in defining the assay conditions. Some cell preparations may also show significant autolysis and appropriate controls are required. Little or no autolytic activity was apparent with lyophilized cells of Lc. lactis strains C2 and C10 suspended in buffer for 30 min at 37°C. Lyophilized cells are highly recommended for use in lysin assay.
One unit of enzyme activity was defined as that concentration of phage lysin which gave a decrease in absorbance of 0.001 units/min under the assay conditions defined previously.
How to cite this article
Mullan, W.M.A. (2005) .
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