270 ENZYMES OF BACTERIA 



as calcium being essential for the action of tryptases and thrombase. The zymogen 

 may be a mixture of an enzyme with an inactivating agent or paralyzer; activation 

 then consists in the destruction of the paralyzer. An activator for enzyme A may 

 be the paralyzer for enzyme B, which acts destructively upon A, as in the action of 

 tryptase of yeast upon zymase. The proteolytic enzyme of the pancreas acts only 

 upon peptone, fibrin, casein, and protamines, but not upon true proteins; when 

 activated by enterokinase, it can act also upon these. This is, however, not a trans- 

 formation of a zymogen into trypsin, as previously assumed, but a stoichiometric 

 and reversible combination of trypsinogen and kinase to give a new enzyme possessing 

 new characteristics. 



The presence of specific nutrients in the medium regulates the quantitative forma- 

 tion of specific enzymes. Thus, the presence of starch will increase the amylolytic 

 power; the presence of proteins, the proteolytic power, etc. The digestive enzymes 

 are usually secreted readily into the medium and are found among the so-called 

 extracellular or exo-enzymes, since the colloidal nutrients (celluloses, starches, pro- 

 teins) cannot enter the cell; the enzymes of metabolism (respiratory enzymes, 

 zymases, etc.) are found among the so-called endo-cellular or endo-enzymes. The 

 same enzyme may be in a biological sense both an exo- and an endo-enzyme, the 

 difference between these two enzymes being one of degree rather than of kind. In 

 some cases, organisms can be cultured so that they produce a certain enzyme which 

 they do not form otherwise, as in the formation of a zymase capable of decomposing 

 galactose into alcohol. The nature of nutrition influences very markedly the nature 

 and abundance of the different enzymes. This is especially true of bacteria and other 

 micro-organisms. 



PREPARATION AND PURIFICATION OF ENZYMES 



The bacterial enzymes which are secreted into the medium can be obtained by 

 filtering the culture free from cells or by using the whole culture as the enzyme 

 preparation. Certain enzymes are obtained only by the destruction of the cell. A 

 slight injury of the cell, resulting in a change in permeability, may be sufficient to 

 allow some of the enzymes to be secreted readily into the surrounding medium. This 

 can be accomplished either: (i) by changing the surface tension of the cell, (2) by 

 bringing about autolysis of the cell, (3) by drying the cells first, then following by a 

 brief autolysis, or (4) by breaking up the cells by mechanical means, as in the use of 

 the Buchner press. The nature of the treatment will depend more upon the nature of 

 the cell than upon the nature of the enzyme. Some enzymes have as yet not been 

 separated from the cell substance, as in the case of lipase of seed or bacterial zymase. 



There are various methods available for the separation, purification, and concen- 

 tration of enzymes. None of these has resulted, however, in the preparation of an 

 enzyme in a pure state. The processes of purification consist in removing as nearly 

 as possible all extraneous matter. Frequently some of the enzyme itself is lost in the 

 process. Since enzymes are very labile, their separation from the accompanying pro- 

 teins, carbohydrates, and salts is accomplished by physical processes of adsorption, 

 elution, and precipitation rather than by chemical processes such as salt formation. 

 The enzyme is first brought into solution, using as a solvent water, dilute salt or very 

 dilute acid and alkali solutions. A phosphate buffer solution of a definite pH has been 



