Concentration upon the Optimum Temperature of a Ferment. 409 



opinion, being of a speculative nature, was not deemed a sufficient answer 

 to the question; its experimental investigation therefore became the more 

 needed. 



The enzyme chosen was the malta.se of Aspergillus oryzce, the same 

 preparation being used as had already been studied in a former communica- 

 tion,* where its optimum temperature, in an action of 16 hours' duration, 

 and H + concentration that of the preparation simply dissolved in redistilled 

 water, is shown to be + 47°. 



Two stages occur in the investigation : (1) A determination of the 

 optimum reaction curve of the enzyme in an action of 16 hours' duration 

 at +47°, for chosen dilutions of substrate and of enzyme, in presence of 

 progressively increasing quantities of acid and of alkali added to the 

 reaction mixture ; (2) separate determinations of the optimum temperature 

 of the ferment under the same conditions of substrate concentration, of 

 enzyme concentration, and of duration of the experiment, for different 

 hydrogen ion concentrations of the medium, corresponding to various points 

 on the above optimum reaction curve. 



The substrate concentration chosen was M/20, or 18 x 10 -3 grm. of 

 hydrated maltose per cubic centimetre of the reaction mixture ; and the 

 enzyme concentration was 6 x 10~ 4 grm. of the enzyme preparation in powder 

 per cubic centimetre of the reaction mixture. 



For the determination of the optimum reaction curve the practical details 

 were as follows : — First, a solution of the enzyme was prepared by dissolving 

 30 mgrm. of the preparation in 10 cm. 3 of redistilled water. A clear pale 

 amber-coloured solution resulted, which, after standing from a half to one 

 hour at the ordinary temperature, was introduced in portions of 1 cm. 3 into 

 a series of eight clean test-tubes (Fischer's extra resistance glass) already 

 containing 90 mgrm. of hydrated maltose and either 4 cm. 3 of pure water 

 or a solution containing a known quantity of acid or alkali. The tubes, 

 after closing with clean sterile corks, were plunged into a water bath 

 regulated at +47°. After 16 hours' incubation they were withdrawn, the 

 corks removed, and each rapidly washed with 1 cm. 3 of water — the washings 

 being carefully added to the contents of the corresponding tube. Next, the 

 tubes were heated for- seven minutes in boiling water to stop the enzyme 

 action, after which they were cooled and the contents diluted to 50 cm. 3 . 

 The proportion of maltose hydrolysed was then determined, by Bertrand's 

 method,f on 20 cm. 3 of the diluted mixture. The numbers obtained are set 

 out in Table I. 



* Ibid. 



t ' Bull. Soc. Chim.,' (3), vol. 35, p. 1285 (1906). 



