PROTEOLYTIC ENZYMES OF SOIL FUNGI 511 



cubic centimeters of the liquid or a portion of the treated myce- 

 hum, equivalent to 0.5 to 0.8 gram of the dried substance per 100 

 cc. of 1 per cent solution of substratum were used. In several 

 cases Griibler's trypsin and pepsin were used for comparison of 

 the enzyme activities. The protein solutions were heated to 

 boiling so as to kill the proteolytic enzymes that they might pos- 

 sibly contain. In all cases a check was made by boiling the pro- 

 tein with the added enzyme, then incubating it in parallel with 

 the enzyme cultures. The proper reaction was always obtained 

 by the use of 0.1 N KOH or HCl. Both chloroform and toluene 

 were used to keep the solutions sterile. The substrata, plus 

 enzyme, as well as the blanks, were incubated at 37°C. After 

 the proper incubation period, 2 cc. portions of the solution were 

 used for the determination of amino nitrogen by the use of the 

 micro-apparatus of Van Slyke (1911, 1913). DupHcate deter- 

 minations were made, but these always checked very well, so 

 that only the averages are given in the tables. The amino nitro- 

 gen content was always figured back to 100 cc. of solution. 



As substrata the following proteins were used: (1) Merck's 

 peptone; (2) casein prepared after Hammarsten, each gram at 

 first being dissolved in 8 cc. of O.In KOH, then brought to the 

 proper reaction by the use of O.In HCl; (3) crystalline egg-albu- 

 men prepared from fresh eggs using the method of Hopkins and 

 Pincus (1898); (4) Merck's fibrin, 1 gram per 100 cc. of water. 

 The concentration of all the substrata was made up in such a 

 manner as to constitute 1 per cent solutions upon the addition 

 of the proper amount of enzyme. 



It appeared desirable to obtain, first, an insight into the 

 nature of the exo- and endoenzymes of several microorganisms. 

 Dox (1910) claimed that the same enzyme may, in the earlier 

 stages of growth of the organism, function within the cell, and 

 later be liberated into the substratum as an extracellular enzyme. 

 Abderhalden and Pringsheim (1910) maintained that the absence 

 of enzymes in the liquid obtained by the Buchner method can- 

 not serve as a criterion for the absence of particular enzymes; 

 the mycelium itself from the culture has to be studied for the 

 presence of the enzyme, which may not have diffused into the 

 liquid medium. 



