POLYSACCHARIDE-SPLITTING ENZYMES 309 



The bacteria are grown in a solution of 2 per cent casein hydrolysate 

 (pH 7.0) at 37 °C. and under conditions of strict aerobiosis; the cells 

 from the 16 hour old culture, separated by centrifugalization, are re- 

 suspended in small amounts of distilled water. 



A medium is prepared consisting of 0.1 per cent capsular polysaccha- 

 ride and 0.1 per cent NaCl in distilled water. This medium is distrib- 

 uted in 25 cc. amounts in large Erlenmeyer flasks (1 liter capacity) to 

 provide for aerobic conditions, and each flask is inoculated with the cells 

 recovered from 500 cc. of the culture of the SHI bacillus in the casein 

 hydrolysate medium. The material is incubated for 12—18 hours at 

 37°C. and the cultures tested to ascertain the disappearance of the spe- 

 cific polysaccharide and the absence of contaminants. The cultures are 

 now frozen and thawed repeatedly to secure the release of the endocel- 

 lular enzyme. 



The enzyme is ultimately separated from the cell debris by filtration. 

 However, since the cell suspension is very viscous, it is first subjected 

 to the following treatment. The cell suspension is made alkaline to pH 

 10.0 by the addition of sodium borate. Equimolecular concentrations of 

 dibasic sodium phosphate and calcium chloride are then added to bring 

 about a heavy precipitate of calcium phosphate which facilitates the 

 clarification of the material by centrifugalization; (it had been estab- 

 lished previously that the enzyme is not adsorbed on the calcium phos- 

 phate at alkaline reaction). The supernatant which contains all the en- 

 zyme in solution is now passed through a Seitz filter, then through a 

 Berkefeld (V) filter. It is important to observe that the enzyme is com- 

 pletely adsorbed on the asbestos pad of the Seitz filter; this, however, 

 can be prevented by washing the filter with nutrient infusion-peptone 

 broth previous to filtration. After this treatment, the enzyme passes 

 through the filter without loss. The potency of this filtrate is such that 

 0.002-0.004 cc. are required to decompose 0.01 mg. of the capsular 

 polysaccharide under the conditions of the test. 



Dubos found that the SHI bacillus grew abundantly in casein- 

 hydrolysate medium, but in this medium the organism did not form 

 any appreciable amount of the enzyme responsible for the decom- 

 position of the polysaccharide. When, however, large numbers of 

 the bacilli grown in the casein medium were re-suspended in a solu- 

 tion of the capsular polysaccharide which constituted the sole 

 source of carbon, the specific carbohydrate substrate was rapidly 



