Preparation and properties of insoluble forms of bacteriophage T4 lysozyme and chicken egg white lysozyme

Bacteriophage T4 lysozyme and chicken egg white lysozyme were covalently bound to cyanogen bromide activated Sepharose and to glutaraldehyde activated polyacrylhydrazido-Sepharose. The latter method seemed less favorable for T4 lysozyme, since the poly-acrylhydrazido-agarose conjugate exhibited low activity compared to the agarose conjugate. Whole bacteria (M.luteus and chloroform-treatedE. coli B cells) and the soluble uncross-linked peptidoglycan polymer fromM. luteus were used as substrates. Both types of conjugates exhibited low specific activity (lytic activity) toward insoluble substrates (cells), but surprisingly high specific activity toward the soluble substrate (hydrolytic activity). Product analysis showed that the enzyme conjugates retained their specificity of action, i.e., the same products were formed, and their rates of production were the same as those observed with the soluble (native) enzyme. The cell wall disaccharide-tetrapeptide GlcNAc-MurNAc-L-ala-D-gIu-(A₂pm-D-Ala) (C₆) inhibits the hydrolytic activity of both the native and the agarose bound T4 lysozyme. Only a slightly increased thermal stability was observed upon immobilization of T4 lysozyme, whereas the stability of the enzyme during storage and handling was greatly improved. The pH optimum of the lytic activity of Sepharose-T4 lysozyme was shifted about 1 pH unit to the alkaline side, compared to that found for the soluble enzyme, whereas no pH shift was observed for the polyacrylhydrazido-Sepharose conjugate. The optimum of the hydrolytic activity of Sepharose-T4 lysozyme was shifted to the acidic side. The pH optima of the lytic activity of the various lysozymes toward the bacterial cells were all very similar (>7), and differed greatly from the pH optima (<6) observed for their hydrolytic activities toward the negatively charged soluble peptidoglycan polymer. It is proposed that the observed differences in pH optima primarily reflect the basically different properties measured, i.e., the β(1–4) cleaving activity (hydrolytic activity), and dissolution process of the damaged cells (lytic activity).