[Reprinted from The Medical News, October 1, 1892.] 
THE ENZYMES OR SOLUBLE FERMENTS 
THE HOG-CHOLERA GERM.1 
By E. A de SCHWEINITZ, Ph. D„ 
BIOCHBMIC LABORATORY, BtrR"E'A'U OF ANIMAL INDUSTRY, DEPARTMENT 
OF AGRICULTURE, WASHINGTON, D. C. 
In the Archivfitr Hygiene, 1890, vol. x, pt. 1, p. 1, 
Fermi describes the detection and isolation of solu- 
ble ferments from the cultures of a number of differ- 
ent germs. Some of these germs belong to the 
class that produce gas (principally CO,2 and H) 
when grown in media containing glucose or glucose- 
forming substances, and as a rule the gas producing 
germs when cultivated upon proper media have been 
found to be ferment builders. 
As has been shown by Smith and other investi- 
gators, the hog-cholera germ produces large quanti- 
ties of gas when grown in peptonized beef-broth 
containing 2 per cent, of glucose. About one-fourth 
of this volume is hydrogen and three-fourths car- 
bon dioxide, the remaining liquid containing acetic 
and succinic acids, as I have found by analysis. 
To detect and isolate the soluble ferments of the 
hog-cholera germ, if any, several different culture- 
media have been tried, but the one that has given 
1 Read before the Chemical Section of the American Associa- 
tion for the Advancement of Science, Rochester, August 17, 1892. 2 
the most satisfactory results is sterilized milk. In 
plain peptonized beef-broth media but very small 
amounts of these ferments were produced, some- 
times none. 
Upon agar-agar the growth of the germ is rapid, 
and from the watery extract of the surface growth a 
mixture of two ferments has been obtained that will 
convert starch into glucose and liquefy gelatin-in 
other words, ferments that act as diastase on the 
one hand and as trypsin on the other. 
If now we take either skimmed or fresh milk, care- 
fully sterilize it, and then inoculate it with the hog- 
cholera germ, after about three weeks the milk has 
become thin and watery in appearance, a curd-like 
coating has formed on the surface, and the reaction, 
if the growth has been normal, has become either 
neutral or alkaline. The germs may now be re- 
moved by filtering the milk through a carefully 
sterilized Pasteur tube, or they may be killed by 
heating the solution to 540 C. for several hours, or 
still better, by the addition of a saturated solution of 
thymol. 
From the cultures sterilized in this way the addi- 
tion of absolute alcohol throws down a voluminous 
precipitate consisting of albumose, a little peptone 
and soluble enzymes. 
A purification of this precipitate and partial sepa- 
ration of enzymes from the accompanying albu- 
minoid matter is attained by a re-solution in water 
and precipitation with basic calcic phosphate. The 
enzymes are carried down mechanically by this, 
and can be dissolved out by water and reprecipitated 
with alcohol. After repeated treatment of this sort 3 
a small quantity of white powder is finally obtained, 
which will liquefy gelatin, digest fibrin and albu- 
min, and convert starch into glucose. 
To separate the diastase-like ferment from the 
liquefying ferment, the mixed ferments were rubbed 
up with glycerin and filtered. The solution in 
glycerin contained the hydrating ferment, while the 
undissolvcd portion taken up again in water and 
precipitated by alcohol does not act as a hydrating 
agent, but as a digestive agent. 
The trypsin-like ferment can also be precipitated 
by means of saturated salt-solution. This latter 
ferment seems to be formed in much smaller amount 
than the former. 
The action of both of these ferments is destroyed 
above 550 C. They contain nitrogen, but when 
pure do not give the general albuminoid reac- 
tions. 
In order to try and obtain these ferments in a 
purer form, and possibly larger quantity, an artifi- 
cial culture-medium proposed by Fermi, in the 
Archiv. fur Hygiene, vol. xiv, for use with other 
germs, was tried for the hog cholera bacillus. This 
contained 1 per cent, ammonium phosphate, o. 1 
per cent, acid potassium phosphate, 0.02 per cent, 
magnesium sulphate, and from 4 to 5 per. cent, 
glycerin. The hog cholera germ grows vigorously in 
this liquid, and after from three to four weeks the 
cultures become alkaline in reaction. They give 
also the peculiar amine odor characteristic of the 
hog-cholera cultures. From these artificial steril- 
ized cultures I have so far been unable to separate 
the diastase-like ferment, but a small amount of the tryptic ferment, showing that the ferment-production 
is dependent upon the medium. 
As for the physiologic effects of these ferments, 
Fermi holds that in general the soluble ferments are 
without great physiologic importance in the process 
of disease. 
The initial experiments that I have made upon 
guinea-pigs show that, so far as this particular dis- 
ease at least is concerned, the ferments are of great 
importance in the production of immunity. 
Guinea-pigs about a pound in weight each have been 
injected with a sterile solution in water of these fer- 
ments, the amount taken varying from 0.003 gram 
up to from 0.02 to 0.05 gram. The injections below 
0.01 gram were without ill-effect, beyond that there 
was a rise of temperature for several days. The 
injection of 0.05 gram was in several instances suffi- 
cient to kill the animals. The autopsies showed 
some injection of the bloodvessels of the intestines, 
but in other respects the animal was normal in con- 
dition. 
A single injection of 0.04 gram of the ferments 
was sufficient to make the animals immune to an 
inoculation with the hog-cholera germ that was suffi- 
cient to cause the checks to die in ten days. 
It is probable, therefore, that the soluble ferments 
exert in their way, at least in some diseases, a potent 
action in rendering the animal insusceptible to 
disease. 
It is possible, too, that to the indirect action of 
these specific ferments we may trace the protective 
and curative influence of the blood-serum of immune 
animals. 
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