EXPERIMENTAL STUDY OF BACTERIAL ENZYMES 29 



The enzymatic activities studied in intact cell suspensions 

 may be complicated by such factors as the rate of passage of 

 the substrate through the cell-wall, the removal of reaction 

 products by other enzyme systems, and differences between 

 the physico-chemical conditions holding inside and outside the 

 cell. Also with complex reactions which may involve many 

 small changes, each catalysed by a specific enzyme, it is 

 difficult to determine whether a given change is the result of 

 the action of one or more enzymes. This can only be decided 

 by preparing the enzymes in a cell-free state, studying their 

 action m vitro, and separating them by methods of protein 

 separation and purification. Where bacteria produce extra- 

 cellular enzymes, these can be easily prepared in the cell-free 

 state by filtering the cells from the medium and then removing 

 the enzyme from the filtered medium by precipitation or by 

 adsorption on to a suitable adsorbant such as alumina or 

 calcium phosphate gel. The study of intracellular enzymes 

 involves rupture of the cell-wall prior to purification pro- 

 cedures. The cell-walls of bacteria are very resistant and 

 special methods have been devised to rupture them in such a 

 way as to liberate the enzymes in an active state. The methods 

 that can be used depend to a certain extent upon the relative 

 resistance of the enzyme concerned and of the cell- wall to the 

 treatment. The following are some of the more common 

 methods used: 



1. Thick washed suspensions of cells are treated with 

 denaturing agents such as toluene, acetone, acetone-ether 

 mixtures, Or by simple drying. If the enzymes survive such 

 treatment they can often be extracted from the denatured 

 cell debris by incubation in buffer solutions. 



2. The cell suspensions are incubated with proteolytic 

 enzymes such as pepsin, trypsin, or papain and the debris 

 extracted with buffer solutions. 



3. Mechanical disintegration in some form of ball-mill. 

 The simple ball-mill is usually ineffective but an effective 

 crushing-mill consisting of steel cylinders rotating in a race 



