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Abstract
This experiment investigated the possibility of using microwave heating as a non-chelating substitute for EDTA in enhancing lysozyme-mediated lysis of Gram-negative bacterial cells. Such a procedure could better preserve the activity of released enzymes. The effectiveness of breakage of Escherichia coli cells by a combination treatment of lysozyme and microwave exposure for various time durations was compared against the traditional lysozyme-EDTA treatment using the Bradford protein assay. Synthetic ONPG substrate was added to the cell lysate supernatants for colourimetric assay of β -galactosidase activity, which was predicted to serve as a general indicator of enzyme activity preservation. Results suggested that protein release correlated inversely with increasing microwave time, and that all microwave treatments released less protein than the traditional method of breakage. However, a transient but significant increase was observed in β -galactosidase activity (over that of the control) in the treatments microwaved for 25 and 30 seconds. Contrary to expectations, β -galactosidase activity was better retained in treatments containing EDTA. The temperature sensitivity of lysozyme was assessed using the lysozyme- Micrococcus assay; no significant loss in lysozyme activity was detected when the enzyme was microwaved for up to 40 seconds. Decreased β -galactosidase activity was observed in heated samples of toluene-permeabilized cells after 2.5 minutes of heating in a 65ºC water bath. Although the results did not suggest that the microwave is a superior substitute for EDTA, a reasonable amount of protein appeared to have been released in a shorter period of time.