Catalytic and Molecular Properties of a Phage-induced Capsular Polysaccharide Depolymerase

Infection of Aerobacter aerogenes with a phage isolated from raw sewage induces the de novo synthesis of a polysaccharide depolymerase active against the capsular polysaccharide of the host organism. The enzyme exists in two forms, soluble and phage-bound. The soluble form has been purified to apparent homogeneity, as judged by disc gel electrophoresis and molecular sieve chromatography, and possesses the following molecular properties: (a) sedimentation coefficient, 10.7 S; (b) Stokes radius, 86.2 A; (c) molecular weight, 379,000; and (d) frictional ratio, 1.77. Treatment of depolymerase with sodium dodecyl sulfate at 42–62° results in dissociation to two apparently nonidentical subunits with molecular weights of 63,200 and 36,400. Depolymerase is a highly specific glycanohydrolase which randomly attacks the galactosyl-α-1 → 3-galactose linkages of the capsular polysaccharide; one susceptible bond occurs in each tetrasaccharide repeating unit of the polymer. Depolymerase activity may be conveniently assayed by the release of reducing groups; the pH optimum of the enzyme is 5.2. Treatment of capsular polysaccharide with NaIO4 and NaBH4 selectively degrades the glucuronate branches; the resultant polysaccharide is digested by depolymerase at less than 1% the rate of native capsular polysaccharide. The smallest oligosaccharide that depolymerase will degrade is a dodecasaccharide, termed C, composed of three tetrasaccharide repeating units. Reduced C is digested by depolymerase at 1% of the rate of capsular polysaccharide and is degraded in a highly specific manner; only the galactosylgalactose linkage immediately adjacent to the terminal, nonreducing tetrasaccharide repeating unit is attacked. The phage-bound and soluble depolymerases are identical with respect to their catalytic properties. Treatment of phage with either 8 m urea or 4 m guanidine·HCl results in quantitative solubilization of the phage-bound depolymerase activity. The solubilized enzyme is indistinguishable from the soluble enzyme isolated from cell lysates as judged by Sephadex G-200 column chromatography.