Studies on the Fire-Blight Organism, Bacillus amylovorus 21 



Carbohydrases 

 Sucrase, or invertase 



Before this work was begun, the only enzyme known to be produced 

 by Bacillus amylovorus was invertase, or sucrase, demonstrated by 

 Stewart (1913). He found a moderate amount of this ferment pro- 

 duced in filtered pond water containing 2 per cent of Witte's peptone 

 and 1 per cent of cane sugar. 



Uschinsky's solution was made up in accordance with the formula 

 given by Duggar (1909), with the following alteration: 2 per cent of 

 sucrose was substituted for the- glycerin, and CaCl 2 was omitted. The 

 pH was adjusted to 6.95. In each of six 150-cubic-centimeter Erlen- 

 meyer flasks, 50 cubic centimeters of the solution was placed. Four 

 flasks were inoculated with fire-blight bacteria, the other flasks being 

 reserved as checks. On testing the solutions in the flasks with Fehling's 

 solution for reducing sugars two weeks after inoculation, the following 

 results were obtained: 



50 cc. 2-per-cent sucrose, with toluol added two days 

 after inoculation 



r>0 cc. 2-per-cent sucrose, inoculated but no toluol added 

 Checks, not inoculated 



Toluol was added to two of the flasks in order to determine whether 

 any large amount of the enzyme invertase was intracellular. More 

 invert sugar was found where the cultures were permitted to grow than 

 where toluol was added. In two weeks of growth many more bacterial 

 cells would develop and disintegrate than in two days. Probably more 

 cells developed and lysed in two weeks than had been formed in the 

 two-days-old culture at the time when toluol was added, so that it 

 cannot be decided from this test how much invertase, if any, is intra- 

 cellular. It is evident, however, that under these conditions invert 

 sugar is produced by the bacteria in larger quantities than is necessary 

 for growth. 



In each of six tubes, 10 cubic centimeters of Uschinsky's solution, 

 with 2 per cent of sucrose added, was placed. After three of the tubes 

 had been inoculated with B. amylovorus, all were incubated as described 

 above for the flasks, and at the end of two weeks the sucrose in all 

 six tubes was inverted and tested with Fcliling's solution, following 

 methods outlined by the Association of Official Agricultural Chemists. 

 It was found that the culture tubes contained 28.2 milligrams less 



