522 SELMAN A. WAKSMAN 



The following experiment was made with the purpose of dem- 

 onstrating the influence of the incubation of the enzyme cultures 

 upon the amino nitrogen production, the reaction of the substrata 

 being neutral to litmus. 



When the velocities of reaction of the different enzymes are 

 compared, one finds in all cases an initial rapid increase in the 

 amounts of amino nitrogen produced, followed by a gradual 

 decrease in velocity. 



The enzyme cultures were incubated in all previous experi- 

 ments at 37°C. which was found to be the optimum temperature 

 for the action of the proteolytic enzymes derived from animal 

 tissues. But since the action of the enzymes of microorganisms 

 differs so much from that of the animal enzymes, it was thought 

 advisable to study the influence of incubation upon the activi- 

 ties of the enzymes used in this work. Both exo- and endo- 

 enzymes of A . niger grown for eight days on the Peptone-Czapek 

 medium were used. Twenty cubic centimeters of the exoen- 

 zyme containing filtrate and 0.8 gram of the endoenzyme con- 

 taining mycelium were added to peptone and casein solutions so 

 as to make the concentration of the substrata just 1 per cent; 

 the reaction was made neutral to litmus and the proper disin- 

 fectants were added. The cultures were incubated at 12°, 23°, 

 29°, 34°, and 39°C. for forty-eight hours; at the end of that 

 period the amino nitrogen was determined in all the solutions. 



The facts brought out in table 8 tend to show that the range 

 of temperature optimum for the action of the enzymes of A. 

 niger is comparatively great. Even at as low a temperature as 

 12°C, the splitting of the proteins still took place, the action 

 increasing with the increase in temperature. The optimum was 

 reached at 29° to 34°C,, followed by a drop in activity at 39°C. 

 We would naturally expect that an organism, whose temperature 

 optimum lies below the optimum for the enzymes of the warm 

 blooded animals, should produce enzymes which will act best at 

 somewhat lower temperatures. 



The question as to the nature of the enzymes of microor- 

 ganisms cannot be definitely answered as yet from the previous 

 experiments. It has been proven that both exo- and endo- 



