PROTEOLYTIC ENZYMES II5 



system eventually contains no more susceptible bonds. 

 Tiselius, on the basis of an electrophoretic analysis of the 

 reaction mixture obtained by the treatment of egg-white 

 with pepsin, supported the former concept [39]. However, 

 chromatographic analysis has revealed that though the sys- 

 tem ultimately contained mostly tripeptides, a few dipep- 

 tides, no free amino-acids and still some undigested pro- 

 tein, in the initial stages of the reaction, deca- and higher 

 peptides were present [31]. 



Proteases of micro-organisms 



Only a small number of the proteases of micro-organisms 

 have been purified and only one has been crystallized [14]. 

 Apart from showing that they produce certain effects, e.g. 

 the liquefaction of collagen and gelatin, there have been few 

 attempts to express activity in terms of the hydrolysis of 

 peptide bonds, and virtually none concerned with their 

 specificity with respect to the bonds attacked [32]. Most of 

 the experiments with synthetic substrates have been con- 

 fined to the peptidases and no bacterial proteinase has been 

 examined in such detail as mammalian pepsin. In addition 

 to intracellular proteases, some micro-organisms also pos- 

 sess extracellular proteolytic enzymes and these enable pro- 

 teins in the environment to be degraded at least to small 

 peptides, if not to amino-acids, and the products are then 

 absorbed and may be further degraded by intracellular 

 enzymes. 



The biological activity of extracellular substances pro- 

 duced by micro-organisms can be detected by incorporating 

 the appropriate substrate in the culture medium or, more 

 usually, by testing the activity of the culture filtrate. The 

 test may be applied directly to the latter, or to a concentrate 

 of the active principle prepared from the culture filtrate by 

 evaporation in vacuo^ by precipitation with ammonium sul- 

 phate or ethanol, by freeze drying, or by an adsorption 

 technique. Enzymic activity of the experimental material is 

 detected by incubating with the appropriate substrate, toxic 

 activity by injecting the material into animals or by pre- 

 cipitation reactions with antisera, and antibiotic activity by 



