Streptococcal bacteriophage 12/12-borne hyaluronidase and its characterization as a lyase (EC 4.2.99.1) by means of streptococcal hyaluronic acid and purified bacteriophage suspensions.

Abstract

Hyaluronic acid was obtained from filtrates of heat-killed cultures of Streptococcus pyogenes group A, strain K56, by simple ethanol precipitation and treatment with an adsorbent. The hyaluronic acid is pure as judged from chemical and sedimentation analyses. Particles of streptococcal bacteriophage 12/12 were isolated from phage-lysed group A streptococci by polyethylene glycol precipitation and isopyenic centrifugation. Electron micrographs of negatively stained preparations showed a typical Bradley group B virus with a long, flexible, cross-striated tail and a knob- or star-like structure at the distal tip of the tail. The hyaluronic acid is depolymerized upon incubation with the phage 12/12 virions. After extensive digestion, a mixture of at least four oligosaccharides is formed, the two smallest of which are a tetra- and octasaccharide terminating in reducing N-acetyl-D-glucosamine. The tetrasaccharide shows an absorption maximum at 231.5 nm with a molar extinction coefficient epsilon = 4820 litres X mole-1 X cm-1, and it is therefore concluded that the bacteriophage-borne hyaluronidase catalyses a beta-elimination. Accordingly it is classified as a hyaluronate lyase (EC 4.2.99.1).