PROTEOLYTIC ENZYMES 121 



well-aerated media in the presence of Ca"^"^. The amount 

 of glucose added to the medium had to be such that the pH 

 at the end of growth had not fallen below pH 7. Gorini con- 

 tends that as well as activating the proteases, Ca"*" "*" also has 

 a stabilizing influence and that in the absence of Ca"*"^ these 

 enzymes are inactivated as fast as they appear in the 

 medium [19]. This view is opposed to that of previous 

 workers who believed that Ca"^ "*" stimulated the actual pro- 

 duction of the enzymes. When proteins and polypeptides 

 were used as a source of nitrogen, the growth of the culture 

 was dependent on its proteolytic activity, and in such condi- 

 tions Ca"*"^ was indispensable for growth [i8]. Sodium 

 fluoride and citrate, substances capable of combining with 

 Ca"^"^, inhibited these proteases, and although Mg"^"^ pro- 

 tected them against such inhibitors and from denaturation 

 by heat, Mg"^"^ could not replace Ca"^"^ as the cationic acti- 

 vator [17]. There is some evidence that the gelatinase activity 

 of cultures of B. suhtilis is dependent on the Mn"^"^ content 

 of the medium [38]. 



Although several workers have regularly demonstrated 

 activity in filtrates from cultures grown in media containing 

 glucose [30], other workers have reported that the presence 

 of fermentable carbohydrate inhibits the formation of pro- 

 teolytic enzymes [3]. Such effects are probably to be attri- 

 buted to the acidic products of carbohydrate catabolism 

 causing the pH of the medium to fall to a value which does 

 not favour the formation of proteases. In buffered media, or 

 in those where the pH does not become acid [cf. 17, 19] the 

 presence of fermentable carbohydrate has no effect [3]. 



Several attempts have been made to ascertain whether the 

 growth of proteolytic bacteria is supported by pure native 

 proteins, or only by those which have been denatured or 

 partially degraded. Such bacteria failed to grow when sub- 

 cultured into a medium containing inorganic salts and a pure 

 protein as a source of carbon and nitrogen [i]. This might 

 be explained on the basis that the synthesis and excretion 

 of an extracellular enzyme involves the utilization of energy, 



