1901.] H. H. Manu— 27ie Mnzymes oj the Tea Leaf. 1G5 



more than one layer of cells. I have not been able yet to more exactly 

 ascertain the nature of these small black bodies. The second means of 

 ascertaining the whereabouts of the enzyme was to kill the leaf in chloro- 

 form vapour, when it became brown in a very few minutes, and then cut 

 sections of the leaf and leaf stalk as before. In the leaf precisely the 

 same occurred as was found by the first method, — the brown colouration 

 always commenced at points in the centre of the tissue. In the stalk the 

 result was very definite. Oxidation always occurred first just outside 

 the fibro-vascular bundles, then it took place just inside the same 

 layer, and thirdly the cells just inside the epidermis were attacked. A 

 third method gave results quite agreeing with this as to the stalk, but no 

 definite results were obtained with the leaf. In examining the sections 

 by this method, they were first left 12 hours in alcohol to extract the 

 tannin and precipitate the enzyme. They were then put in a drop of 

 water on a slide, a drop of guaiacum tincture immediately added and 

 the preparation then again washed with water. The blue compound 

 is soluble in alcohol, and the enzyme is soluble in water so that it is 

 necessary to do these operations as rapidly as possible. 



The result obtained showed a general blueing of the section, but 

 on leaving a short time in glycerine the parts to which the enzyme had 

 merely spread faded, and left the rest quite heavily stained. The 

 fibro-vascular bundles were quite free from blue colour, and as for the rest 

 it was most intense first in the cells just outside this layer, second in the 

 point just inside it, and third just inside the epidermis. So far as the 

 stalk is concerned then, the several methods agree as to the points at which 



he greatest amount of enzyme is to be found, and this distribution is 

 almost exactly the same as that of the largest quantity of the tannic 

 acid. 



In general, therefore, with regard to the question already considered 



it has been established — 



(1) That an oxidase occurs in the leaf of the tea-plant used for 

 manufacturing tea. 



(2) That this oxidase is the principal agent in bringing about the 

 fermentation and colouring of the leaf. It is most active^ below 55°C. 

 and is destroyed about 80°0., is very sensitive to acids, and also to 

 alkalies, but not to quite the same extent. There is distinct evidence 

 that part of it usually occurs as a pro-enzyme in the leaf. 



(3) That it occurs in greatest quantity in the unopened tip leaf 

 of the shoot, and that the quantity decreases as the leaves get older, but 

 that the stalk contains at least the same amount as the tip leaf. 



(4) That leaf, taking into consideration gardens of the same type, 

 which contains the most enzyme makes the most highly flavoured tea. 



