22 ENZYME ACTION [CH. 



atmospheres. The phosphate and co-enzyme can also be removed from 

 zymin by washing with water. The washed residue is then found to be 

 incapable of fermentation, as also are the washings. If, however, the 

 boiled washings are added to the washed residue, the system is synthesized 

 and can now carry out fermentation again. The chemical nature of the 

 co-enzyme, which is thermostable, and the precise part played by it in 

 the process, are as yet unknown (Harden, 4). 



Expt. 10. Preparation of zymin. (a) By alcohol and ether. For the following 

 experiments fresh yeast should be used which has been washed several times with 

 distilled water and dried on a filter-pump. Weigh out 25 gms. of yeast and stir 

 it up well in a mixture of 300 c.c. of absolute alcohol and 100 c.c. of ether for 4-5 

 minutes, and then filter on a filter-pump. Next wash the yeast with 50 c.c. of 

 ether. Then spread it out on a piece of filter-paper and allow it to dry for one hour. 

 (6) By acetone and ether. Take 50 gms. of yeast and stir it into 300 c.c. of acetone. 

 Continue stirring for 10 minutes, and filter on a filter-pump. The mass is then 

 mixed with 100 c.c. of acetone for 2 minutes and again filtered. The residue is 

 roughly powdered, well-kneaded with 25 c.c. of ether for 3 minutes, filtered, drained 

 and spread on filter-paper for an hour in the air. It can be finally dried at 45 C. for 

 24 hours. 



Expt. 11. Action of zymase. Fill a small test-tube with 40% glucose solution. 

 Add 2 gms. of the zymin (from Expt. 10) for each 10 c.c. of solution and a little 

 toluol, and shake. Then fit a slightly larger test-tube over the mouth of the first 

 tube. Now invert. A small quantity of air will rise to the top of the first tube 

 which, however, will remain filled with the sugar solution. Place the tubes either in 

 an incubator or water-bath at 22-25 C. A control set of tubes should also be arranged 

 containing sugar solution and zymin which has been previously well boiled in a little 

 water. Bubbles of carbon dioxide will be evolved from the unboiled zymin. 



Expt. 12. Action of maltase. (Harden and Zilva, 12.) Into two test-tubes, (a) and 

 (6), put the following : 



(a) 20 c.c. of a 2 % solution of maltose -f 0*5 gm. of zymin. 



(6) 20 c.c. of the same solution of maltose + 0'5 grn. of zymin which has been 

 well boiled in distilled water. 



Plug both tubes with cotton -wool, after adding a few drops of toluol, and place 

 in an incubator at 38 C. for 12-24 hrs. Test the liquid in both tubes by making the 

 osazone (see p. 49). Glucosazone will crystallize out in tube (a), maltosazone in 

 tube (6). 



Expt. 13. Action of peroxidase. (Harden and Zilva, 12.) Into four small 

 evaporating dishes, (a), (6), (c] and (d), put the following : 



(a) A suspension of 0'5 gm. of fresh yeast in 10 c.c. distilled water + 1 c.c. of 

 benzidine solution (1 / in 50% alcohol) + 2-3 drops of hydrogen peroxide (20 vols.). 



(b) A suspension of 0'5 gm. of zymin in 10 c.c. of distilled water + 1 c.c. of 

 benzidine solution + 2-3 drops of hydrogen peroxide. 



(c) A suspension of 0'5 gm. of washed zymin in 10 c.c. of distilled water + 1 c.c. 



