in] PLANT ENZYMES 23 



From zymin some of the enzymes, e.g. invertase and the glucoside- 

 splitting enzyme, can be extracted with w«ater: other enzymes, e.g. zymase 

 and maltase, are not so readily extracted. From the living cells the 

 enzymes are only obtained with difficulty, the extraction of yeast juice, 

 containing zymase and other enzymes, needing, by Buchner's method, 

 a pressure as great as 500 atmospheres. 



In connexion with alcoholic fermentation by zymase, the following 

 point is of special interest. For carrying out this process, another sub- 

 stance is necessary in addition to the phosphate and enzymes already 

 mentioned, i.e. a thermostable co-enzyme of unknown nature. The sepa- 

 ration of zymase from the co-enzyme has been accomplished by filtering 

 expressed (Buchner) yeast juice through a special form of gelatine filter 

 under a pressure of 50 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. Take 50 gms. of bakers 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, (a) Detection of carbon dioxide. It has been shown 

 (Harden, 4) that the greater the volume of sugar solution used with a given weight 

 of zymin, the weaker is its action. To demonstrate its activity, therefore, it is best 

 to use not more than 5-10 c.c. of 10 7o glucose solution for every 2 gms. of zymin. 

 Place the mixture in a test-tube and fit it with a cork and glass tubing, the latter 

 dipping under a solution of lime water in a test-tube. Place the test-tube containing 

 the zymin and glucose solution in a beaker of water and warm to 35-40° C. Bubbles 

 of carbon dioxide will be evolved and will produce a precipitate of calcium carbonate 

 in the lime water. A control experiment should be made using boiled zymin. 

 (6) Detection of alcohol. Into a small flask put 8 gms. of zymin, 20 c.c. of 10 7o glu- 

 cose solution and a little toluol. Keep the flask in an incubator at 37-40° C. for 

 12 hours. Then filter through filter-paper (or linen) into a small distilling flask. 

 Distil over one half or two-thirds of the original volume. Add to the distillate in a 

 test-tube, 3-5 c.c. of iodine in potassium iodide solution and then 5 % caustic soda 

 until the colour vanishes. Shake up and warm gently in a beaker of water to 60° C. 

 A smell of iodoform will be detected and a yellow crystalline deposit of the same 

 substance will appear in the tube on cooling and standing. Examine the crystals 

 under the microscope and note their characteristic star-like shape. 



