160 H. H. Mai.n— 77/.' Enzymes of the Tea Leaf. [No. 2, 



effect after 2 hours, but after 4 hours only a slight reaction was ob- 

 tained until Hydrogen Peroxide was added, when a fairly intense blue 

 colour was produced with guaiacum tincture. After 18 hours there was 

 still the diiference, though even with Peroxide the colour was much less 

 intense. This is a sfcronc? indication that tlie latter reao;ent liberates the 

 enzyme from a compound (a pro-enzyme) in which it was much less 

 easily attacked by Alkalies tlian when already free. 



That we have here to deal with an oxidising enzyme was made 

 clear by its action with hydroquinone and with pyrogallol. In the for- 

 mer case darkening, indicating oxidntion, was very rapid in presence of 

 the enzyme, and much more so than in check solutions to which either 

 no addition was made, or to which even a boiled solution of the ferment 

 had been added. I was not able to isolate the product of oxidation. 

 The same rapid darkening took plnce in presence of the enzyme with 

 pyrogallol. In three hours the colour had become very dark brown, 

 while both the duplicates were hardly tinted brown. After 18 hours the 

 difference was extreme, the pyrogallol being almost entii ely oxidised in 

 the one case, only a light brown colour having been produced with boiled 

 ferment or with none at all. Gallotannic Acid behaved differ-ently 

 and showed itself far more resistant to oxidation than either of the above 

 substances, very slight change having taken place even after 18 hours. 



The reaction with Hydroquinone was so striking that it was used 

 to determine the optimum temperature for the activity of the enzyme. 

 Three solutions of Hydroquinone were prepared. No. 1 was kept at 

 ordinary temperature (26" C.) N^o. 2 at 50-55° C. No. 3 at 60-62° C. 

 After 1| hours No. J was hardly changed, while oxidation was proceeding 

 rapidly in Nos. 2 and 3 and no difference could be detected between 

 them. After 4 hours however while No. 1 still showed hardly any alter- 

 ation, No. 2 was far and away ahead of No. 3 in the progress of the 

 reaction. It was regrettable that there seemed no means of measuring 

 exactly this progress, but the experiment clearly shows that the best 

 temperature for the action of the ferment does not exceed 53° 0. and that 

 it is much more rapid at this temperature than at the usual temperature 

 at which the operation is carried out. 



The crucial test, however, as to the relation of this oxidase to the fer- 

 mentation of tea was whether when a solution of the enzyme was added 

 to tea juice, the colour which forms the mark of fermented tea was 

 produced more quickly than in a normal case. An experiment on this 

 ine was therefore made, and the colour was produced considerably 

 more quickly than in the untreated juice. An attempt was made to 

 utilise sterilised tea juice for this purpose, but the process of sterilising 

 to destroy the enzyme (as the ferment cannot be removed by any filtra- 



i 



