INVESTIGATIONS OF THE INTERACTIONS OF SACCHARIDES WITH THE LYSOZYME FROM BACTERIOPHAGE-LAMBDA

The bacteriophage lambda R gene has been isolated into an Escherichia coli expression system and the R gene product, a lysozyme, has been ov erexpressed and purified to homogeneity using an efficient purificatio n procedure. A turbidimetric assay utilizing chloroform-treated E. col i cells has been optimized to assess the bacteriolytic activity of the purified enzyme. Using this assay, oligomers of beta(1 --> 4) N-acety l-D-glucosamine at high concentrations were shown to inhibit lysozyme but were not cleaved by the enzyme. Differential scanning calorimetry revealed that the thermal denaturation of lysozyme was found to increa se in the presence of (GlcNAc)(3) and (GlcNAc)(5). The lysozyme was al so expressed in an E. coli strain auxotrophic for methionine, allowing for the incorporation of [methyl-C-13]methionine into the enzyme. An alteration of the [H-1-(13)]IHMQC NMR spectra of the labelled enzyme w as observed in the presence of(GlcNAc)(5). Commercially available nitr ophenyl glycosides did not act as substrates for lambda lysozyme, The results indicate that lambda lysozyme has specific interactions with o ligosaccharides of N-acetylglucosamine, but is incapable of hydrolyzin g these sugars. The relevance of the structure of peptidoglycan to the activity of lambda lysozyme is discussed.