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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 Å; (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.

NCBI PubMed ID: 5096084
Journal NLM ID: 2985121R
Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
Institutions: Department of Physiological Chemistry, Johns Hopkins University School of Medicine, Baltimore, USA, Department of Biochemistry and Biophysics, University of California, Davis, USA, Department of Biochemistry, Phalavi University School of Medicine, Shiraz, Iran, Department of Microbiology, University of Virginia School of Medicine, Charlottesville, USA, Department of Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, USA
Methods: amino acid analysis, radiolabeling, UV, extraction, cell growth, enzymatic assay, spectrophotometry, centrifugation, gel electrophoresis

A capsular polysaccharide produced by Aerobacter aerogenes has been purified and shown to consist of galactose, mannose, and glucuronic acid in a molar ratio of 2:1:1, respectively. Partial acid hydrolysis liberated an aldobiouronic acid identified as 2-O-β-D-glucuronosyl-D-mannose. Analysis of capsular polysaccharide by Smith periodate degradation further indicated that all of the galactose residues are linked through C-3, all of the mannose residues are linked through both C-2 and C-3, and all of the glucuronic acid is at terminal, nonreducing positions. Digestion of capsular polysaccharide with a specific phage-induced polysaccharide depolymerase resulted in its nearly quantitative conversion to two limit oligosaccharides, termed A and B. Partial acid hydrolysis, methylation analysis, and periodate degradation indicated that A is the tetrasaccharide, Gal-1→3-(GlcUA-1→2)-Man-1→3-Gal. B was characterized as an octasaccharide composed of two units of A by the following procedure: Smith periodate degradation of B yielded the pentasaccharide mannosyl-α-galactosyl-α-galactosyl-α-mannosyl-α-lyxitol. This pentasaccharide was characterized and the anomeric configuration of the glycosidic bonds determined by sequential enzymatic degradation with specific exoglycosidases. Further analyses indicated that B is the octasaccharide, Gal-1α→3-(GlcUA-1β→2)-Man-1α→3-Gal-1α→3-Gal-1α→3-(GlcUA-1β→2)-Man-1α→3-Gal. A third oligosaccharide, termed C, was present in small amounts in extensive depolymerase digests of capsular polysaccharide. This oligosaccharide was shown to be a dodecasaccharide composed of three units of A. Exhaustive depolymerase digestion of C resulted in its slow degradation to yield equimolar amounts of A and B. These results indicate that Aerobacter aerogenes capsular polysaccharide is composed of repeating sequences of the following tetrasaccharide. Moreover, they also indicate that the phage-induced polysaccharide depolymerase is a glycanohydrolase which specifically cleaves the galactosyl-1 (α)/→ 3-galactose linkages in the capsular polysaccharide.

NCBI PubMed ID: 4328830
Journal NLM ID: 2985121R
Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
Institutions: Department of Physiological Chemistry, Johns Hopkins University School of Medicine, Baltimore, USA, Department of Biochemistry and Biophysics, University of California, Davis, USA, Department of Biochemistry, Phalavi University School of Medicine, Shiraz, Iran, Department of Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, USA
Methods: periodate oxidation, acid hydrolysis, GC, Smith degradation, paper chromatography, radiolabeling, methanolysis, enzymatic digestion, extraction, methylation analysis, paper electrophoresis, reduction, CC, cell growth, derivatization, evaporation, anthrone-sulfuric acid assay, carbazole method