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Studies on purification and characterization of an autolytic enzyme from cell walls of Bacillus subtilis



Graduation date: 1968The purpose of this investigation was to purify and characterize\ud the autolytic enzyme from cell walls of Bacillus subtilis 168. The\ud crude enzyme was obtained by autolysis of purified cell walls in\ud buffer at 37° C. Two purification methods were developed. The first\ud involved fractional precipitation with ammonium sulfate. The enzyme\ud activity was found in the 30-85% fraction. Purification by this\ud method was 3.2 fold while the recovery was 35%. A second, more\ud efficient, method was developed using ethanol as a precipitant in the\ud presence of 0.1 M NaCl. Crude autolysates were precipitated with\ud 75% cold ethanol. The precipitate was dissolved and exposed to 33%\ud ethanol. This fraction was further purified by chromatography on\ud Bio Gel A50m. Active fractions were pooled and concentrated. This\ud scheme resulted in a purification of 14 fold and a yield of 31%.\ud With heat-inactivated cell walls as substrate the partially\ud purified autolytic enzyme was active at temperatures from 30°C to\ud 62°C with maximum activity at 54°C. The pH optimum was broad\ud (7-10); maximum activity occurred at pH 9-9.5.\ud Divalent cations were required for activity. Activation\ud occurred with Ba⁺⁺, Ca⁺⁺, Mg ⁺⁺, and Mn ⁺⁺. The reaction was\ud inhibited by Fe⁺⁺ and Cu⁺⁺. Solutions of enzyme were stable for several hours at room\ud temperature and for at least two months at -20°C. Activity was unaffected\ud by freezing and thawing during this period. Lyophilization\ud caused a 50% reduction in activity.\ud No evidence for proteolytic activity was found.\ud The partially purified enzyme contained 3% tightly bound organic\ud phosphorus which was assumed to be in teichoic acid. This complex\ud was not dissociated by several physical methods such as electrophoresis,\ud ion-exchange chromatography, and gel filtration. These\ud findings permitted tentative characterization of the enzyme as an\ud acidic glycoprotein

Year: 1967
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