518 



SELMAN A. WAKSMAN 



substrata plus enzymes were incubated for forty-eight hours at 

 37°C. At the end of that period, the amino nitrogen content 

 of the cultures was determined; one set of cultures was then 

 made neutral to phenolphthalein (no. 2 in table 5), while the 

 others were left untouched (no. 1 in the table) ; the cultures were 

 then further incubated for forty-eight hours and amino nitrogen 

 determined. The results are given in table 5. 



It is apparent that, if the proteolytic enzymes of A. niger 

 are a mixture of several enzymes, the possibility of the existence 

 of one enzyme which may act in a reaction optimum for tryptic 

 activities is excluded, unless that enzyme was destroyed while the 



culture was incubated for the first forty-eight hours; which is 

 very doubtful, since the medium itself, where the enzyme is 

 produced, is very acid. The H-ion concentration of the filtrate 

 of the eight-day-old culture of A. niger grown on the Peptone- 

 Czapek medium was often as high as pH =2.0. The fact is 

 that the neutralization of the substratum to phenolphthalein, 

 after forty-eight hours of incubation at a neutral reaction, 

 checked the further activity of the enzyme, as is seen in table 5, 

 where the amount of amino nitrogen is found to increase in the 

 neutral culture, while the culture made alkaline gave no further 

 increase; the slight decrease found is due to the dilution of the 

 substratum through the addition of the alkali for change of re- 



