DISSOLVING ENZYMES. 79 



A very easy method of obtaining the enzym in large quantity in a solid state is by precipita- 

 tion from the juice of decayed plants with 5 to 6 times the quantity of absolute alcohol. By this 

 method the slime, swollen in the fluid, is precipitated in white particles which contain the enzym 

 and the bacteria. The precipitate is purified by 5 or 6 decantings with alcohol allowed to stand for 

 a day in ether, changing the latter often, then filtered and dried. There results a gray white mass, 

 easily breaking up in a mortar to a fine powder which, when water is added, swells up again to a 

 viscous fluid, the latter when filtered furnishes a very clear filtrate with only a few dead bacteria. 

 When this filtrate is used in the same dilution as the original juice its dissolving power is as great as 

 that of the latter. This enzym solution, whether from potatoes, carrots, or onions, is neutral to 

 litmus. The activity of the dry enzym powder is not diminished after it has been kept for four 

 months. A quicker way of obtaining this enzym solution is by the addition of a disinfectant to the 

 juice to check the action of the bacteria without essentially diminishing the power of the enzym. 

 The substances tried were ether, chloroform, cyanide of potash (2 per cent), mercuric chloride (o. i and 

 0.2 per cent) and formalin (o. i and 0.2 per cent). The most satisfactory are formalin and chloroform, 

 a 0.2 per cent solution of the former kills the bacteria without noticeably diminishing, at least in the 

 beginning, the action of the enzym. A o. i per cent solution does not always produce a sterile 

 solution. Chloroform is usually as sure as 0.2 per cent formalin, and has no effect on the enzym 

 action at the expiration of a longer time. On the other hand ether only restricts the bacteria in their 

 development without killing them. Potassium cyanide was less sure in its effect. One-tenth per cent 

 mercuric chloride did not always kill all the bacteria; 0.2 per cent was safer, although resulting, 

 especially in the potato juice, in the throwing down of large quantities of quicksilver in the form of 

 mercuric sulphide. Two-tenths per cent sublimate had a very injurious effect on the enzym. 



Juice which is kept for 24 hours without the addition of any disinfectant has a very offensive 

 odor. In 3 days, however, it still has the same tissue-dissolving power as in the beginning. After 

 6 days this was very much diminished and in 15 it had vanished altogether. Juice to which chloro- 

 form and ether had been added, had at first the same dissolving power as that containing no disin- 

 fectant. Diminution of this power in the juice treated with chloroform was evident first after 15 days, 

 while in that treated with ether there was no change, although the bacteria in the latter were not 

 killed but only checked in development. A o. i per cent solution of formalin did not make the juice 

 sterile; when 0.2 per cent was added, at the end of 19 hours the dissolving power was as great as 

 in the juice not treated. At the end of 43 hours it was noticeably diminished, but at the end of 6 

 days it was greater than in that of the juice not treated; at the end of 15 days it was very slow but 

 still evident. One-tenth per cent solution of mercuric chloride did not kill the bacteria, but clearly 

 restricted their development. At the end of 19 hours the action of the enzym was essentially weakened 

 then gradually in the solution which contained sulphur compounds the mercury was thrown down 

 as mercuric sulphide, and the solution showed at the end of 15 days a renewed strong action. Two- 

 tenths per cent sublimate killed the bacteria but weakened very much from the beginning the action 

 of the enzym, although at the end of 15 days the latter was not completely destroyed. 



In another series of experiments in which the disinfectant was added to a solution free of bacteria 

 that is, to the alcoholic precipitate of potato juice and water, the result was the same. At the end 

 of three weeks the solution which was kept without any disinfectant and those treated with ether 

 and with chloroform showed very powerful enzym action. This was also true at the breaking off 

 of the experiment two weeks later. Solutions treated with o. i and 0.2 percent formalin and sublimate 

 had at the end of three weeks only very weak dissolving power; at the end of 5 weeks this was 

 completely lost in the solution treated with formalin. 



To test the dissolving power of the enzym on the middle lamella in tissues from various sources, 

 thin razor sections were used. These were immersed in the enzym solution and from time to time 

 examined under the miscroscope ; they were also tested by a light pressure on the cover glass. 



The potato juice acted almost twice as quickly as the onion juice while the action of carrot juice 

 was intermediate. In general, thin sections of carrot immersed in potato juice were completely 

 broken up in ten minutes 20 minutes at the latest so that only with difficulty could they be 

 lifted out of the fluid, and the slightest pressure on the cover glass broke them up completely into 

 individual cells. With the onion juice this result was obtained in 30 to 40 minutes. Neutralized 

 sap worked no better, so that the poorer dissolving power is to be attributed to the less quantity of 

 the enzym, since the growth of bacteria on onion is less luxuriant and rapid than on potato, as the 

 result of the greater acidity of the former. These sections, when examined under the miscroscope, 

 showed the same phenomena described as occurring in the diseased plants: middle lamella dissolved, 

 cells separated one from another, membrane somewhat thicker in the cells of the diseased portion 

 than in normal tissue, outer contour indistinct. Further changes of the membrane after a longer 

 immersion in the enzym solution have never been observed. The membrane always gave the cellulose 

 reaction. 



