THE CO-ENZYME OF YEAST-JUICE.
In the previous chapter reference was made to the fact that the
addition of boiled yeast-juice greatly increases the amounts of carbon
dioxide and alcohol formed from sugar by the action of a given volume
of yeast-juice.
When the boiled juice is dialysed the substance or substances to which
this effect is due pass into the dialysate, the residue being quite
inactive. In order to ascertain the effect on the process of alcoholic
fermentation of the complete removal of these unknown substances
from yeast-juice itself, dialysis experiments were instituted with
fresh yeast-juice, capable of bringing about an active production of
carbon dioxide and alcohol from sugar. It was already known from the
experiments of Buchner and Rapp [1898, 1] that dialysis in parchment
paper for seventeen hours at 0° against water or physiological salt
solution only produced a diminution of about 20 per cent. in the total
amount of fermentation obtainable, and in view of the less permanent
character of the juice from top yeasts a more rapid method of dialysis
was sought. This was found in the process of filtration under pressure
through a film of gelatin, supported in the pores of a Chamberland
filter candle, which had been introduced by Martin [1896].
In this way it was found possible to divide the juice into a residue
and a filtrate, each of which was itself incapable of setting up the
alcoholic fermentation of glucose, whereas, when they were reunited,
the mixture produced almost as active a fermentation as the original
juice [Harden and Young, 1905, 1; 1906, 2].
The apparatus employed for this purpose consists of a brass tube
provided with a flange in which the gelatinised candle is held by
a compressed india-rubber ring, and is shown in section in Fig. 5.
Two such apparatus are used, each capable of holding about 70 c.c.
of the liquid to be filtered. The tubes, after being filled with the
yeast-juice, are connected by means of a screw joint with a cylinder
of compressed air and the filtration carried out under a pressure of
50 atmospheres, [p060] the arrangement employed being shown in Fig.
6. In the earlier experiments 25 to 50 c.c. of yeast-juice were placed
in each tube and the filtration continued until no more liquid passed
through. The residue was then washed several times /in situ/ by adding
water and forcing it through the candle. The time occupied in this
process varied from six to twelve hours with different preparations of
yeast-juice. The candle was then removed from the brass casing and the
thick, brown-coloured residue scraped off, dissolved in water, and at
once examined. It was subsequently found to be possible to dry this
residue /in vacuo/ over sulphuric acid without seriously altering the
fermenting power, and this led to a slight modification of the method,
which is now conducted as follows. Two quantities of 50 c.c. each of
yeast-juice are filtered, without washing, and the residues spread
on watch-glasses and dried /in vacuo/. Two fresh quantities of 50
c.c. are then filtered through the same candles and the residues also
dried. The 200 c.c. of juice treated in this way give a dry residue
of 17 to 24 grams. The residue is then dissolved in 100 c.c. of water
and filtered in quantities of 50 c.c. through two fresh gelatinised
candles and the residue again dried. A considerable diminution in
weight occurs, partly owing to incomplete removal from the candle
and brass casing, and the final residue only amounts to about 9 to
12 grams. Occasionally it is necessary to repeat the processes of
dissolving in water, filtering, and drying, but a considerable loss
both of material and fermenting power attends each such operation.
[Illustration FIG. 5. (Filter Apparatus, Tube/Flange Detail--Ed.)]
The sticky residue dries up very rapidly /in vacuo/ to a brittle,
scaly mass, which is converted by grinding into a light yellow powder.
The filtrate was invariably found to be quite devoid of fermenting
power, none of the enzyme passing through the gelatin.
[Illustration: FIG. 6. (Filter Apparatus--Ed.)]
/Properties of the Filtered and Washed Residue./--The residue
prepared as described above consists mainly of the protein, glycogen,
and dextrins of the yeast-juice, and is almost free from mineral
phosphates, but contains a certain amount of combined phosphorus. It
also contains the enzymes of the juice, including the proteoclastic
enzyme, and the hexosephosphatase (p. 54). Its solution in water is
usually quite inactive to glucose or fructose, but in some cases
produces a small and evanescent fermentation. When the original
filtrate or a corresponding [p061] quantity of the filtrate from
boiled fresh yeast-juice is added, the mixture ferments glucose or
fructose quite readily. The following table shows the quantitative
relations observed, the sugar being in all cases present in excess:--
──────────────────────┬────────┬──────────┬────────┬────────┬──────────
│ │ Filtrate │ Boiled │ Water │ CO{2}
No. Material. │ Volume.│ added. │ Juice │ added. │ evolved.
│ │ │ added. │ │
│ │ c.c. │ c.c. │ c.c. │
──────────────────────┼────────┼──────────┼────────┼────────┼──────────
1 Undried and │ │ │ │ │
unwashed residue │ 15 c.c.│ 0 │ 0 │ 15 │ 0 g.
│ 15 " │ 15 │ 0 │ 0 │ 0·035 "
2 " " │ 15 " │ 0 │ 0 │ 15 │ 0·024 "
│ 15 " │ 0 │ 15 │ 0 │ 0·282 "
3 Undried and washed│ │ │ │ │
residue │ 25 " │ 0 │ 0 │ 0 │ 0·4 c.c.
│ 25 " │ 0 │ 25 │ 0 │268 "
4 " " │ 20 " │ 0 │ 0 │ 0 │ 8·3 "
│ 20 " │ 20 │ 0 │ 0 │ 90·3 "
5 Washed and dried │ │ │ │ │
residue │ 1 gram │ │ │ │
│ in │ │ │ │
│ 15 c.c.│ 0 │ 0 │ 0 │ 0 "
│ " │ 0 │ 12 │ 0 │108 "
6 " " │ 1 gram │ │ │ │
│ in │ │ │ │
│ 25 c.c.│ 0 │ 0 │ 0 │ 0 "
│ " │ 0 │ 25 │ 0 │364 "
──────────────────────┴────────┴──────────┴────────┴────────┴──────────
[p062]
These experiments lead to the conclusion that the fermentation of
glucose and fructose by yeast-juice is dependent upon the presence,
not only of the enzyme, but also of another substance which is
dialysable and thermostable.
Precisely similar results were subsequently obtained by Buchner and
Antoni [1905, 2] by the dialysis of yeast-juice. One hundred c.c. of
juice were dialysed for twenty-four hours at 0° against 1300 c.c. of
distilled water, and the dialysate was then evaporated at 40° to 50°
to 20 c.c. The fermenting power of 20 c.c. of the dialysed juice was
then determined with the following additions:--
(1) 20 c.c. of dialysed juice + 10 c.c. of water gave
0·02 gram CO{2}.
(2) 20 c.c. of dialysed juice + 10 c.c. of evaporated dialysate gave
0·52 gram CO{2}.
(3) 20 c.c. of dialysed juice + 10 c.c. of boiled juice gave
0·89 gram CO{2}.
It was shown in the previous chapter that phosphates are essential to
fermentation, and hence it becomes necessary to inquire whether the
effect of dialysis is simply to remove these. Experiment shows that
this is not the case. Soluble phosphates do not confer the power of
producing fermentation on the inactive residue obtained by filtration.
Moreover, when yeast-juice is digested for some time before being
boiled, it is found, as will be subsequently described, that the
boiled autolysed juice is quite incapable of setting up fermentation
in the inactive residue, although free phosphates are abundantly
present [Harden and Young, 1906, 2].
The filtration residue is never obtained quite free from combined
phosphorus, but the production from this of the phosphate necessary
for fermentation to proceed, may be so slow as to render the test for
co-enzyme uncertain, owing to the absence of sufficient phosphate.
When a filtration residue is being tested it is therefore necessary
to secure the presence of sufficient phosphate to enable the
characteristic reaction to proceed, and at the same time to avoid
adding phosphate in too great concentration, as this may, in the
presence of only small amounts of enzyme or co-enzyme, inhibit the
fermentation (p. 71). The proof that a filtration residue or dialysed
juice is quite free from co-enzyme is therefore a somewhat complicated
matter, and not only involves the experimental demonstration that
the material will not ferment sugar, but also that this power is not
imparted to it by the addition of a small concentration of phosphate.
As it has been found (p. 73) that the fermentation of fructose is
less affected than that of glucose by the presence of excess of
phosphate, the practical method of examining a filtration residue
for co-enzyme is to test its action on a solution of fructose (1)
alone and (2) in presence of a small concentration of phosphate. If
the residue produces no action [p063] in either case, but produces
fermentation when a solution of co-enzyme is added in the presence of
the same concentration of phosphate as was previously employed, it may
be concluded that this sample was free from co-enzyme but contained
enzyme; such an experiment also affords a definite proof that the
co-enzyme does not consist of phosphate.
This dialysable, thermostable substance, without which alcoholic
fermentation cannot proceed, has been provisionally termed the
co-ferment or co-enzyme of alcoholic fermentation. This expression
was first introduced by Bertrand [1897], to denote substances of
this kind, and he applied it in two instances--to the calcium salt
which he considered was necessary for the action of pectase on
pecten substances, and to the manganese which he supposed to be
essential for the activity of laccase. Without inquiring whether these
substances are precisely comparable in function with that contained
in yeast-juice, the term may be very well applied to signify the
substance of unknown constitution without the co-operation of which
the thermolabile enzyme of yeast-juice is unable to set up the process
of alcoholic fermentation. The active agent of yeast-juice consisting
of both enzyme and co-enzyme may be conveniently spoken of as the
fermenting complex, and this term will occasionally be employed in the
sequel.
The co-enzyme is present alike in the filtrates from fresh yeast-juice
and from boiled yeast-juice, and is also contained in the liquids
obtained by boiling yeast with water and by washing zymin or dried
yeast with water.
Practically the only chemical property of the co-enzyme, other than
that of rendering possible the process of alcoholic fermentation,
which has so far been observed, is that it is capable of being
decomposed, probably by hydrolysis, by a variety of reagents,
prominent among which is yeast-juice. This was observed by Harden and
Young in the course of their attempts to prepare a completely inactive
residue by filtration. In many cases a residue was obtained which
still possessed a very limited power of fermentation, only a small
amount of carbon dioxide being formed and the action ceasing entirely
after the expiration of a short period; on the subsequent addition
of boiled juice, however, a very considerable evolution of carbon
dioxide was produced. This was interpreted to mean that the residue
in question contained an ample supply of enzyme but only a small
proportion of co-enzyme, and that the latter was rapidly destroyed,
so that the fermentation soon ceased. The boiled juice then added
provided a further proportion of co-enzyme by the aid of which the
surplus enzyme was [p064] enabled to carry on the fermentation. This
view was confirmed by adding to a solution of a completely inactive
filtration residue and glucose successive small quantities of boiled
juice and observing the volumes of carbon dioxide evolved after each
such addition. Thus in one case successive additions of volumes of
3 c.c. of boiled juice produced evolutions of 8·2, 6, and 6 c.c. of
carbon dioxide. In another case two successive additions of 15 c.c.
of boiled juice produced evolutions of 54 and 41·2 c.c. On the other
hand, the enzyme itself also gradually disappears from yeast-juice
when the latter is incubated either alone or with sugar (p. 20).
The cessation of fermentation in any particular mixture of enzyme and
co-enzyme may, therefore, be due to the disappearance of either of
these factors from the liquid. If the amount of co-enzyme present be
relatively small it is the first to disappear, and fermentation can
then only be renewed by the addition of a further quantity, whilst the
addition of more enzyme produces no effect. If, on the other hand,
the amount of co-enzyme be relatively large, the inverse is true; the
enzyme is the first to disappear, and fermentation can only be renewed
by the addition of more enzyme, a further quantity of co-enzyme
producing no effect. It has, moreover, been found that the co-enzyme,
like the enzyme, disappears more rapidly in the absence of glucose
than in its presence, incubation at 25° for two days being as a rule
sufficient to remove all the co-enzyme from yeast-juice from top
yeasts in the absence of sugar, whilst in the presence of fermentable
sugar co-enzyme may still be detected at the end of four days.
In all the experiments carried out by Harden and Young with juice
from English top yeast it was found that when a mixture of the juice
with glucose was incubated until fermentation had ceased, the further
addition of co-enzyme in the form of boiled juice did not cause any
renewal of the action; in other words, the whole of the enzyme had
disappeared.
On the other hand, Buchner and Klatte [1908], working with juice and
zymin prepared from bottom yeast, observed the extremely interesting
fact that after the cessation of fermentation the addition of an equal
volume of boiled juice caused a renewed decomposition of sugar, and
that the processes of incubation until no further evolution of gas
occurred and re-excitation of fermentation by the boiled juice could
be repeated as many as six times. Thus in one experiment the duration
of the fermentation was extended from three to a total of twenty-four
days, and the total gas evolved from 0·73 gram to 2·19 grams. The
phenomenon has been found to be common to yeast from Munich and
[p065] from Berlin as well as to zymin and maceration extract, and
it was further observed that the boiled juice from one yeast could
regenerate the juice from another, although the quantitative relations
were different.
In these samples of yeast-juice, therefore, there is present a
natural condition of affairs precisely similar to that obtaining in
the artificial mixtures of inactive filtration residue and co-enzyme
solution made by Harden and Young. The balance of quantities is such
that the co-enzyme disappears before the enzyme, leaving a certain
amount of enzyme capable of exercising its usual function as soon as
sufficient co-enzyme is added. This establishes an interesting point
of contrast with the juice prepared from top yeast in England, in
which the enzyme does not outlast the co-enzyme [Harden and Young,
1907]. The difference may be due to some variation in the relative
proportions of enzyme and co-enzyme or of the enzymes to which
the disappearance of each of these is presumptively due, or to a
combination of these two causes. It was, however, found, even in the
juice from bottom yeast, that incubation for three days at 22° without
the addition of sugar caused the disappearance of the enzyme as well
as of the co-enzyme, and left a residue alike incapable of being
regenerated by the addition of co-enzyme or of restoring the power of
producing fermentation to an inactive mixture containing enzyme and
sugar.
If the fermenting power of the juice is to be preserved by repeated
regeneration for a long period, it is absolutely necessary to add the
co-enzyme solution each time as soon as fermentation has ceased, since
the enzyme in the absence of this addition rapidly disappears, even in
the presence of sugar.
This result is probably to be explained, at all events in the main, by
the presence in the co-enzyme solution of the antiprotease to which
reference has already been made [Buchner and Haehn, 1910, 2]. This
agent, the constitution of which is still unknown, protects proteins
in general from the action of digestive enzymes, and on the assumption
that the alcoholic enzyme of yeast-juice belongs to the class of
proteins, may be supposed to lessen the rate at which this enzyme is
destroyed by the endotryptase of the juice. This antiprotease is, like
the co-enzyme (p. 68), destroyed by lipase but is more stable than the
co-enzyme towards hydrolytic agents, and can be obtained free from
co-enzyme by boiling the solution for some hours alone or by heating
with dilute sulphuric acid. Such a solution possesses no regenerative
power, but still retains its power of protecting proteins against
digestion and of preserving the fermenting power of yeast-juice.
[p066]
It must, however, be remembered that the addition of a phosphate alone
may greatly prolong the period of fermentation of yeast-juice (p.
55), and sugar is well known to exert a similar action. It appears,
therefore, that the existence of the enzyme is prolonged not only
by the presence of the antiprotease but also by that of sugar and
hexosephosphate, into which phosphate passes in presence of sugar.
Similar effects are exerted on the co-enzyme by sugar and probably
also by hexosephosphate.
The fermenting complex, therefore, in the presence of these
substances, either separately or together, falls off more slowly in
activity and is present for a longer time, and for both of these
reasons produces an increased amount of fermentation. It seems
probable also that the hexosephosphatase is similarly affected,
so that the supply of free phosphate is at the same time better
maintained, and the rate of fermentation for this reason decreases
more slowly than would otherwise be the case.
It is in this way that an explanation may be found of the remarkable
increase in total fermentation, which is produced by the addition to
yeast-juice and sugar of boiled yeast-juice, containing free phosphate
(which passes into hexosephosphate) as well as co-enzyme, of boiled
autolysed yeast-juice, containing free phosphate but no co-enzyme, or
of phosphate solution alone.
In no case is the original rate of fermentation greatly increased
after the initial acceleration has disappeared, but in every case
the total fermentation is considerably augmented, and this is no
doubt mainly to be attributed, as just explained, to the diminished
rate of decomposition of the fermenting complex and probably of the
hexosephosphatase.
Although both enzyme and co-enzyme are completely precipitated from
yeast-juice, as already described (p. 38), by 10 volumes of acetone,
the co-enzyme is less easily precipitated than the enzyme, and a
certain degree of separation can therefore be attained by fractional
precipitation [Buchner and Duchaček, 1909]. The enzyme cannot,
however, be completely freed from co-enzyme in this manner, and the
process is attended by a very considerable loss of enzyme. This is
probably due to the fact that only small quantities of acetone can
be added (1·5 to 3 volumes), in order to avoid precipitation of
co-enzyme, and that the precipitates thus formed contain a large
proportion of water, a condition which appears to be fatal to the
preservation of the enzyme.
It is, however, not quite certain whether it is the zymase or the
hexosephosphatase which is destroyed in these cases, as no attempt
[Pg 067] was made to distinguish between them. In any case the
precipitates obtained by fractional treatment with acetone, even when
reunited, produce a much smaller fermentation than the original juice
or the powder prepared by bringing it into 10 volumes of acetone.
Attempts to isolate the co-enzyme from boiled yeast-juice have also
been hitherto unsuccessful. It has, however, been found possible to
remove a considerable amount of material from the solution without
affecting the co-enzyme. When 1 volume of alcohol is added to boiled
yeast-juice, a bulky precipitate, consisting largely of carbohydrates,
is produced, and the filtrate from this is found to contain the
co-enzyme and can be freed from alcohol by evaporation. Further
precipitation with alcohol has not led to useful results.
When a solution which has been treated in this way is precipitated
with lead acetate and kept neutral to litmus, the free phosphate
and hexosephosphate are thrown down and the co-enzyme remains in
solution. The filtrate can be freed from lead by means of sulphuretted
hydrogen and neutralised, and then forms a solution of co-enzyme free
from phosphate and hexosephosphate but still containing combined
phosphorus. More complete purification than this has not yet been
accomplished. Occasionally the precipitate of lead salts retains some
of the co-enzyme, apparently by adsorption, but usually the greater
part remains in the solution (Harden and Young).
The co-enzyme is partially removed from yeast-juice by means of a
colloidal solution of ferric hydroxide (Resenscheck). A precipitate
is thus obtained which contains phosphorus and resembles boiled
yeast-juice in its regenerative action on yeast-juice rendered
inactive by fermentation. It has not, however, so far been found
possible to isolate any definite compound from this precipitate. There
are also indications that when yeast-juice, either fresh or boiled,
is electrolysed, the co-enzyme tends to accumulate at the cathode
[Resenscheck, 1908, 1, 2].
Buchner and Klatte [1908] made use of yeast-juice rendered free from
co-enzyme by incubation with sugar solution to examine the nature
of the agent by which the co-enzyme is destroyed. This agent is
certainly an enzyme, since boiled yeast-juice can be preserved with
unimpaired powers for a considerable length of time, and suspicion
fell naturally, in the first instance, on the endotryptase of the
yeast cell. Direct experiment showed, however, that yeast-juice,
which, when fresh, rapidly destroyed the co-enzyme of boiled juice,
lost this power on preservation, but retained its proteoclastic
properties without diminution, so that the tryptic enzyme could not be
the one concerned. The direct action of commercial trypsin on boiled
yeast-juice also yielded [p068] a negative result, although this
cannot strictly be regarded as an indication of the effect of the
specific proteoclastic enzymes of yeast-juice. On the other hand, it
was found that when boiled juice was treated for some time with an
emulsion containing the lipase of castor oil seeds, the co-enzyme was
completely destroyed. This is a result of great importance, inasmuch
as it probably indicates that the co-enzyme is chemically allied to
the class of substances hydrolysable by lipase, i.e. to the fats and
other esters.
Further, observations by Buchner and Haehn [1909] have shown that
digestion with potassium carbonate solution containing 2·5 grams per
100 c.c. also brings about the destruction of the co-enzyme, and that
this is also slowly accomplished by the repeated boiling of the juice.
The co-enzyme is also destroyed both by acid and alkaline hydrolysis,
and when the solution is evaporated to dryness and the residue ignited.
Beyond this general indication nothing is known of the chemical
nature of the co-enzyme. The intimate relation of phosphoric acid
to the process of fermentation renders it not impossible that the
co-enzyme may contain this group, but there is no definite evidence
for such a belief. Purely negative results have been obtained
with all the substances of known composition which have yet been
tested, among these being soluble phosphates, hexosephosphates and
a number of oxidisable and reducible substances, such as quinol,
p-phenylenediamine, methylene blue, peptone beef broth, etc. (Harden
and Young; Harden and Norris [1914]; see also Euler and Bäckström
[1912]), glycero-phosphates (Buchner and Klatte).
The precise function of the co-enzyme is even more obscure than
its chemical nature. The system of reacting substances consisting
of fermentable material, enzyme and co-enzyme, bears, however, an
obvious superficial resemblance to many of the systems required for
the accomplishment of chemical changes in the animal or vegetable
organism. Such a triad of substances is, for example, requisite for
the process by which the red blood corpuscles of an animal are broken
up by the serum of a different animal into the blood of which the red
corpuscles of the first animal have been injected. This effect is only
produced when two substances are present, the amboceptor or immune
body and the complement. The analogy may be carried to a further
stage since the amboceptor is, like the co-enzyme, more thermostable
than the complement, which therefore corresponds with the enzyme.
Immune serum can, in fact, be freed from complement by being heated at
57-60° for half an hour, whereas the amboceptor is unaffected by this
treatment. On the other hand, the complement and amboceptor do not
[p069] appear to act like enzymes but rather like ordinary chemical
reagents, remaining in combination even after the blood corpuscle has
been broken up, whereas the enzyme and co-enzyme of yeast-juice are
again liberated when the reaction between sugar and phosphate has been
completed. [Pg 070]